Conformations of Stereoisomeric Base Adducts to 4

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Conformations of Stereoisomeric Base Adducts to 4-Hydroxyequilenin Shuang Ding,† Robert Shapiro,† Nicholas E. Geacintov,*,† and Suse Broyde*,‡ Department of Chemistry and Department of Biology, New York University, 100 Washington Square East, New York, New York 10003 Received February 11, 2003

Exposure to estrogen through estrogen replacement therapy increases the risk of women developing cancer in hormone sensitive tissues. Premarin (Wyeth), which has been the most frequent choice for estrogen replacement therapy in the United States, contains the equine estrogens equilin and equilenin as major components. 4-Hydroxyequilenin (4-OHEN) is a phase I metabolite of both of these substances. This catechol estrogen autoxidizes to potent cytotoxic quinoids that can react with dG, dA, and dC to form unusual stereoisomeric cyclic adducts (Bolton, J. L., et al. (1998) Chem. Res. Toxicol. 11, 1113-1127). Like other bulky DNA adducts, these lesions may exhibit different susceptibilities to DNA repair and mutagenic potential, if not repaired in a structure-dependent manner. To ultimately gain insights into structurefunction relationships, we computed conformations of stereoisomeric guanine, adenine, and cytosine base adducts using density functional theory. We find near mirror image conformations in stereoisomer adduct pairs for each modified base, suggesting opposite orientations with respect to the 5′ f 3′ direction of the modified strand when the stereoisomer pairs are incorporated into duplex DNA. Such opposite orientations could cause stereoisomer pairs of lesions to respond differently to DNA replication and repair enzymes.

Introduction Excessive exposure of women to estrogens is linked to an increased risk of developing breast and endometrial cancer (1-5). One source of such exposure is estrogen replacement therapy. Premarin (Wyeth), which has been the most frequent choice for estrogen replacement therapy in the United States, contains the equine estrogens, equilin and equilenin, as major components (6, 7). 4-OHEN,1 a phase I metabolite of both of these substances (8-10), is a catechol estrogen, which autoxidizes to potent cytotoxic quinoids that can cause a variety of DNA lesions (11, 12). The nature of estrogen-induced carcinogenesis is complex and multifaceted (13-15), but one possible pathway for cancer initiation involves the modification of DNA by 4-OHEN (16). Experimental evidence has shown that 4-OHEN can induce DNA damage and apoptosis in breast cancer cell lines (17) and cellular transformation in vitro (18), as well as DNA damage in vivo when it is injected into the mammary fat pads of rats (19). Among the DNA lesions observed in rats were oxidized bases, apurinic sites, single strand breaks, and stable adducts produced by alkylation (19). Such adducts, if not repaired efficiently, may cause mutations that in turn, may initiate carcinogenesis (20). The quinoids produced by 4-OHEN oxidation react with dG, dA, and dC to form cyclic adducts. Chemical structures of these adducts have been determined previ†

Department of Chemistry. Department of Biology. Abbreviations: 4-OHEN, 4-hydroxyequilenin; 4-OHEN-G, 4-hydroxyequilenin-guanine; 4-OHEN-A, 4-hydroxyequilenin-adenine; 4-OHEN-C, 4-hydroxyequilenin-cytosine; PAHs, polycyclic aromatic hydrocarbons. ‡

1

ously by electrospray MS and NMR methods (Figure 1) (8, 21, 22). These reveal three chiral centers in each case, leading to the possible existence of eight stereoisomeric adducts. However, only four of the possible eight stereoisomers have been detected so far using LC coupled nanoES-MS experiments (23). For the guanine adducts, stereoisomers with H and OH at the 2′- and 3′-positions in trans were not found, presumably because these orientations would strain the five-membered E ring. In the case of the adenine and cytosine adducts, stereoisomeric adducts containing the H and OH at the 1′- and 3′positions in trans were not found, also presumably due to strain, in this case of the E and adjacent A rings (23). These experiments actually found only three of the expected four stereoisomers for the adenine and cytosine adducts; however, a fourth was inferred in each case by analogy with the guanine adducts and was assumed to have coeluted with another adduct (23). In the present work, we are concerned with a study of the experimentally observed isomers for each base. There is a lack of information about the conformations of the stereoisomeric 4-OHEN adducts, except for a previous conformational analysis of one of the stereoisomeric adducts of 4-OHEN-G, namely, 4-OHEN-G1 (24). In the current study, we have computed the conformations for four stereoisomers of 4-OHEN-A and 4-OHEN-C and the conformations of the three 4-OHEN-G adduct stereoisomers that were not investigated previously (24). A computational investigation was carried out employing quantum mechanical geometry optimization using density functional theory (DFT), which delineated conformational possibilities for each stereoisomeric base adduct. Results of our previous study for 4-OHEN-G1 are also included here to permit ready comparison with the other

