Free radicals from single crystals of 2′-deoxyuridine

JOURNAL

OF MAGNETIC

RESONANCE

(1983)

%,225-246

Free Radicals from Single Crystals of 2’-Deoxyuridine K.VOITANDJ.HUTTERMANN Institut

fiir

Biophysik Postfach

und Physikalische Biochemie, Universittit 397, D-8400 Regensburg, West Germany

Regensburg,

Received April I, 1983; revised June 16, 1983 X-irradiation of single crystals of 2’deoxyuridine (UdR) between 8 K and room temperature gives rise to a variety of different radical structures, seven of which are described here. The dominant low-temperature species are the ** anion on the uracil base and alkoxy radicals. The former is characterized by the usual a-proton interaction at C6(- 1.24, -0.83, -2.1 mT) but displays additional interactions with the nitrogens on Ni and Ns. For the alkoxy radicals, there is marked variability in structures yielding two primary and one secondary species. Upon warming, an a-hydroxy alkyl radical at C, of the deoxyribose grows in showing an (Ycoupling (-0.84, - 1.42, -2.36 mT), a B interaction (3.1 mT) and an exchangeable OH coupling (-0.3 mT). Upon irradiation at room temperature, a 5yl and a 6-yl radical are the main radical centers both formed by net gain of a hydrogen at the 6 and 5 position of the base, respectively. Resides an (Ycoupling, the former species shows inequivalent fl protons (3.9, 4.9 mT) and interaction with the Ns-H group. The 6-yl radical has equivalent fl couplings of 3.3 mT. Storage of crystals for several months at 300 K results in loss of both 5- and 6-yl radicals and formation of two different species, one characterized by two &type interactions (4.2, 1.8 mT) and one exhibiting an a-type coupling (-0.53, -1.88, -0.98 mT). Tentative assignments for these species are given and the solid-state radiation chemistry of uracil derivatives with respect to nucleic acid radiation damage is discussed. INTRODUCTION

There are two main aspects of interest involved in the solid-state radiation chemistry of 2’-deoxyuridine (UdR, structure I), although this deoxyriboside derivative of the

OH

H I

RNA base uracil is neither a RNA nor a DNA constituent. For one thing, UdR is the unsubstituted reference compound for a series of Shalogen substituted deoxy225

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VOIT AND HiiTTERMANN

ribosides 5-chloro- (ClUdR), 5-bromo- (BUdR), and 54ododeoxyuridine (IUdR). Replacement of the DNA constituent thymidine (TdR), the 5-methyl substituted uracil deoxyriboside, by one of the 5-halogen substituted compounds in DNA enhances its in viva radiosensitivity (for a review about the biological and medical implications cf. (I)). An understanding of the molecular mechanisms of this sensitization at the free radical level necessitates the comparison of radical structures, concentrations, and reactions in the substituted vs the normal compounds. Having studied the 5halogen substituted deoxyribosides in considerable detail (2-6) over the past years and with several investigations on TdR available in the literature (7-9), we intend the present study on UdR to complete this series of deoxyuridine derivatives. The other aspect involved is the influence of the sugar or the sugar phosphate group on radical formation and decay by comparing free radicals in the deoxyriboside UdR with recent results from the riboside uridine (UR, (IO)), the ribotide uridine5’-phosphate (UMP, (11-14)) and the arabinofuranoside (UAra, (15)) containing the same base uracil. EXPERIMENTAL

Aqueous solutions of 2’-deoxyuridine (UdR, Pharma Waldhof) produced single crystals either by slow evaporation at room temperature or by cooling a saturated solution in a thermostatted bath. The monoclinic crystals are plates or needles elongated along b and show two different habits depending on the velocity of growth along a or c* as shown in Fig. 1. Both habits exhibit pronounced cleavage planes perpendicular to a. This feature, together with the elongation along b, served for identification of the habit which was, in addition, controlled by optical goniometry. Rahman and Wilson (16) have determined the space group as P2,with four molecules per unit cell. There are two molecules of UdR in the asymmetric unit (denoted (A) and (B)). The uracil bases of the dimer form an angle of 21’; the planes through C,,, C3,, Or and C4, of the two sugars are tilted by 77’. For both molecules the sugar is in the C&end0 conformation.

FIG. 1. External appearance of 2’-deoxyuridine (UdR) single crystals showing main growth habit (left) and minor modification (right) together with orthogonal axes a, b, c* used for ESR measurements.

RADICALS

FROM 2’-DEOXYURIDINE

227

X irradiation and ESRdata acquisition employing an a, b, P reference axis system with a and b coinciding with the crystallographic axes and data evaluation were performed as described previously (I 7). RESULTS

