J. Phys. Chem. 1995, 99, 3867-3874
3867
Structure and Relative Stability of Deoxyribose Radicals in a Model DNA Backbone: Ab Initio Molecular Orbital Calculations Anny-Odile Colson and Michael D. Sevilla* Department of Chemistry, Oakland University, Rochester, Michigan 48309 Received: October 26, 1994; In Final Form: December 7, 1994@
Ab initio molecular orbital calculations have been performed in this study to determine the stability of five deoxyribose centered radicals embedded in a short DNA segment. The effect of phosphate groups on the sugar radical conformation, energetics, and electronic properties are evaluated through a comparison of models with and without phosphate groups. Geometry optimization performed at the ROHF/3-21G level reveals the C1’ centered radical is the most energetically favored in all the DNA fragments considered in this study, while the C2’ radical is the least stable and maintains a near planar configuration All energy minima calculated correspond to deoxyribose radicals with a pseudorotation phase angle lying in the S quadrant of the pseudorotation cycle. The phosphate groups significantly affect the puckering mode of the C2’ and C3’ radicals and energetically destabilize C3’ radical relative to the other sugar radicals. Isotropic hyperfine coupling constants significantly differ between models with and without phosphate groups, most particularly in the C3’ and C4’radicals. Owing to the nonplanarity of the sugar ring in the C4’ radical, the proton couplings are found to have a significant cos 0 dependence in the relation a = BO B1 cos 0 B2 cos2 0.The trend in oxidizing power based on the calculated HOMO energies is predicted to follow the order 421’ < C4’ < 4-22’ < C3’ < C5’. Cytosine attachment to the C1’ and C4’ deoxyribose radicals does not appear to affect the relative energies nor the isotropic hyperfine couplings of these two species. In both deoxycytidine radicals, the base maintains an anti conformation, which therefore does not disrupt the hydrogen bonding pattern in the base pair.
(3).
+
Introduction It is widely accepted that ionizing radiation results in cell death through damaging the DNA of living cells. The most serious lesions observed in the macromolecule are those responsible for altering the genetic code,’ via strand breaks2g3 and chemical alterations of the DNA bases. Although many of these damages are repaired, it is the double strand break that often leads to biologically impaired cells and ultimately to cell death. In this study, we focus on radicals on the sugar moiety of the DNA molecule for they are known to be precursors in mechanisms leading to strand breakage.“,5 There is evidence for a definite role of sugar radicals in the indirect effect of ionizing radiations?. 6-12 but some controversy as to whether deoxyribose radicals are produced by the direct effect have arisen.13 Radicals produced in the backbone can result from radiation-induced radicals formed on the DNA bases, and since sugar centered radicals are not apparent in the EPR spectra of as of yet there is no direct evidence that they are among the primary radicals formed by the direct effect. However, thorough ENDOR studies performed on X-irradiated single crystals of 5’-dGMP13 and deoxyadenosine monohydrate’* reveal a variety of sugar-centered radicals at 10 K, even after low radiation doses, indicating the potentiality for sugar radicals to be considered as primary radicals. In addition, a recent ESR investigation of y-irradiated frozen solutions of dAMP reveals the presence of the C1’ centered sugar radical upon annealing at 190 K.19 These results strongly suggest that sugar radicals are likely produced by the direct effect of radiation on DNA. Indirect effects mechanisms through which sugar radicals are formed include hydrogen abstraction reactions by OH’ and H’ a t t a ~ k ? , ~ , ~ , ~oxidation ~-” reactions involving peroxyl and alkoxy1 radicals precursors,’* and transfer of the radical site from *Abstract published in Advance ACS Abstracts, February 15, 1995.
+
OH
I HO-P=O
OH
I
HO-PI0
I
II
?