10.1021/tx0340246 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/29/2003

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Figure 1. Structure of 4-OHEN and structures and stereochemistry of linkage site of 4-OHEN-G, 4-OHEN-A, and 4-OHEN-C adducts. Torsion angle definitions: δ′, N1-C2′-C3′-N2; ′, C1′-C2′-C3′-C4′; ζ′, C3′-C4′-C5′-C10′; η′, C9′-C10′-C1′-O1′; γ′, O1′-C1′-C2′-N1; and θ′, C1′-N1-C6-N6 for 4-OHEN-A and θ′, C1′-N3-C4-N4 for 4-OHEN-C.

stereoisomeric adducts. Near mirror image conformations in stereoisomer adduct pairs for each base were found. Moreover, our results suggest that in double-stranded DNA, the equilenin ring system is unlikely to intercalate into the B-DNA helix, and is likely to reside at the helix exterior on the major groove side. This stems from the nonplanar and conformationally restricted nature of these adducts, which obstruct the Watson-Crick base pairing edge. The near mirror image stereoisomer pairs are thus likely to adopt opposite orientations with respect to the 5′ f 3′ direction of the modified strand, along the DNA helix exterior.

Materials and Methods Using INSIGHTII (from Accelrys Inc., a subsidiary of Pharmacopeia, Inc.), we constructed chemical structures for each of the four stereoisomers of 4-OHEN-G, 4-OHEN-A, and 4-OHENC (Figure 1) (8, 22, 23), employing the established chemical structure and stereochemistry of each 4-OHEN base adduct and a crystal structure that contained equilenin (25) for the geometry of the B, C, and D rings. Then, to create starting conforma-

tions for each stereoisomer, restrained energy minimization calculations were carried out, using the Merck molecular force field (MMFF94) (26-29) implemented in SPARTAN 5.1. These calculations were restrained to produce conformational families for the E ring and the cyclohexene A ring (Figure 1) that are chemically feasible for these molecules (see Results). In the next, quantum mechanical stage, the conformational families derived from SPARTAN were subjected to geometry optimization, using the quantum mechanical DFT method (Becke3LYP functional), with the 6-31G* basis set (30-33). The quantum mechanical calculations were performed with Gaussian 98 (34). We also made several specific additional searches for conformers that appeared to be missing from the results of this set of trials, as we were concerned with the multiple minimum problem. Specifically, a mirror image conformer with inverted dihedral angles was constructed for 4-OHEN-G2 from optimized conformation 1b of 4-OHEN-G1, for 4-OHEN-G1 from optimized conformation 2a of 4-OHEN-G2, and for 4-OHEN-G4 from optimized conformation 3b of 4-OHEN-G3. These were then subjected to the same two stage minimization as the other initial conformational models. These trials produced, respectively, optimized conformers 4-OHEN-G2c, 4-OHEN-G1d, and 4-OHEN-G4b (see Results, Table 3, and Discussion).

Conformations of 4-Hydroxyequilenin Base Adducts

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Figure 2. Linkage site starting conformations for 4-OHEN-G adducts in stereo view: (a) 4-OHEN-G1, (b) 4-OHEN-G2, (c) 4-OHEN-G3, and (d) 4-OHEN-G4. All stereo images are constructed for viewing with a stereo viewer.

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Table 1. Torsion Angle Values in Starting Conformations of 4-OHEN-G Adducts I (°)

II (°)

III (°)

IV (°)