1. The IP Anion Radical Upon irradiation at both 8 and 77 K, one of the prominent features obtained is an anisotropic doublet with splittings ranging from about 0.8 to 2.1 mT which is assigned to the ?r* anion (II). An additional, anisotropic substructure is observed at 0

orientations close to the direction normal to the uracil bases. A series of spectra as obtained upon rotation of the crystals about c* is shown in Fig. 2 (top). Along a, the main doublet is seen to be split into an eight-line structure. Flanking the doubletoctet group is a broader feature two lines of which are clearly visible. From the variation of the doublet in this and the other two planes it follows that these outer two lines are part of a different radical, perhaps the a-hydroxy alkyl radical at carbon Cy of the deoxyribose to be discussed below. The experimental data for the variation of the g factor and the main doublet splitting of the anion are shown in Fig. 2 (middle and bottom, respectively) together with the calculated variation derived from the final tensors. These are given in Table 1. Figure 3 (top) displays an experimental spectrum for the orientation HIJa, i.e., normal to the planes of the bases, which was taken in the second-derivative mode for enhanced resolution. The corresponding spectrum for a crystal grown from D20 is shown in the middle part of Fig. 3. Clearly, the center part belonging to the anion displays a discernible substructure which, moreover, is different between crystals grown from Hz0 and D20. For the latter, after several candidates were tested, spectra simulation using the a-proton interaction for that orientation together with the coupling of two equivalent nitrogens of 0.44 mT each reproduces the experimental feature most satisfactorily as can be judged from the simulation given in the bottom part. Since the couplings are anisotropic and resolved well only along a, we feel the assignment to the two nitrogens at N1 and N3 is feasible and have, therefore, included these parameters in Table 1. The spindensity distribution calculated for the anion which is shown in Table 6 places indeed, after geometry optimization, equal spin on N, and N3 both in the UHF + CI and RHF treatment. The magnitude of the 14N

228

VOIT AND HOTTERMANN

,

2mT

:

c

-b

-a 7r*-anion

t

I

[Lll I

. .

. .

,

b

(

(

C”

,

,

(

Q

I

(

,

b

[mTl

0.6

j

7

b

b

FIG. 2. Set of experimental ESR spectra (first derivative, rotation about c*, -9.5 GHz) obtained at low microwave powers (~0.01 mW) from UdR crystals irradiated at 77 K (top) and spectral parameters from three planes of data of the ?y* anion assigned to the main doublet splitting; for the hyper6ne coupling (bottom), the variation derived from final tensor values includes second-order calculation to fit data in bc* plane.

couplings expected from these values of 0.06, however, is about half the value derived from the experimentally observed interaction. Nevertheless, the trend in spin densities obtained from the experimental values is reproduced by the calculations.

RADICALS

229

FROM 2’-DEOXWRIDINE TABLE 1

SPECTRALPARAMETERSOF THE ANION IN 2’-DEOXYURIDINE Direction cosines Angular correlation with molecular direction

Tensor

Elements

a

b

g

2.0015 2.0028 2.0036 2.0026

0.9662 -0.2156 -0.1408

0.0732 0.7542 -0.6524

0.2468 0.6201 0.7446

I( base normal; (A) 13”, (B) 9”

K

-1.24 mT -0.83 mT -2.10 mT

0.989 1 0.0488 -0.1386

-0.1301 0.7307 -0.670 1

0.0685 0.6809 0.7291

)I base normal, (A) lo”, (B) 18” 11C-H bond; (A) lo’, (B) 18” i C-H bond, in plane; (A) 12”, (W 4”

Iso.

-1.39 mT

N,

0.44 mT -0.0 -0.0 0.15 mT

-1.0

-0.0

-0.0

0.44 mT -0.0 -0.0 0.15 mT

-1.0

-0.0

-0.0

ISO.

Iso. N3 ISO.

C*

With a spin density of about 10% on N3 one would expect, following the results of several studies of pyrimidine 5-yl radicals (2, 3, 18, 28), a coupling of about 0.4 mT, in a crystal grown from H20, of the proton attached to that position. This expectation is corroborated by the present findings since the nine-line pattern of Fig. 3 (top) as compared to the octet in the deuterated crystal can be explained by the additional proton interaction of that magnitude. The anion lines decay between 140 and 180 K without forming a successor species. Bleaching of the anion with red light for about 3 hr at 77 K produces at most 1 to 2% conversion into the 5-yl radical which is discussed below. 2. Alkoxy Radicals There is a pronounced variability in the spectra observed after irradiation at 77 K under conditions (e.g., high microwave powers) which are known to favor detection of alkoxy radicals RCH26 or RIR2CH6 (I I, 24, 15, 19, 20). It was impossible to get consistent results allowing for the extraction of tensor data. For example, of five different crystals irradiated simultaneously in the same vessel, only one showed line correlations from which we were able to reach conclusions on the presence and type of alkoxy radical. In a test using polycrystalline powders of UdR we obtained a rising baseline in the appropriate g-factor range (-2.1 to 2.00) but never resolved spectra. It appears that the variability in results obtained from the single crystals is inherent to the crystal structure and not a result of impurities present or other artifacts.

230

VOIT AND HiiTTERMANN Experimental

Experimental

Simulation

FIG. 3. Experimental ESR spectra (second derivative, -9.5 GHz) from UdR crystals for H 11a showing subsplitting of main CYcoupling of # anion comprising nine lines in a crystal from Hz0 (top) and an octet in a D20-grown specimen (middle). The bottom spectrum is a simulation of the 40 case using two equivalent nitrogen couplings.

Two examples can be given for primary alkoxy radicals RCH& involving the Cs,OS, site. The upper part of Fig. 4 displays a spectrum obtained upon rotation of a single crystal about the b axis. The magnetic field direction is nearly parallel to a. Two different quartets of doublets which are indicated as stick spectra beneath the experimental patterns can be discerned from their g-factor separation. For the lowfield species (Al) the major couplings are 7.6 and 5.7 mT. There is an additional doublet subsplitting amounting to about 1 mT. The other species (A2), the lines of which flank the anion lines at g - 2.00, displays one coupling of 9.5 mT, another one of 2.5 mT, and a subsplitting of 0.6 mT. Since this spectrum is part of a full set of data obtained from rotation about b, a simulation of the line variation was performed for the two different CsP-O=,,directions of the dimer in the asymmetric unit. It was

RADICALS Primary

alkoxy

species

231

FROM 2’-DEOXYURIDINJ!