HO-+=O
I OH
I
11
HO--P=O
I
OH
m
Figure 1. Structures investigated in this study at the ROHF/3-21G (11) level: (I) (R)-2-Amino-(s)4-hydroxy-(s)-5-methyltetrahydrofuran, 2’-deoxyribose 3’,5’-biphosphate, and (ID)2’-deoxyribose 1’-amino 3’,5‘-biphosphate.
the base to the sugar.3,7-8*25 The sugar lesions generated in these reactions will largely depend on experimental conditions such as, for instance, P H , oxic, ~ ~ and anoxic media.15,27 Schuchmann et aL2*determined that hydrogen abstraction by OH’ in D-glucose occurs with nearly equal probability (ranging from 10 to 30%) at any of the six carbon atoms. The lack of selectivity of the reactive OH’ radical toward any particular carbon site of the sugar ring was later demonstrated by Fitchett et al.*O Furthermore, Pardo et al. show a clear correlation between the C-H bond strength and the barrier to hydrogen abstraction in methanol and ethanol.2g This group further established a relationship between the relative rate constant and the bond strength and later used this technique to determine the energetics of hydrogen abstraction in deoxyribose radicals modeled by (R)-2-amino-(S)-4-hydroxy-(S)-5-methyltetrahydrofuran (structure I in Figure l).30In addition, these workers thoroughly investigated the structures and energetics of the four
0022-365419512099-3867$09.00/0 0 1995 American Chemical Society
3868 J. Phys. Chem., Vol. 99,No. 11, 1995
Colson and Sevilla
TABLE 1: Structural Parameters of Deoxyribose and Its Radicals in Structures 11 and III Calculated at 3-216 (deg)
rn
I1
e x p e B-DNA nonrad
nonrada
C1’
C2’
C3’
C4’
C5‘
nonradicalb
C1‘
422’
C3’
C4‘
C5’
-34.8 33.3 -18.6 -4.0 24.7 120.5 36.6
-40.6 43.0 -30.6 6.3 21.2 133.8 44.2
-15.2 27.5 -28.8 21.1 -4.0 168.8 29.4
17.6 -5.8 -7.0 17.5 -22.5 251.2 21.1
-25.0 33.2 -30.2 16.4 5.8 152.9 33.9
-25.4 31.3 -35.6 21.7 2.4 158.4 37.6
-25.2 31.6 -35.9 21.9 2.0 158.8 38.6
-24.0 35.4 -33.9 19.9 2.8 157.9 36.6
-16.8 28.9 -29.7 20.8 -2.8 166.4 30.6
11.3 -1.4 -7.9 14.4 -16.5 240.8 16.2
3.5 8.4 -16.9 19.2 -13.9 208.5 19.2
-22.5 35.7 -36.1 23.5 -0.5 162.9 37.8
1.3 18.4 -30.3 31.3 -20.4 200.4 32.3
vo vl v2 v3 v4 P Urn
Torsion angles fixed in a B-DNA conformati~n.~~ Fully geometry optimized.
ring carbon centered radicals. In DNA, the sugar moiety is bonded to two phosphate groups at the 3’- and 5’-positions, which may affect the puckering of the ring and hence the energetics of the various radicals. Furthermore, since the Cl’, C2’, C3‘, and C5’ centered radicals have been observed experimentally,’3J8 theoretical investigation of their structure and relative stability is of importance. In this work, through the use of ab initio molecular orbital calculations performed on a DNA fragment composed of one sugar and two phosphate groups, we present the conformations and properties of the five carbon centered radicals which result from hydrogen abstraction. Our previous theoretical efforts investigated charge localization after ionization of the DNA bases31 and the sugar-phosphate backbonee3* In this work, we investigate the neutral radicals likely produced after proton transfer from the radiation produced holes. Methods All calculations were conducted using the Gaussian 92 programs33implemented on an IBM RS 6000 and a Cray C 90. The split valence basis set ROHF/3-21G34was employed for geometry optimization of the structures described below. The 6-31G leveP5 was used for calculating the isotropic hyperfine constants derived from the Fermi contact analysis of the UHF wave function.36 No higher basis set was considered for geometry optimization, for studies on furanose rings3’ showed the results between the 3-21G and 6-31G* basis sets to be qualitatively comparable. In this work, we present the relative energies of five neutral deoxyribose radicals resulting from hydrogen abstraction at carbons C1’ through C5’. The sugar moiety is considered with two phosphate groups bound at the 3‘ and 5‘ carbons, respectively. In a previous work,32we report the neutral nonradical structure of this system (2’-deoxyribose 3‘,5‘-biphosphate (structure 11 in Figure 1)) for which bond distances and bond angles were optimized at the 3-21G basis set, keeping all torsion angles fixed in a B-DNA configuration. This optimized structure is used as a starting configuration for complete optimization of the five neutral radicals investigated in this study. After abstracting the hydrogen atoms at the carbon sites of interest, all bond distances, bond angles, and torsion angles were varied. Exception was made of the four hydrogens of the phosphate groups which were fixed in the geometry previously obtained for the neutral nonradical system. Furthermore, full optimization of the C5’ centered radical led to a significant bend in the backbone allowing for the 5’ phosphate group to hydrogen bond with the oxygen of the sugar ring. Since such a conformation is not permissible in DNA, the C4’-C5’-O-P (Le., p) torsion angle was fixed at 174.41”, this value being the average of the p torsion angles obtained in the optimization of the Cl’, C2’, C3’, and C4’ centered radicals. The resulting geometries were then empioyed to fully optimize the parent and all five carbon centered radicals in which one of the HI’
B E f k c t ofphosphate
.c2: 11,
.C3’:111 .CV: I11
.C3: I1 44’:
n, 111
.CV: I1 Figure 2. Positions of deoxyribose radicals in structures I, II, and III (Figure 1) on a pseudorotation cycle. =E, and ,”r define envelope (E) and twist (T) conformers in x’-endo and/or y’-exo pucker mode. The arrows show the effects of the 3’- and 5’-phosphate groups on the puckering of the sugar: C3’ is the most affected; C1’ is not affected. Radicals in parentheses are of the North type pucker from Maskiewicz and Osman30and according to the present study cannot occur in B-DNA without significantly affecting the local structure of the macromolecule (Figure 4).