-2.0 2.4 -37.4 102.2 -166.2

-5.7 -4.2 30.4 34.9 -83.0

δ′ ′ ζ′ η′ γ′

-33.2 -30.1 -2.2 36.1 -97.9

4-OHEN-G1 27.2 35.3 14.9 69.3 -144.1

δ′ ′ ζ′ η′ γ′

-30.1 -44.0 -2.4 -84.3 170.2

4-OHEN-G2 32.2 36.8 10.0 -56.3 100.1

2.4 -0.3 36.0 -99.8 163.8

3.3 1.0 -26.5 -38.1 91.1

δ′ ′ ζ′ η′ γ′

-30.9 -30.3 -3.6 -73.2 28.0

4-OHEN-G3 25.8 39.8 9.9 -51.9 -30.3

-0.8 -1.0 34.8 -99.0 40.5

-1.5 2.5 -37.9 1.1 -63.1

δ′ ′ ζ′ η′ γ′

-33.1 -49.1 0.7 23.0 69.9

4-OHEN-G4 32.1 34.7 7.0 65.8 -19.1

0.8 0.9 -39.5 99.8 -41.0

1.5 -2.6 39.9 -0.3 63.4

Results Initial Models. In this stage of the work, our goal was to survey the feasible conformational families for each of the 4-OHEN base adducts. Although their stereochemical characteristics are different, all stereoisomers of each base adduct have similar chemical and structural characteristics, namely, their hybridization and bond nature, which limit their possible conformations. Initial conformational models were created for the four stereoisomers of each base adduct (Figure 1) using SPARTAN 5.1 (Wavefunction, Inc.). The geometries of the connecting E ring and the adjacent cyclohexene A ring, together with the assistance of hand models for each stereoisomer, served as guides to delineating possible conformational families. Preliminary energy minimizations were carried out, which then provided the starting conformations for the DFT calculations. 1. 4-OHEN-G. Four starting conformational families for each stereoisomer of 4-OHEN-G were generated (Figure 2), based on the geometrical properties of the fivemembered ring generated by adduct formation and of the adjacent cyclohexene A ring of the equilenin moiety (Figure 1). The possible conformations of this molecule are restricted because of the constrained nature of the linking five-membered E ring and the cyclohexene A ring. In addition, N1, C2, and N2 cannot pucker, because of the sp2 hybridization at N1 and C2 and the primarily sp2 hybridization at N2 (Figure 1). As a result, we considered conformational flexibility only at C2′ and C3′. For each of the four stereoisomers, we obtained four starting conformations for subsequent quantum mechanical optimization. As illustrated in Figure 1, the dihedral angle δ′ (N1-C2′-C3′-N2) describes the conformation of the linking five-membered E ring, ′ (C1′-C2′-C3′-C4′)

and ζ′ (C3′-C4′-C5′-C10′) define the cyclohexene A ring, η′ (C9′-C10′-C1′-O1′) defines the orientation of the O1′-HO1′ hydroxyl, and γ′ (O1′-C1′-C2′-N1) describes the spatial relationship between O1′ and N1. The torsion angle values in these initial models are summarized in Table 1. In starting conformation I, the linking five-membered E ring adopts an envelope conformation with a negative δ′ value; we define this as having a negative envelope conformation (Figure 2 and Table 1). In this conformer, the cyclohexene A ring, whose conformation is correlated to that of the E ring, adopts a sofa conformation with only one atom, namely C2′, out of the plane and negative ′. In starting conformation II, the linking five-membered E ring exhibits an envelope conformation, which is the opposite of that of starting conformation I. It has a positive δ′ value, and we define this as having a positive envelope conformation. The cyclohexene A ring displays a sofa conformation with C3′ as the only atom out of the plane, producing a positive ′. In addition to the two envelope conformations, this five-membered ring can also adopt a planar form. In this case, the cyclohexene A ring can have two possible opposite boat conformations with O1′-HO1′ axial (starting conformation III) and O1′-HO1′ equatorial (starting conformation IV). 2. 4-OHEN-A and 4-OHEN-C. The unsaturated bicyclo[3.3.1]nonane type linkage site makes the possible conformations of 4-OHEN-A severely restricted (Figure 1). Because of the rigid bridge comprised of C1′, C2′, and C3′ at the linkage site, the conjugated ring system of the adenine moiety, and the cyclohexene A ring, there is only one starting conformational family. The dihedral angle θ′ (C1′-N1-C6-N6) is around zero in the starting conformation of each 4-OHEN-A stereoisomer (Table 2). Because of the similar bicyclo[3.3.1]nonane type linkage site, 4-OHEN-C has a similar starting structure for each stereoisomer. Each stereoisomer has only one starting conformation in which the dihedral angle θ′ (C1′N3-C4-N4) is around zero (Table 2). Optimized Conformations of 4-OHEN-G Adducts. The geometries of the starting conformations for the 4-OHEN base adduct stereoisomers were energy-optimized quantum mechanically using the DFT method (Becke3LYP functional), with the 6-31G* basis set. Table 3 summarizes torsion angle values and energies in the optimized conformers of the 4-OHEN-G stereoisomers. 1. 1′R,2′S,3′R-4-OHEN-G1. As described in the previous study of this stereoisomer (24), the above four starting conformations converge to three optimized conformers. They are characterized by differing conformations of their linking five-membered E rings, namely, negative envelope, positive envelope, and planar (Figure 3). Starting conformations I and IV converge to optimized conformation 1a, in which the linking five-membered E ring adopts the negative envelope conformation and the cyclohexene A ring adopts a half-chair conformation. Starting conformation II yields optimized conformation 1b, in which the linking five-membered E ring adopts a positive envelope conformation (opposite to that of opti-