RCH,6

H II a

L

I

J

1 I

Al Secondary

alkoxy

H in ab-plane,

species

A2 R, R2CH6

20° off b axis

---Y

,L mT: I

I

L-f-

FIG. 4. ESR spectra (first derivative, -9.5 GHz) from different UdR crystal specimen irradiated at 77 K and measured at high microwave powers (20 mW) showing patterns of two primary alkoxy radicals (top) and of one secondary species (bottom).

found that, although the angle between the C-O bonds amounts to 60”, the lines for a primary alkoxy radical at these positions nearly coincide upon rotation about b and, moreover, closely agree with the experimental trend displayed by species (A2). This good correlation allowed us to reconstruct a g tensor with elements of 2.08, 2.00, and 2.00 where the direction of the value of 2.08 coincides approximately with the CY-OY bond in the undisturbed crystal lattice. The additional splitting of each of the quartet lines into a doublet of 0.6 mT spacing can be explained by the y-proton interaction of the C,, proton. The corresponding dihedral angle calculated according to Box and co-workers (21) is sufficiently small (22” for (A) and 26” for (B)) to warrant this assumption. There is no agreement, however, between the crystal structure and the line variation for species (Al). Nevertheless, its assignment to a primary alkoxy radical is unambiguous. We are unable, however, to estimate the degree of reorientation of the CY-05, bond for this species since we never obtained correlated data in three orthogonal planes. We note that the g factor, if we assume that it attains its maximum value in the ac* plane, is fairly large, 2.11. The bottom part of Fig. 4 gives a spectrum obtained from another crystal upon rotation about the a axis about 20” away from b. It shows a doublet of 5.6 mT spacing without substructure centered at a g factor of 2.13. This latter value is so large as to suggest the presence of another doublet upfield to yield a total quartet with a reduced g factor. The gamut of lines obtained for the rotation, however, definitively excludes this possibility. We have to assign, therefore, the doublet group to a secondary alkoxy species R,R$HC) with one P-proton interaction only. The simulation of the corre-

232

VOIT AND HOTTERMANN

sponding features for the only possible site in UdR, the OY position, yields a reasonable agreement for a g tensor with elements 2.13, 1.99, and 2.00. Comparison of the present data with those available for alkoxy radicals in the literature shows that the g factor for the primary species (A2) fits in well with the range of maximum tensor elements which are between 2.08 and 2.09 (19) whereas only one other substance, inositol (20), exhibits a g factor as large as that of species (Al). Although there are now data for several secondary alkoxy radicals RIR2CH6 available (14, 22-23), the maximum value of 2.13 for the g factor found here is considerably higher than values in any other crystal. It is interesting to note that a variability in the type of alkoxy radical stabilized has also been observed in 3’cytidilic acid which exhibits a primary species at 77 K (19) but a secondary one at 4.2 K (21). The decay of the alkoxy radicals in UdR follows, comparable to the radical structures, a complex pattern. Mostly, the alkoxy lines were lost between 90 and 120 K but in several experiments they vanished already at 77 K during a few hours. There is no single successor-type radical of the alkoxy species but it appears that the lines which flank the anion lines at 77 K (cf. Fig. 2, top) are gaining in intensity upon alkoxy decay. 3. The a-Hydroxy

Alkyl Radical at C,

Upon warming of crystals irradiated at 77 K there are several spectral changes discernible in the center part of the spectra but no definitive information can be gained below about 200 K at which temperature the lines of the anion (II) have vanished. Due to the variability in the decay pattern of alkoxy species and anion we were unable to obtain three complete planes of data but could, from several plots around the b axes, ascertain that at about 200 K there exists a quartet feature at

R, R,CH&H,OH-

FIG. 5. Set of experimental ESR spectra (second derivative, rotation about b axis, -9.5 GHz) obtained from UdR crystals after irradiation at 300 K and storage of specimen for 1 week at that temperature. Stick spectrum beneath orientation H(lc* indicates lines of a-hydroxy alkyl radical at carbon Cs comprising OL-and @-proton coupling. The OH coupling is not present in the D20-grown crystal.

RADICALS

FROM 2’-DEOXYURIDINE

233

8 -

2.0026 with intensities of about 1:2:2:1 spanning altogether about 5.0 mT and showing a subsplitting of about 0.5 mT. Subsequent to irradiation at room temperature, although there was severe overlap from lines of other radicals, we could extract more data for this species. Figure 5 exhibits one plane of data obtained at X band from a crystal grown from D,O which is rotated about b approximately one week after irradiation at which time the center

x*

19: 2.0035

2.0025

2.0020 I

b

!

I

I

r x \./

C*

2.0 -

b 6. Spectral parameters of a-hydroxyalkyl radical in UdR single crystals. Variation of experimental data for g factor (top), (I- (middle), and &proton coupling in whydroxy alkyl radical at C5’. Solid lines give variation calculated from final tensor values. FIG.