hydrogens was substituted for an amine group (2’-deoxyribose 1’-amino 3’,5’-biphosphate (structure 111in Figure 1)) to mimic the effect of a DNA base. For the reason discussed above, the p torsion angles were not allowed to vary; all other variables (with the exception of those involving the four hydrogens of the phosphates) were optimized. Coordinates (x,y,z) for the optimized structures are available in the supplementary material. In a second set of calculations, the neutral nonradical3-21G optimized structure of cytosine38 was bound to the most energetically stable sugar radicals of the optimized DNA fragments (111) obtained above. In this set of calculations, cytosine was chosen among the four DNA bases for its small size (in terms of electron count) relative to the purines and thymine. Three parameters were allowed to vary: the glycosyl bond length, the angle Cl’-Nl-C2 and the torsion angle C2’C 1’-N 1-C2. Results and Discussion
I. 2’-Deoxyribose 3’,5’-Biphosphate (11) and 2’-Deoxyribose 1’-Amino 3’,5’-Biphosphate (In)Radicals. 1. Puckering Mode. Sugar ring puckering is believed to play a major role in determining rates of hydrogen abstraction reactions as well as the relative stability of the resulting r a d i c a l ~ . ~We ,~~ therefore present this aspect first. The 3-21G optimized torsion
Ab Initio Molecular Orbital Calculations
J. Phys. Chem.. Vol. 99, No. 11, 1995 3869
Figure 3. Sugar puckering in the parent Ill and its radicals. For clarity, the hydrogen atoms have been omitted
predict that whichever site a hydrogen is abstracted from in a deoxyribose sugar moiety of the B-DNA, the resulting radical will be of the S-type, although the C2' and C4' radicals might be less stable than their counterparts of the N-type resulting from hydrogen abstraction at these sites in A-DNA. To further verify this, we have geometry optimized the C4' radical of I11 employing a starting geometry composed of a B-DNA backbone and a N-type sugar puckering30 (zE). This structure is shown in Figure 4a from which it clearly appears that steric hindrance will not allow sufficient space for the DNA base. Upon optimization of this structure (Figure 4b), the puckering mode (y4 + 5 )- (v3 + Yo) v2 tanP= and v,=of the optimized sugar remains unchanged, whereas the cos P 2v2(sin 36" sin 72") backbone significantly reorganizes forming a hairpin-like structure which would allow for base attachment at C1'. In Table 1, we note that the torsion angles u's and the angle However, such structure is no longer of a B-DNA type. This of pseudorotation P obtained in the B-DNA configuration of result suggests that this conformation with a N-type sugar, the neutral parent fragment (It) investigated in a previous study32 although found to be more stable by ca. 5 kcaVmol at the 3-21G compare relatively well to data obtained from fiber diffraction level than the fragment in which the sugar puckers in the S-type, in B-DNA.40 Both experimental and theoretical (11) parent is nevertheless unlikely to occur in B-DNA unless substantial structures are in a ,E puckering mode, with a maximum outlocal reorganization occurs in the backbone resulting in a kink of-plane pucker of ca. 40.0". This value of u, decreases upon in the B-DNA structure. Further, we are not confident in this radical formation, indicating a flattening of the ring. The most predicted greater stability of the kinked structure in which the significant change occurs for the CY centered radical which sugar has a N-type conformation, as we believe internal basis becomes nearly planar, while urnis only slightly affected upon set superposition in this kinked structure and at the 3-21G level radical formation in (11) at CY, C4', and C5'. Following the of calculation might be substantial. pseudorotation cycle shown in Figure 2, we find the sugar ring With