Table 2. θ′ Torsion Angle Values in Starting Conformations of 4-OHEN-A and 4-OHEN-C Adducts θ′

1′S,2′S,3′R-4-OHEN-A1 -1.3°

1′R,2′R,3′S-4-OHEN-A2 0.8°

1′S,2′R,3′R-4-OHEN-A3 -1.5°

1′R,2′S,3′S-4-OHEN-A4 0.8°

θ′

1′S,2′S,3′R-4-OHEN-C1 -1.9°

1′R,2′R,3′S-4-OHEN-C2 1.9°

1′S,2′R,3′R-4-OHEN-C3 -2.6°

1′R,2′S,3′S-4-OHEN-C4 2.6°

Conformations of 4-Hydroxyequilenin Base Adducts Table 3. Torsion Angle Values and Energies in Quantum Mechanically Optimized Conformations of 4-OHEN-G Adductsa

Chem. Res. Toxicol., Vol. 16, No. 6, 2003 699 Table 4. θ′ Torsion Angle Values in Quantum Mechanically Optimized Conformations of 4-OHEN-A and 4-OHEN-C Adducts θ′

4-OHEN-A1 -7.3°

4-OHEN-A2 7.4°

4-OHEN-A3 -8.7°

4-OHEN-A4 8.3°

θ′

4-OHEN-C1 -3.0°

4-OHEN-C2 3.8°

4-OHEN-C3 -5.3°

4-OHEN-C4 5.9°

4-OHEN-G1 ∆E (kcal/mol) δ′ ′ ζ′ η′ γ′

1a

1b

1c

1d

0 -28.2° -26.2° -8.0° 55.8° -101.6°

3.8 21.7° 30.1° -19.5° 92.7° -172.7°

3.0 -3.6° 6.8° -35.2° 111.7° -170.5°

2.7 -8.4° 2.0° -33.4° 99.6° -165.5°


2a

2b

2c

2.4 7.2° -3.1° 33.2° -101.7° 167.8°

0 28.4° 26.8° 7.9° -56.3° 101.8°

3.7 -21.0° -29.2° 19.0° -92.9° 170.4°

3a

3b

3c

1.6 -23.2° -19.7° -0.5° -71.0° 19.4°

2.4 -1.8° 0.0 20.7° -79.3° 23.4°

0 16.9° 27.1° -29.3° -0.7° -80.0°



4a

4b

0 -15.9° -26.0° 29.2° -0.3° 79.6°

1.6 23.5° 20.0° 0.5° 69.8° -18.0°

a ∆E is the energy relative to the lowest energy optimized conformation for each 4-OHEN-G stereoisomer. These values are -965738.255, -965738.305, -965736.890, and -965736.748 kcal/ mol for the G1, G2, G3, and G4 stereoisomers, respectively.

mized conformation 1a) and the cyclohexene A ring exists as a distorted boat. Starting conformation III converges to optimized conformation 1c, in which the linking fivemembered E ring retains a planar conformation and the cylohexene A ring adopts a boat conformation. Optimized conformation 1d (Table 3) is a slightly lower energy variant of optimized conformation 1c that was derived from a special search, as described in the Materials and Methods section. 2. 1′S,2′R,3′S-4-OHEN-G2. Starting conformations I and III converge to optimized conformation 2a (Figure 4). The linking five-membered E ring is almost planar, and the cyclohexene A ring has a boat conformation with an axial O1′-HO1′ hydroxyl group (η′ ) -101.7°). Starting conformations II and IV converge to optimized conformation 2b, in which the linking five-membered E ring adopts a positive envelope conformation. The cyclohexene A ring becomes a half-chair with pseudoaxial O1′-HO1′ (η′ ) -56.3°). Optimized conformation 2c resulted from a special search (see Materials and Methods). In this conformer, the five-membered E ring adopts a negative envelope conformation and the cyclohexene A ring exists as a distorted boat. 3. 1′S,2′S,3′R-4-OHEN-G3. The four SPARTANgenerated starting conformations converge to three optimized conformers (Figure 5). Starting conformation I