234

VOIT AND HOTTERMANN

part becomes somewhat more discernible. The quartet assigned to the cu-hydroxy alkyl radical at C5, (structure III) is indicated in stick-spectrum form under the spectrum along c*.

OH

H

III Figure 6 shows the variation of the spectral parameters, the g factor (top), the (Yand the P-proton interaction (middle and bottom, respectively). The tensor data derived from these curves are collected in Table 2 which also shows the angles between the relevant direction cosines and the molecular directions expected for the R,R2CH,&H,0H radical fragment at carbon CY on the deoxyribose to which these data are assigned. Although the angular correlation is only approximate, we feel the assignment is fairly safe due to the fact that for a base x symmetry of the radical a fairly large angular mismatch is obtained from the data and that an exchangeable OH coupling has been ascertained. Similar cy-hydroxy alkyl radicals and, if we accept that the alkoxy radicals are the precursor radicals in the present case, similar reaction pathways in single crystals have been detected in several pyrimidine nucleoside and -tide derivatives (14, 24TABLE 2

Direction cosines Tensor

Elements

g

2.0020 2.0035 2.0027

Iso.

2.0027

mT mT mT

H,

-1.42 -2.36 -0.84

Iso.

-1.50 mT

HP 1% )IOH

mT 3.96 mT 2.50 mT 3.10 mT 2.83

-0.2-0.4

mT

a

Angular correlation with molecular directions

b

c*

0.8619 -0.5040 0.0540

0.4965 0.8609 0.1105

-0.1022 0.0684 0.9924

1)base normal, (A) 30”, (B) 14’ (1C-H bond; (A) 64”, (B) 16” I C-H bond, in plane; (A) 60”, (B) 18”

0.8342 -0.5894 0.5904

-0.0117 0.7021 0.7119

0.5512 0.3984 -0.3801

11base norm&, (A) 28”, (B) 36” )I C-H bond; (A) 32”, (B) 35” I C-H bond, in plane; (A) 13’, (B) 22”

0.9311 -0.1216 0.3437

-0.0348 0.9088

-0.3625

0.4197

-0.399 1 0.8420

RADICALS

FROM 2’-DEOXYURIDINE

235

26) a purine nucleoside (27) and in carbohydrates (22,23). In one case, the mechanism could be shown to involve intermolecular abstraction of a CY-methylene hydrogen from a neighboring molecule by a primary alkoxy radical (25) whereas intramolecular abstraction by a secondary alkoxy species has been suggested in another compound (24). Another mechanism proposed in deprotonation of the primary oxidation product on the oxygen at the adjacent carbon (23) which might account, in the present case, for the population of radicals possibly related to the cY-hydroxy alkyl radical obtained directly after irradiation at 77 K mentioned in conjunction with the anion. 4. The 5-yi Radical There are two different sets of data at room temperature which can be assigned to a 5-yl radical at the uracil base resulting from net gain of a hydrogen atom at carbon C6 (structure IV). 0 H H H

IV

These differ in their lifetime at 300 K. The short lived species can be observed only after irradiation at 77 K and subsequent warming. It decays within days at room temperature whereas the other fraction remains stable for several weeks. Figure 7 (top) shows one plane of data obtained upon rotation about b after irradiation at 300 K under conditions optimized for the presentation of 5-yl radicals by uv conversion (A 3 320 nm at 77 K) of the 6-yl species (see below). The eight lines arising ftom an LYproton and two inequivalent methylene /3 couplings are indicated as stick spectrum along c*. The high resolution of the second-derivative display of the spectra allows one to discern a small doublet subsplitting of each of the octet lines amounting to about 0.2 mT. This interaction is lost when crystals grown from D20 are studied. Since, in addition, for the direction parallel to the base normal a 1: 1: 1 coupling of 0.1 mT due to a 14N interaction is found, this substructure can safely be assigned to the N3-H group by comparison with the corresponding couplings in other 5-yl pyrimidine radicals (2, 3, 18, 28). The spectral parameters collected for radical IV are listed in Table 3. We have managed to extract the two different a-proton tensors for the two molecular sites (A) and (B) of the UdR dimer but not the different g-tensor elements or the &proton interactions. These latter data have been derived from the long-lived species on site (B). The calculated spin densities included in Table 6 roughly reproduce the values derived from the (Yproton and the N3 couplings by the usual relations.

236

VOIT AND HiilTERMANN

[mTl 3.0

1.6

-

0.6

-

FIG. 7. Set of experimental ESR spectra (second derivative, rotation UdR crystals after irradiation at 300 K and subsequent uv bleaching radical as indicated as stick spectrum beneath Hllc* (top) and variation (middle), and the two inequivalent fl couplings (bottom), the small, stick spectrum (top) is assigned to the N,-H proton.

about b, -9.5 GHz) obtained from to enhance spectral features of 5-yl of main interactions, the OLcoupling additional splitting indicated in the

As mentioned above, uv bleaching of crystals irradiated at 300 K enhances the yield of 5-yl radicals by converting 6-yl species. The reverse process, frequently described in the literature for uracil and cytosine derivatives (29, 30) could not be observed. Instead, radical IV transforms upon heating into a 2Bproton radical (VI) (see below). It is interesting to note that after that transformation, IV can be regained from VI by renewed uv bleaching.