regard to the phase angles, we have exchanged the of structnre (11) to pucker in the envelope 2E mode for the Cl', phosphate groups of the optimized radicals in structure I11 with C3', C4' and C5' centered radicals, while the C2' radical puckers an OH at the 3'-position and a CH, group at the 5'-positions to into the twist form Similar puckering modes are obtained obtain structure I. Geometry optimization of the CI', CY.C3'. upon geomeuy optimization of the CI', CY, and C4' radicals and C4' radicals resulted in calculated phase angles (represented of structure (111) (Figures 1 and 2). while both C3' and C5' in Figure 2) that were identical to those determined for the centered radicals rotate into the 3E mode. The resulting S-type conformers by Miaskiewicz and OsmanJo This result geometries are presented in Figure 3. therefore suggest the 3' and 5' substituents play an important It is important to note that all conformers observed in this study are of the S-type (South), while Miaskiewicz and O ~ m a n ~ ~role in determining the puckering of the sugar ring. Indeed, with the exception of the C1' radical which is unaffected by reported the N-type (North) to be slightly more stable than the the phosphate groups addition, the phase angles of the remaining S-type conformers for the C2' and C4' radicals of structure I. three species rotate clockwise in the pseudorotation cycle (see In addition the phase angles P of all but the C1' radical differ Figure 2). Not surprisingly, the C3' radical is the most affected, between the S-type conformers of the two models. This with a AP of ca. 120'. followed by C2' and C4'. apparent disagreement can be explained on the following basis. B-DNA structures prefer the C2'-endo pucker (S-type), while 2. Consequences of Puckering on Bond Angles and Bond the C3'-endo pucker (N-type) is mostly observed in A-DNAJO Lengths. Westhof et al.4' have plotted the average endocyclic In this study, all the sugar radicals result from optimizations bond angle against the square of the amplitude of puckering employing a B-DNA model as a staning geomeuy. Hence we for a variety of five-membered rings utilizing X-ray crysta-
angles of the deoxyribose ring in the parent structures I1 and I11 and their corresponding neutral radicals are presented in Table 1. Selected optimized angles and bond distances are tabulated in Table 2. In DNA, the most commonly observed puckering modes of the sugar are CY-endo and CY-endo, while RNA generally maintains a C3'-endo configuration. These modes can be described in terms of the phase angle of pseudorotation P and the maximum torsion angle u,. P and u, are defined as39
+
3.
3870 J. Phys. Chem., Vol. 99, No. 11, 1995
Colson and Sevilla
Q ,,‘,‘,‘,’I , I , ,
Figure 4. 3-21G geometries of the N-type C4’ sugar radical of structure III before (a) and after (b) geometry optimization. a is composed of a B-DNA backbone and a N-type sugar ring. In b, the sugar retains its N conformation whereas the backbone kinks.
TABLE 2: 3-21G Calculated Endocyclic Angles (deg) and Selected Bond Distances (A) I11
I1 Ol-C1’-C2’ Cl’-CY-C3’ C2’-C3’-C4’ C3’-C4’-01 C4’-01 -C1’ Cl‘-C2’ C2‘-C3’ C3‘-C4’ C4’-01 01-C1‘ C1‘-N
nonrad
€1’
€2’
€3’
101.2 102.3 103.8 106.5 106.7 1.51 1.53 1.53 1.45 1.48
108.7 101.8 104.4 105.8 110.4 1.51 1.53 1.53 1.45 1.40
104.8 110.1 103.7 106.6 110.4 1S O 1.49 1.54 1.44 1.45
104.8 101.1 107.2 105.3 110.2 1.54 1.51 1.52 1.45 1.46
€4’ 103.0 101.0 102.5 108.8 109.5 1.54 1.53 1S O 1.39 1.47
Planar
10s
1
f
3 107
4
P
106
a
I
105
$”
‘t
104
0
50
100
150
200
u,2/10
Figure 5. Plot of the average endocyclic bond angle vs the tenth of the square of the maximum torsion angle for I1 and 111. The relations are y = 108.03 -0.020368 vm2/10(R2= 0.99) and y = 108.04 0.020577 vm2/10(R2= 0.99), respectively.The underlined designations are for structure 11.