converges to optimized conformation 3a. The linking fivemembered E ring still adopts the negative envelope conformation, and the cyclohexene A ring retains a sofa conformation. Starting conformations II and III converge to optimized conformation 3b, in which the linking fivemembered E ring is planar and the cyclohexene A ring adopts a boat conformation. Starting conformation IV converges to optimized conformation 3c, in which the linking five-membered E ring adopts a positive envelope conformation, opposite to optimized conformation 3a. The A ring in conformer 3c has a distorted boat conformation. 4. 1′R,2′R,3′S-4-OHEN-G4. The four initial conformations converge to two optimized conformational families (Figure 6). Starting conformations I and IV converge to optimized conformation 4a. The linking five-membered E ring adopts a negative envelope conformation with C3′ out of plane, and the cyclohexene A ring becomes a distorted boat conformation with gauche conformation between O1′ and N1 (γ′ ) 79.6°). The O1′-HO1′ group stays equatorial with η′ ) -0.29°. Starting conformations II and III converge to optimized conformation 4b, in which the linking five-membered E ring adopts a positive envelope conformation that is opposite to optimized conformation 4a. The A ring in conformer 4b has a sofa conformation with a pseudoaxial O1′-HO1′ hydroxyl group. Optimized Conformations of 4-OHEN-A and 4-OHEN-C Adducts. The starting conformations of each of the stereoisomeric 4-OHEN-A and 4-OHEN-C adducts retain their initial conformational types after DFT minimization (Figures 7 and 8). As shown in Table 4, the dihedral angle θ′ is around zero in each DFT optimized structure. Relative Stabilities of Stereoisomers. Table 5 gives relative stereoisomer stabilities. We note that the 4-OHEN-G1 and G2 pair are lower in energy than the G3 and G4 pair. We also see that 4-OHEN-A4 and 4-OHEN-C4 are the most stable isomers for adenine and cytosine adducts, respectively.

Discussion In this work, we have investigated the conformations of stereoisomeric adducts of 4-OHEN to guanine, cytosine, and adenine. We considered four experimentally deduced stereoisomers for each base adduct. In the case of the more flexible guanine adducts, we find two or three possible conformational families for each stereoisomer, while the more rigid adenine and cytosine adduct stereoisomers are each restricted to a single conformational family. Most interestingly, we find that stereoisomeric adduct pairs adopt near mirror image conformations (Figure 9) reminiscent of the orientations adopted by stereoisomeric adducts derived from metabolically activated PAHs (37). Conformational Preferences in 4-OHEN-G Adducts. Possible features contributing to the energetic stabilities of the conformers in the four stereoisomers of 4-OHEN-G include the following: (i) Allylic strain: An

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Figure 3. Stereo views of optimized conformations for 4-OHEN-G1. The linkage site is also shown separately as an inset. Table 5. Relative Stabilities of 4-OHEN Adduct Stereoisomersa ∆E (kcal/mol)

4-OHEN-G1a 0.05

4-OHEN-G2b 0

4-OHEN-G3c 1.42

4-OHEN-G4a 1.56

∆E (kcal/mol)

4-OHEN-C1 0.63

4-OHEN-C2 0.31

4-OHEN-C3 0.46

4-OHEN-C4 0

∆E (kcal/mol)

4-OHEN-A1 0.08

4-OHEN-A2 0.20

4-OHEN-A3 0.002

4-OHEN-A4 0

a The lowest energy conformer for each guanine adduct stereoisomer was selected from Table 3 and is indicated by name. cytosine and adenine adduct stereoisomers each have only one optimized conformation. For each base adduct, the stereoisomer with the lowest energy is assigned ∆E ) 0, and energies are relative to this lowest energy stereoisomer. Energies may be compared only for stereoisomers of a given base adduct. Absolute energies for the guanine adduct stereoisomers are given in Table 3. For the cytosine adduct stereoisomers, ∆E ) 0 corresponds to an absolute energy of -873102.630 kcal/mol, and for the adenine adduct stereoisomers, ∆E ) 0 corresponds to an absolute energy of -918511.755 kcal/mol.