RADICALS

231

FROM 2’-DEOXWRIDINE

TABLE 3 SPECTRALPARAMETERSOF THE 5-yl RADICAL (IV) IN 2’-DEOXYURIDINE Direction cosines Tensor g

ISO.

Elements 2.0018 2.0031 2.0035 2.0028

H(B) (I

-1.91 -0.71 -2.84

mT mT mT

ISO.

-1.82

mT

H(A) OI

-2.18 -0.73 -2.95

mT mT mT

a

b

0.9489 0.2184 -0.2275

-0.1724 0.9633 0.2056

0.2640 -0.1559 0.9518

0.9616 0.0328 -0.2724

-0.1377 0.9164 -0.3757

0.2373 0.3989 0.8857

C*

Angular correlation with molecular directions 11base normal; (A) 15”, (B) 6”

11base normal; (A) 14”, (B) 5” 11C-H bond; (A) 6”, (B) 5” I C-H bond, in plane; (A) 14”, (W 3"

Iso.

Hi?,

Iso. Ho* lso.

-0.1467 0.9052 -0.3989

0.0046 -0.4026 -0.9153

11base normal; (A) 5”, (B) 18” 11C-H bond; (A) 6”, (B) 7” I C-H bond, in plane; (A) 6”, (B) 22”

-1.95 mT 3.87 3.53 4.22 3.87

mT mT mT mT

0.9223 0.3520 0.1590

-0.2668 0.8783 -0.3967

-0.2793 0.3235 0.9040

4.73 4.87 4.96 4.85

mT mT mT mT

0.7763 0.3513 0.5234

-0.5308 0.8122 0.2422

-0.3401 -0.4658 0.8169

-0.0

-0.0

0.3

mT

Iso.

-0.0 -0.0 -0.1

mT

HN,

iso. -0.2 mT

N3

-0.9892 -0.1361 0.0545

-1.0

5. The 6-yl Radical The radical formed by net gain of a hydrogen at the opposite site of the 5,6 double bond, CS (structure V), is usually present in the spectra of crystals irradiated at 0 H

HAN A O

.

?f ';'

V

H H

238

VOIT

AND

HOTTERMANN

room temperature where it accounts for about 30% to 40% of the total radical concentration. Its lines are also observed, but with less relative contribution (< 10%) upon warming of crystals to 300 K after irradiation at 77 K. Usually, there is severe overlap with lines of IV and center part species but data can be extracted with sufficient accuracy upon measuring at 77 K of crystals irradiated at room temperature. A series of spectra obtained upon rotation under b under these conditions is shown in Fig. 8 (top) together with the variation of the cy-and the two @-proton interactions (bottom) in the three crystal planes. The corresponding tensor data are listed in Table 4; the comparison between calculated and experimental spin densities is given in Table 6. There exists a slight uncertainty as to which UdR site, (A) or (B), is responsible. We have indications from higher-order derivative spectra, e.g., for Hlla, that both sites could be involved and thus propose that the data are for a “mean” between the two configurations. One notes that the p couplings are nearly equivalent, in contrast to the situation in the 5-yl species. Features similar to those described here for V have been assigned to a radical at C4, of the ribose moiety in a single crystal of UMP (12), an interpretation not unequivocally accepted (24). Testing the angular correlation of the present tensor data for that position gives an angle of over 80” for the direction of g,;, and the assumed 7~orbital on Cr. We thus have to conclude that structure V is the only feasible candidate for the spectral features in UdR. 6. Radicals from Storage of Crystals at Room Temperature

There appear to be several superimposed reactions of radicals initially observed subsequent to irradiation at 300 K or of species detected after warming of crystals to that temperature. In the former case, the total spin concentration decreases over a period of 5 months by only about 20% whereas the spectral changes occurring in the first 10 weeks indicate a significant amount of radical transformations. Subsequent to irradiation at 77 K and warming to 300 K, there is a fast initial decrease in radical concentration due to loss of the short-lived 5-yl fraction (cf. Section 4), but afterwards the picture is roughly identical with that obtained from crystals irradiated at 300 K. Although it is very difficult to single out specific reactions, two possible routes of radical transformations can tentatively be extracted from the data. One involves the decay of the 5-yl species (IV) on molecule (B) (the long-lived intermediate) and the concomitant formation of a radical characterized by a fairly isotropic g factor and two /3 protons of about 4.2 and 1.8 mT. Figure 9 (top) shows a plane of spectra obtained upon rotation about b from crystals which were uv-bleached to produce more 5-yl radicals (TV) from 6-yl species (V) and were subsequently treated with ir radiation to promote the conversion of the former into the 2/3 fragment. The quartet feature is indicated as stick diagram under the bottom spectrum. The remaining lines in the center are from one or two other species (see below). The bottom part of Fig. 9 gives the angular variation of the two couplings in the three planes of measurement. Due to the isotropic behavior of the spectral parameters we have no definitive clue for assigning this line group to a specific molecular site. It is interesting to note, however, that additional uv bleaching of crystals treated as described above brings back some of the 5-yl population apparently from the 2/3 species. This is a feature ascribed to a radical comprising three p-proton interactions in uridine, which was

RADICALS

239

FROM 2’-DEOXYURIDINE

ZmT,

-

[mTl( 3.0

2.L

1

1.8

j I

1.2

P

36-

b FIG. 8. Set of experimental ESR spectra (first derivative, rotation about b, -9.5 GHz) obtained from UdR crystals at 77 K after irradiation at 300 K showing pattern of 6-yl radical as indicated by the stick spectrum beneath Hllc* (top) and experimental and calculated variation of main hyperiine interactions, the (Y(middle), and the two nearly equivalent j3 protons (bottom).