lography data. They demonstrated that the straight line resulting from such a plot fits the relation 0 = 108” - 0.001 86vm2.It is interesting to note that the average endocyclic angles calculated from data in Table 2 for the I1 and I11 radical and parent species fit a straight line shown in Figure 5 (0= 108.03 - 0 . 0 0 2 0 4 ~(11) ~ ~and 0 = 108.04 - 0 . 0 0 2 0 6 ~(111)) ~ ~ when
€5’
104.2 101.2 103.9 105.8 109.8 1.53 1.53 1.53 1.45 1.46
nonrad
€1’
c2’
€3’
103.3 102.7 104.0 106.0 110.7 1.53 1.52 1.54 1.44 1.47 1.42
107.3 102.4 104.3 105.9 110.5 1.51 1.53 1.54 1.44 1.43 1.39
103.4 111.5 103.8 106.8 112.0 1S O 1.48 1.54 1.44 1.47 1.42
105.6 104.8 108.6 105.3 112.2 1.55 1.51 1.51 1.44 1.48 1.42
€4’ 102.0 102.2 102.4 108.6 110.2 1.53 1.52 1.51 1.39 1.49 1.41
€5’ 105.2 104.3 104.0 104.8 111.2 1.54 1.52 1.53 1.44 1.48 1.42
plotted against urn2. These plots further illustrate the concept of ring flattening upon radical formation. In both I1 and I11 models, the C2’ radical is nearly planar as previously observed in other systems,30while C4’ and C5’ centered radicals are the most puckered. Selected calculated bond lengths are presented in Table 2. From these data, it is clear that radical formation and subsequent puckering have a significant impact on several bond distances. In general, the bonds about the radical centers are the most affected. We note that the C1’-0 bond, although overestimated in the nonradical species (1.48 &theory) vs 1.42 &experiment)), is affected by the presence of both the amine group as previously postulated30 and the phosphate groups. Indeed, in the radical species, the C1’-0 bond lengthens by 0.02 8, upon amine group attachment and by 0.03 8, upon phosphate groups (this effect is most likely dominated by the 5’ substituent) attachment. 3. Relative Energies. Figure 6 presents the relative ROW/ 3-21G energies of the S-type sugar radicals of structures I, 11, and I11 investigated in this study. All three models (in the presence or absence of the phosphate groups) predict C1’ to be the favored site for H-abstraction and hence radical formation in the sugar moiety, while the C2’ radical is the least likely to occur. In addition, Figure 6 very clearly shows the effects of the phosphate groups on the relative energies of the various radicals. As expected, the C2’ radical is nearly unaffected by such addition, while the C3’ radical is substantially destabilized by the phosphates (most probably the 3’-phosphate). A similar
Ab Initio Molecular Orbital Calculations
J. Phys. Chem., Val. 99,No. 11, 1995 3871
6
5
-
8 4
R
3”,
!.
3
B
W
t
‘32
3 1
0
Figure 6. Relative stabilities (kcdmol) of the five sugar radicals in I, II, and 111 (Figure 1) calculated at ROHF/3-21G.
TABLE 3: Isotropic Hyperfine Couplings of the Sugar Radicals in Structures I, 11, and III Calculated from the UHF/6-31G// 3-21G Fermi Contact Analysis ( M H Z ) ~ &2’
&1’
expb IC
this work I,II,III
expb
I‘ north
&4’
C3‘ this work
I, II, III
ex$
IC
IC
this work
north
I,II,III
C5’
expb
thisworkII,III
90.9
HI’
Hzd 45.0 41.7 35.5,44.5,34.8 Hz[ 78.4 97.7 91.2, 107.9, 90.6 H;
94.2 95.9, 103.0, 86.7d -64.8 -146.W -140.2, -150.1,-149.8c
this work I,II,III
106.6
46.8 52.2 53.9;7.0; 19.7; 106.9 95.7 89.3; 49.3;73.4
30.0,48.8,49.5
85.7 13.2,7.4,6.2 77.1 74.7 73.3, 17.4,18.3
Hq’ HSd HS