axial O1′-HO1′ hydroxyl group would avoid steric crowding due to allylic strain between O1′-HO1′ on the A ring and the C11′-H2 methylene group on the C ring (35). (ii) The relationship between O1′ and N1: The gauche conformation of O1′ and N1 could avoid the steric crowding and electrostatic repulsion between these two atoms, which occurs when they are in the syn configuration. (iii) Hydrogen bonding: A hydrogen bond between HO1′ and O6(G) could stabilize the structure. (iv) Conformation of the cyclohexene A ring: Both theoretical and experimental studies on cyclohexene and its derivatives have shown that the half-chair conformation is more stable than the boat conformation (36). We analyzed the conformational

stabilities for the conformers of each stereoisomer of 4-OHEN-G, considering the above features. 1. 4-OHEN-G1. The O1′-HO1′ group is in a pseudoequatorial position, with a dihedral angle η′ ) 36.1° in starting conformation I and η′ ) 34.9° in starting conformation IV (Table 1, Figure 2a). These two starting conformations converge to optimized conformation 1a with a pseudoaxial η′ ) 55.8° (Table 3, Figure 3). The three optimized conformations 1a, 1b, and 1c have a pseudoaxial or axial O1′-HO1′ hydroxyl group (the dihedral angle η′ is 55.8°, 92.7°, and 111.7° in optimized conformations 1a, 1b, and 1c, respectively). These optimized conformations avoid the steric crowding due to



Figure 4. Stereo views of optimized conformations for 4-OHEN-G2. The linkage site is also shown separately as an inset.

allylic strain between the equatorial O1′-HO1′ hydroxyl on the A ring and the C11′-H2 methylene group that was present in the starting conformation. Optimized conformation 1a is more stable than optimized conformations 1b and 1c by ∼3-4 kcal/mol (Table 3). This appears to stem from a hydrogen bond and the conformation of the cyclohexene A ring. Only optimized conformation 1a has a hydrogen bond between HO1′ on the 4-OHEN moiety and O6(G), and the half-chair conformation of the cyclohexene A ring in this conformer is more preferred than the boat type conformation in optimized conformations 1b and 1c. 2. 4-OHEN-G2. Starting conformations I and III converged to optimized conformation 2a with almost a fully axial O1′-HO1′ hydroxyl group. Starting conformations II and IV converge to optimized conformation 2b. The O1′-HO1′ group changes from a pseudoequatorial orientation (η′ ) -38.1°, Table 1) in starting conformation IV to a pseudoaxial one (η′ ) -56.3°, Table 3) in optimized conformation 2b. Again, this change from a pseudoequatorial to a pseudoaxial conformation upon optimization relieves the allylic strain present in the starting conformation. The O4′ to N2 distance increased from 2.8 Å in both starting conformations I and IV to 3.1 Å in optimized conformations 2a and 2b, which avoids steric crowding between these atoms. Optimized conformation 2b is more stable than optimized conformation 2a by 2.4 kcal/mol (Table 3). This energy difference probably stems from a hydrogen bond and the conformation of the cyclohexene A ring, as in 4-OHEN-G1. The hydrogen bond between HO1′ and O6-

(G) and the half-chair conformation of the cyclohexene A ring makes optimized conformation 2b more preferred than optimized conformation 2a whose A ring has a boat type conformation. 3. 4-OHEN-G3. Starting conformations II and III converge to optimized conformation 3b, which alleviates the allylic strain (η′ changes from -51.9° in Table 1 to -79.3° in Table 3) and the steric crowding between O1′ on the equilenin moiety and O6(G) in starting conformation II. All three optimized conformers have a hydrogen bond between HO1′ and O6(G). The energetically most favorable conformer is 3c (Table 3). This has a gauche conformation between O1′ and N1(G) whereas the orientation is cis in optimized conformations 3a and 3b. Optimized conformation 3a is somewhat more stable than optimized conformation 3b, probably because of the greater stability of the sofa conformation of the cyclohexene A ring in conformer 3a as compared to the boat conformation in conformer 3b. 4. 4-OHEN-G4. Starting conformations I and IV converge to optimized conformation 4a. The O1′-HO1′ hydroxyl group remains in the equatorial orientation, and the gauche conformation between O1′ and N1(G) is also maintained. Starting conformations II and III converged to optimized conformation 4b, with pseudoaxial O1′-HO1′ group and cis orientation between O1′ and N1(G). The steric crowding between O4′ on the equilenin moiety and N2(G) in starting conformations I and III decreased after DFT optimization (distance increased from 2.8 to 3.0 Å). Both optimized conformers have a hydrogen bond between HO1′ and O6(G).