assigned to a radical at C,, of the ribose moiety (IO), the protons at carbons CT and Cg’ giving rise to the observed couplings (structure VI). If we accept that one of the protons forms a dihedral angle with the unpaired electron orbital so as to decrease

240

VOIT

AND

HtiTTERMANN TABLE

SPECTRAL

PARAMETERS

OF THE 6-yl Diion

4 RADICAL

(V) IN 2’-DEOXYURIDINE

cosines Angular correlation with molecular directions

Tensor

Elements

a

b

c*

g

2.0023 2.0035 2.0030 2.0029

0.9316 0.0057 0.3632

-0.0932 0.970 1 0.2237

-0.3511 -0.2423 0.9044

11base normal,

0.9823 -0.1295 0.1351

0.1870 0.7036 -0.6854

-0.0062 0.6986 0.7154

I( base normal, (A) 5”, (B) 22” 11C-H bond; (A) 7”, (B) 14” I C-H bond, in plane; (A) 9”, (B) 11”

Isa H,

-1.72 -0.93 -2.87

mT mT mT

Iso.

-1.84

mT

%I

Iso. H8l

1%

3.31 2.98 3.57 3.29

mT mT mT mT

0.7618 0.6375 0.0170

-0.5724 0.6606 0.4855

0.3032 -0.3797 0.8740

3.49 3.04 3.43 3.32

mT mT mT mT

0.6495 -0.6728 -0.3543

0.4649 0.7201 -0.5150

0.6016 0.1697 0.7806

H

(A)

15”, (B) 9“

OH

VI the interaction, the present data for what is denoted a 2j3 radical could fit in with the features of VI. There is a third coupling of about 0.7 mT visible at some orientations which, however, is mostly obscured by the fact that the lines of the 20 fragment are fairly broad. A second radical obtained upon storage of crystals gives rise to the multiplet in the center part of the spectra of Fig. 9 (top). At Q-band frequencies, the spectra lose some of the multiplet structure indicating that second-order effects have prevailed-in the X-band spectra. A spectrum obtained at Q band for the magnetic direction along c* is shown in Fig. 10 (top). The species in question gives rise to the center doublet indicated as stick diagrams. The nature of the underlying and flanking multiplet is unclear. For the doublet which, in this orientation, exhibits an additional subsplitting probably due to the two molecular sites in the asymmetric unit, an a-type hyperhne coupling can be extracted from the Q-band spectra which is given in the bottom half

RADICALS

FROM 2’-DEOXYLJRIDINE

241 2 mT

‘7-l

,

L+

Z

2/3-species

b FIG. 9. Set of experimental ESR spectra (first derivative, rotation about b, -9.5 GHz) obtained ftom UdR crystals irradiated at 300 K afler uv bleaching and ir warming treatment to promote transformation of 5-yl radical into 2 fl species (top) and variation of the two @proton interactions in the three planes of measurement (bottom).

of Fig. 10 together with the variation of the g factor in the three planes of measurement. The corresponding tensor data are listed in Table 5 which also contains the parameters of the 2P radical. The assignment of this (Yradical is not straightforward. The isotropic coupling of 1.13 mT and the relatively high value of 2.0050 for the maximum element of the g tensor indicate a radical fragment of the type

VOIT AND HiiTTERMANN

242

ImT 2.0

I I

+

site 2

, a-species

1.6

‘I b

2.0056

2.002L

I

b

I

r

I

c*

I

I

I 0

1

1

b

FIG. 10. ESR spectrum (Q band, second derivative, Hllc*, -34.5 GHz) showing doublet-type pattern of IY species of each UdR site in the asymmetric unit of crystals of UdR irradiated and stored at 300 K (top) for 3 months and variation of a-type coupling (middle) and g factor (bottom) in the three planes of measurement.

with about 50% spin density on the a-carbon and a considerable degree of carbonyl conjugation. A fi-agment of that type has been assigned to a deoxyribose radical on

RADICALS

FROM

TABLE RADICALS

FORMED

UPON STORAGE Direction

Tensor

Elements

a

cosines

Iso. K

2.0023 2.0024 2.0050 2.0032 -0.98 -1.88 -0.53

mT mT mT

SINGLE

Tentative radical candidates

C*

“-Type

g

5

OF 2’-DEOXWRIDINE

b

-1.13

CRYSTAIS

AT 300 K

Angular correlation with molecular directions

Radical

0.9 143 -0.0522 0.4016

-0.3641 0.3278 0.8717

0.1771 0.9432 -0.2807

VII

)I base normal;

VIII

1) ?r orbital;

0.8976 0.1137 -0.4298

-0.1418 0.9348 -0.2600

0.3684 0.3364 0.8666

VII

11C-H bond; (A) 25”, (B) 13” I( base normal, (A) 40”, (B) 35” I both; (A) 33”, (B) 30” (I C-H bond; (A) 25” (B) 13’ )I A orbital; (A) 26”, (B) 40” I both; (A) 20”, (B) 17”

VIII

ISO.

243

2’-DEOXYURIDINE

(A) 26”,

(A) 23”,

(B) 13”

(B) 15”

mT 2p Radical

g

HP,

ISO.