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The energy difference (1.6 kcal/mol) between optimized conformations 4a and 4b may come from the preference for the gauche over the cis conformation of O1′ and N1(G), as in 4-OHEN-G3. Conformational Preferences in 4-OHEN-A and 4-OHEN-C Adducts. The conformational flexibility in

all 4-OHEN-A and 4-OHEN-C stereoisomers is severely limited. This stems from the combined effects of the rigid C1′-C2′-C3′ bridge, the adenine or cytidine-conjugated ring system, and the sp2 hybridization at C4′, C5′, and C10′ in the cyclohexene A ring. In the optimized conformers (Table 4, Figures 7 and 8), the adenine-conjugated



Figure 7. Stereo views of optimized conformations for 4-OHEN-A.

ring system, and the conjugation of the carboxyl group on the cyclohexene A ring with the aromatic B ring, could contribute to the stabilization of these conformations. Relative Stereoisomer Stabilities. The computed relative stereoisomer stabilities are for the gas phase, and would be altered in solution. Hence, these quantities must be interpreted cautiously, particularly in those cases where the energies are close. However, we can

rationalize the relative stabilities structurally in those cases where the energy differences are more substantial. The stereoisomer pair of 4-OHEN-G1 and G2 is lower in energy than the G3 and G4 pair (Table 5); this may stem from the preference for the trans configuration between the N1 and the O1′-HO1′ hydroxyl group in the G1 and G2 pairs, over the cis configuration in G3 and G4. 4-OHEN-A4 and 4-OHEN-C4 are the most stable

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Figure 8. Stereo views of optimized conformations for 4-OHEN-C.

isomers for the adenine and cytosine adducts, respectively, because of the anti O2′-HO2′ hydroxyl group in the bicyclo[3.3.1]nonane type linkage site and because the C18′ methyl group is far away from the base moiety, which avoids crowding. Relative stabilities of guanine vs adenine vs cytosine adducts cannot, however, be assessed from these computations since these are not alternate conformers for stereoisomers of the same base adduct. Near Mirror Image Relationships in Stereoisomeric Adduct Pairs. Consideration of the chemical

structures of the stereoisomeric adducts under investigation shows that they are paired, with inverse R and S configurations (Figure 1). Our studies reveal that these adduct pairs adopt near mirror image conformations. The two covalent bonds formed between 4-OHEN and guanine have a cis configuration, since the five-membered E ring would be highly strained in the trans configuration. With a fixed position of the guanine base, the spatial arrangement of the 4-OHEN moiety is determined by the orientation of the O3′-HO3′ hydroxyl group at the C3′



Figure 9. Lowest energy optimized conformation for each 4-OHEN base adduct. Adduct names are as given in Tables 3 and 4 and Figures 3-8.

atom (Figure 9). In 4-OHEN-G1 and 4-OHEN-G3, O3′-HO3′ is below the plane of guanine, and the equilenin moiety is oriented above this plane. On the other hand, in 4-OHEN-G2 and 4-OHEN-G4, the equilenin moiety is oriented below the plane of the guanine base with the O3′-HO3′ hydroxyl group located above the plane. Thus, the 4-OHEN-G1 and 4-OHEN-G2 adducts are almost mirror images of one another except for the D ring with the C18′ methyl group on the equilenin moiety; a similar relationship is observed in the case of the adducts 4-OHEN-G3 and 4-OHEN-G4 (Figure 9). The D ring with the C18′ methyl group is rather far from the linkage region and generally would not be expected to have a large effect on the conformations of these adducts. Indeed, our results do show that the stereoisomer pairs usually adopt essentially mirror image conformations. This may be seen from a comparison of 4-OHEN-G1 and 4-OHEN-G2 and 4-OHEN-G3 and 4-OHEN-G4 (Table 3), which show inverse dihedral angles that are characteristic of mirror image conformers. Figures 3-6 show this structurally. For 4-OHEN-G3 and 4-OHEN-G4, optimized conformations 3c and 4a, respectively, are the energetically most favored conformers and have inverse dihedral angles (Table 3). Optimized conformations 3a and 4b are also characterized by inverse dihedral angles and similar energy values. The highest energy conformer, optimized conformation 3b, however,