%*

Iso. H3

-2.002-2.003 1.68 1.62 1.86 1.72

mT mT mT mT

0.8385 0.5440 0.0120

-0.5438 0.8374 0.0535

0.0191 -0.05 14 0.9984

4.41 4.62 4.23 4.42

mT mT mT mT

0.9128 0.4090 -0.0204

-0.3994 0.8776 -0.2650

-0.0905 0.2499 0.9640

-0.7-0.8

mT

carbon CY in the nucleotide UMP (14). The corresponding species in the nucleoside UdR, structure VIII has a disadvantage that there should be an a-hydroxy group attached to Cg’ for which an exchangeable proton interaction should be detected, in contrast to the experimental observations. OH

244

VOIT AND HiiTTERMANN TABLE 6 SPIN DENSITIES

OF VARIOUS

RADICALS

IN 2’-DEOXYLJNDINE

Spin densities

NI Anion (II) Experimental Calculated RHF + Cl UHF 5-yl Radical (IV) Experimental calculated RHF + CI UHF 6-yl Radical (V) Experimental Calculated RHF + CI UHF

c2

N3

0.11 0.06 0.06

c4

0.11 0.0 -0.01

0.06 0.05

-0.004 0.005

0.010 -0.042

G 0.55

0.15 0.10

-0.04 0.002 -0.004

CS

0.08 0.03

0.47 0.57

0.71 -0.079 -0.260

0.593 0.658

0.72

0.09 0.09

-0.026 0.015

0.003 0.0

0.011 0.002

-

0.743 0.622

Another candidate to be considered is the 04-protonated species (VII) which also would give rise to a predominant o-type coupling from 50% spin density at carbon Cg. This radical offers the advantage of explaining a possible decay pattern of the primary anion without producing the 5-yl radical (IV). The reaction pattern, though not very clear, however, would have the species in question rise very probably upon decay of the 6-yl species (V) at 300 K. For this reason, we prefer structure VIII or a related one on the sugar over VII since, moreover, for the latter we would expect a better agreement between the tensor directions and the molecular geometry which is given for both structures and the two molecular sites (A) and (B) in Table 5. The angles show that for both VII and VIII the mismatch is approximately identical. DISCUSSION

The low temperature radiation chemistry of crystals of UdR is in good agreement with what presently appears to be the mechanistic picture of the main pathways of radiation damage in uracil nucleoside and -tide derivatives (6). The dominant primary radicals are base ?r* anions and alkoxy radicals on the sugar as is found for most of the other uracil derivatives like the nucleotide uridine-S-phosphate (UMP) (II, 12, 24) the arabinofuranoside UAra (15) and also, at least to a major degree, the range of 5-substituted uracil nucleosides thymidine (TdR) (31) 5-chloro- (ClUdR), 5-bromo(BUdR), and 5-iododeoxyuridine (IUdR) (5, 19). However, the specific features for both radicals in UdR differ somewhat from those in the other compounds. For one thing, the coupling due to N1 and the Ns-H group in addition to the main C6-H (Yproton interaction in the anion has so far not been resolved in any other substance

RADICALS

FROM

2’-DEOXYURIDINE

245

except perhaps IUdR (5) for which, however, only a coupling to N1 was detected. More striking is the marked variability in the chemical and molecular structures of the alkoxy radicals for which two primary ones were detected with only one of them related to the crystal structure and one secondary. In other uracil nucleosides or -tides always only one type and a clear relation to the crystal structure is observed. This difference can, at least in part, be accounted for by the specific feature that crystals of UdR contain two molecules in their asymmetric unit. The fate both of the anion to decay, upon warming, into a diamagnetic product and of the alkoxy radicals to transform into a-hydroxy alkyl radicals at carbon Cs of the deoxyribose are reactions common to the nucleotide UMP (II, 14). The latter radical conversion is also the main mode of alkoxy decay in ClUdR and BUdR (25). The room temperature radicals, the 5-yl and 6-yl species formed from net gain of a hydrogen atom at the respective sites of the uracil base 5,6 double bond in UdR, as well as the conversion of the latter into the former radical by bleaching with longwavelength uv only partially agree with what is known from other uracil derivatives. In the pure base, the 6-yl radical is formed at 300 K, the 5-yl species being the result of subsequent uv bleaching only (32). In UAra (15) only one type of radical appears to constitute the spectra at room temperature for which the authors leave open the decision between the two possible structures. With the present knowledge available about the difference in the magnitude of B interactions in 5-yl and 6-yl radicals of uracil, we suggest that the spectra in question are due to the 5-yl species. Differing assignments have been advanced for the nucleotide UMP. The 5-yl species is not debated but there is disagreement on the presence (1 I) or the spectral parameters, if present (13, 14), of the 6-yl radical. Sagstuen (II) proposes a C,,-located sugar radical as alternative to the 6-yl species which, however, is also converted into the 5-yl radical by uv. The existence of this bleaching effect in the absence of a base 6yl radical is also reported for the riboside uridine (10) in which still another C4’located radical is the source of 5-yl species. It thus appears that in nucleosides and -tides containing the uracil base the 5-yl radical is consistently formed at 300 K whereas the 6-yl species, which is the main product in the base (32, 33), exhibits a variable probability of being stabilized. There are at least three other free radical species formed or present at room temperature. For two of them, the data were extracted but no definitive assignment is possible. The significant amount of radical transformation upon storage of crystals at room temperature is also observed in UMP (13, 24). One cannot be certain, however, whether this can be attributed to the molecular structure of the compound or to a specific crystalline environment. The former, however, is definitively one of the reasons the solid-state radiation chemistry of UdR at 300 K somewhat is more complex than that of the 5-substituted derivatives TdR, ClUdR, and BUdR (2, 3, 7-9) for which, generally, not more than three radicals are present at room temperature which remain stable for years. The lack of reactivity comes from the fact that 5-substitution of uracil effectively suppresses the formation of the 6-yl radical which appears, in UdR and UMP (24), to be a major source of radical transformations. It is this feature which renders UdR to be less suitable as a reference compound for the comparative radiation chemistry of the series of 5-methyl- and 5-halogen substituted uracil deoxyribosides than was anticipated initially.