has no corresponding mirror image conformer in the case of 4-OHEN-G4. Therefore, we searched specifically for such a conformation for 4-OHEN-G4 by creating it as a starting conformation and optimized it as was done for the other starting conformations (see Materials and Methods). However, our search for a mirror image conformer to optimized conformation 3b did not produce such a conformation. Instead, the starting conformation of this type converged to optimized conformation 4b (see below). Mirror image relationships between the optimized conformers of 4-OHEN-G1 and 4-OHEN-G2 are like those of 4-OHEN-G3 and 4-OHEN-G4. In the case of 4-OHENG2, the 1b near mirror image conformer was located as an energy minimum from a specific search for such a conformer (see Materials and Methods) (Table 3). Also, in a specific search for the mirror image to optimized conformation 2a, we obtained optimized conformation 1d. This conformation is a closer mirror image than the conformer 1c, which had been located in the original search, and is also lower in energy. The 4-OHEN adenine and cytosine adducts also have near mirror image relationships between their stereoisomers (Figure 9). The dihedral angle θ′ is inverse in the corresponding near mirror image isomers (Table 4). Mirror Image Symmetry Breakage by the D Ring. Conformations of stereoisomer pairs are essentially mir-

706


ror image except for the methyl-containing D ring. In all cases except one, energy minima were found in which the conformations were near mirror images except for the D ring with the C18′ methyl group. However, in the case of 4-OHEN-G4, the near mirror image of conformer 3b converged to a related form. The mirror image breaking methyl group on the D ring (Figure 1) appears to have had a cascade effect that disfavored the mirror conformation, likely through the interactions involving the O1′-HO1′ and O3′-HO3′ hydroxyl groups. Conformations of Adducts in DNA. The structures of the 4-OHEN base adducts are sufficiently unique to allow for some consideration of the conformations they may adopt in double-stranded DNA. These cyclic adducts obstruct the Watson-Crick hydrogen-bonding edge of the base. Therefore, the modified base residue could not adopt a Watson-Crick hydrogen-bonded conformation in a B-DNA duplex. Furthermore, the bulk of the equilenin moiety of the adduct is oriented at a sharp angle to the modified base plane (Figure 9), has only one aromatic B ring, and has a C18′ methyl group that is out of the plane of the equilenin moiety. These bulky and conformationally restricted structures do not appear to be favorable for intercalation into the DNA double helix, since a massive local disruption of the DNA duplex would be required to accommodate the large, nonplanar ring system that is near perpendicular to its attached base. Instead, the modified base may adopt the syn glycosidic bond conformation, which would place the equilenin moiety into the major groove region. Another possibility, retention of the anti conformation in the modified base with displacement of the partner base into a groove, as suggested by a reviewer, is possible but would appear to require disruption of the base pair adjacent to the modification due to the sharp angle between the base and the equilenin ring system. Another interesting point is the expected orientation of the stereoisomer adduct pairs with near mirror image conformations. Such stereoisomeric pairs would be expected to adopt opposite orientations with respect to the 5′ f 3′ direction of the modified strand. This is illustrated in Figure 9, which shows how these pairs align oppositely with respect to the modified bases. It is anticipated that such opposite adduct orientations might elicit different susceptibilities to excision by nucleotide excision repair enzymes and bypass of these stereoisomeric pairs of lesions by DNA polymerases. Such stereoisomer-dependent biological responses (e.g., translesion bypass catalyzed by some bypass polymerases (38) and DNA repair (39-41)) have been observed for stereoisomeric adducts derived from PAHs. It will be interesting to elucidate such possibilities through experimental investigations.

Acknowledgment. This research was supported by NIH Grants CA-75449 and CA-28038 to S.B., NIH Grant CA-76660 to N.E.G, and NIH CA 73638 to Judy L. Bolton (University of Illinois at Chicago). Computations were carried out on SGI workstations in our laboratory and at the Information Technology Services of New York University, as well as at the DOE NERSC and the NSF SDSC Supercomputer Centers. We appreciate stimulating discussions and suggestions with Judy Bolton and Minsun Chang (University of Illinois at Chicago).

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Conformations of Stereoisomeric Base Adducts to 4

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