246

VOIT AND HfiTTERMANN ACKNOWLEDGMENT

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Hu 248/6-2). REFERENCES 1. W. SZYBALSKI, Cancer Chemother. Rep. Part 158,539 (1974). 2. J. HUTTERMANN, W. A. BERNHARD, E. HAINDL, AND G. SCHMIDT, Mol. Phys. 32, 11 I1 (1976). 3. E. HAINDL AND J. HOTTERMANN, J. Magn. Reson. 30, 13 (1978). 4. J. HOTTERMANN, G. W. NEILSON, AND M. C. R. SYMONS, Mol. Phys. 32,269 (1976). 5. M. H&IN AND J. HfFrmmmm, Int. J. Radiat. Biol., in press. 6. J. HUTTERMANN, Ultramicroscopy 10, 25 (1982). 7. B. PRUDEN, W. SNIPES, AND W. GORDY, Proc. Nat. Acad. Sci. NY 53, 917 (1965). 8. W. FLOSSMANN, H. ZEHNER, AND A. MULLER, Z. Naturjbrsch. C 35C, 20 (1980). 9. J. N. HERAK AND C. A. MCDOWELL, J. Magn. Resort. 16,434 (1974). 10. E. SAGSTUEN, J. Magn. Reson. 44, 518 (1981). 11. (a) E. SAGSTUEN, Radiat. Res. 81, 188 (1980); (b) E. SAGSTUEN, Radiat. Res. 84, 164 (1980). 12. H. C. Box, W. R. POTTER, AND E. E. BUDZINSKI, .I. Chem. Phys. 62,3476 (1975). 13. B. RAKVIN AND J. N. HERAK, Int. .I Radiat. Biol. 38, 129 (1980). 14. G. RADONS, H. OLOFF, AND J. HOTTERMANN, Znt. J. Radiat. Biol. 40, 245 (1981). IS. R. BERGENE AND R. A, VAUGHAN, Int. J. Radiat. Biol. 29, 145 (1976). 16. A. RAHMAN AND H. R. WILSON, Acta Crystallogr. Sect. B 28, 2260 (1972). 17. H. OLOFF, E. HAINDL, J. HU~ERMANN, Radiat. Res. 80, 447 (1979). 18. R. BERGENE, T. H. JOHANNIXY EN, AND T. HENRIKSEN, Int. J. Radiat. Biol. 29, 541 (1976). 19. W. A. BERNHARD, D. M. CLOSE, J. HOTTERMANN, AND H. ZEHNER, J. Chem. Phys. 67,12 11 (1977). 20. H. C. Box AND E. E. BUDZINSKI, J. Chem. Phys., 67, 4726 (1977). 21. H. C. Box, H. G. FRUEND, AND E. E. BUDZINSIU, J. Chem. Phys. 74,2667 (1981). 22. K. P. MADDEN AND W. A. BERNHARD, J. Chem. Phys. 70,243l (1979). 23. K. P. MADDEN AND W. A. BE-, J. Phys. Chem. 83,2643 (1979). 24. J. HOTTERMANN, E. HAINDL, G. SCHMIDT, AND W. A. BERNHARD, Int. J. Radiat. Biol. 32, 431 (1977). 25. J. HOTTERMANN,

W. A. BERNHARD, E. HAINDL, AND G. SCHMIDT, J. Phys. Chem. 81,228 (1977). W. A. BERNHARD, D. M. CLOSE, K. R. MERCER, AND J. C. CORELLI, Radiat. Res. 66, 19 (1976). 27. C. ALEXANDER AND C. FRANKLIN, J. Chem. Phys. 54, 1909 (1971). 28. J. N. HERAK AND A. VELENIK, J. Magn. Reson. 49,64 (1982). 29, W. ROSSMANN, E. WESTHOF, AND A. MOLLER, .I Chem. Phys. 64, 1688 (1976). 30. E. WESTHOF, W. FLOSSMANN, H. ZEHNER, AND A. MOLLER, Faraday Discuss. Chem. Sot. 63,248 26.

(1977). 31. H. C. Box 32. H. ZEHNER,

E. E. BUDZINSKI, J. Chem. Phys. 62, 197 (1975). W. FLOSSMANN, E. WESTHOF, AND A. MULLER, Mol. Phys. 32,869 33. W. FLOSSMANN, J. HQrrmmrw, A. MUELLER, AND E. WESTOF, Z. Naturjbrsch. AND

(1976). C 28, 523 (1973).