Cyclohexene Ring and Fjord Region Twist Inversion in Stereoisomeric

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Cyclohexene Ring and Fjord Region Twist Inversion in Stereoisomeric DNA Adducts of Enantiomeric Benzo[c]phenanthrene Diol Epoxides Min Wu,† Shixiang Yan,† Dinshaw J. Patel,‡ Nicholas E. Geacintov,*,† and Suse Broyde*,§ Chemistry and Biology Departments, New York University, New York, New York 10003, and Cellular Biochemistry & Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Received September 18, 2001

The sterically hindered, nonplanar fjord region polycyclic aromatic hydrocarbons (PAHs) have been of great interest because of the exceptionally high mutagenic and tumorigenic activity of certain of their metabolically activated diol epoxides. Benzo[c]phenanthrene (B[c]Ph), a representative fjord region PAH, is metabolically activated to a pair of enantiomers, 1S,2R,3R,4S-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene, (+)-anti-B[c]PhDE, and the corresponding 1R,2S,3S,4R enantiomer, (-)-anti-B[c]PhDE. Both of these can bind covalently to the amino group of purines in DNA via trans addition. In the present work we carry out an extensive computational investigation of the 1R(+) and 1S(-)-trans-anti-B[c]Ph adducts to the base guanine, with the goal of delineating the conformational possibilities for the fjord region and the adjacent cyclohexene-type benzylic ring and their relevance to DNA duplexes. We created 10 369 starting structures for each adduct and minimized the energy using AMBER 5.0. A limited set of conformational families is computed, in which the R isomer structures are near mirror images of the S isomer. The benzylic rings are essentially all halfchair-type. Cyclohexene-type ring inversion as well as fjord region twist inversion are possible for each isomer and are correlated. DNA duplexes modified by fjord region adducts select conformers from the allowed families that optimize stacking interactions, which contributes to the stability of the carcinogen-intercalated DNA duplex structures [Cosman et al. (1993) Biochemistry 32, 12488-12497; Cosman et al. (1995) Biochemistry 34, 1295-1307; Suri et al. (1999) J. Mol. Biol. 292, 289-307; Lin et al. (2001) J. Mol. Biol. 306, 1059-1080]. In turn, this stability could contribute to the resistance to repair by the human nucleotide excision system observed in fjord region adducts [Buterin et al. (2000) Cancer Res. 60, 1849-1856].

Introduction The sterically hindered fjord region (1) polycyclic aromatic hydrocarbons (PAHs)1 have been of great interest because of the exceptionally high mutagenic (2-4) and tumorigenic activities (5-10) of certain of their metabolically activated diol epoxides (11). These types of PAHs are present in the environment in automobile exhaust, tobacco smoke, and as food contaminants (1216). The steric hindrance across the fjord region leads to nonplanar structures in the parent PAH (17), and it has long been speculated that this distortion in the aromatic ring system may play a role in the tumorigenicity of their activation products (7, 18, 19). Benzo[c]phenanthrene (B[c]Ph), a representative fjord region PAH, is metabolically activated to two pairs of * To whom correspondence should be addressed. Phone: (212) 9988407. Fax: (212) 998-8421. E-mail: [email protected]. Phone: (212) 9988231. Fax: (212) 995-4015. E-mail: [email protected]. † Chemistry Department. ‡ Cellular Biochemistry & Biophysics Program. § Biology Department. 1 Abbreviations: PAH, polycyclic aromatic hydrocarbon; B[c]Ph, benzo[c]phenanthrene; (+) and (-)-anti-B[c]PhDE, 1S,2R,3R,4S-3,4dihydroxyl-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene and the corresponding 1R,2S,3S,4R enantiomer, respectively; B[a]P, benzo[a]pyrene; B[g]C, benzo[g]chrysene.

diastereomeric diol epoxides, namely syn and anti benzo[c]phenanthrene diol epoxide (B[c]PhDE) (20-23). Both (+) and (-)-anti-B[c]PhDE can react with purines in DNA (24-26) via cis and trans addition to form the corresponding (+) and (-) cis and trans-anti adducts, with trans addition predominant (27). Both adenine and guanine amino group adducts are obtained in vitro and in vivo (5, 24, 25, 27-30). While adenine adducts are found in higher proportion (25), the mutational burden imposed by all adducts (31, 32) can contribute to mutagenesis and cancer initiation via alteration in function of key oncogenes or tumor suppressor genes (33-36). High-resolution NMR solution structures of 1R(+) and 1S(-)-trans-anti-B[c]Ph-N6-dA (37, 38) and N2-dG (39) adducts in DNA have revealed that the B[c]Ph moiety adopts an intercalated position in the DNA duplex; the 1R(+) isomer is placed on the 5′-side of the modified adenine, while it is on the 3′-side of the modified guanine. The 1S(-) isomer is intercalated on the 3′-side of the modified adenine, but it is on the 5′-side of the modified guanine. There is distortion but not disruption of the A-T or G-C base pair at the lesion site. Interestingly, these structures suggest opposite twists in the distorted, nonplanar fjord aromatic regions in the two stereoisomers,

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Figure 1. Structures of B[c]Ph, (+)/(-)-anti-B[c]PhDE, and 1R(+)/1S(-)-trans-anti-B[c]Ph-N2-G base adducts. Torsion angles R′, β′, γ′1, γ′2, δ′ are defined as follows: R′, N1-C2-N2-C1(B[c]Ph); β′, C2-N2-C1(B[c]Ph)-C2(B[c]Ph); γ′1, C1-C2-C3-C4; γ′2, C18-C1-C2-C3; δ′, C15-C16-C17-C18.

which serve to optimize stacking of the carcinogen with the adjacent base pairs (37-40). Also, these structures are characterized by heterogeneity in their cyclohexenetype benzylic ring conformations. Over the years, simple cyclohexene-type inversion has been studied extensively (41-45) but has never been investigated in a complex system such as the B[c]PhDE adducts. We have been intrigued by the multiplicity in the benzylic ring conformations and in the distortion from planarity of the aromatic ring systems in the vicinity of the fjord region. Our goal in the present work is to elucidate the fundamental structural principles that govern the benzylic ring conformations and fjord region twists, which occur in hindered fjord region adducts, and to relate the results to experimental observations in DNA duplexes in solution and to the biological consequences induced by these lesions. We have carried out a comprehensive computational investigation of benzylic ring conformation and fjord

region twist, employing the 1R(+) and 1S(-)-trans-antiB[c]Ph-N2-G adducts, without the sugar residues (Figure 1), as prototypes. We performed 10 369 energy minimization trials for each adduct, using AMBER 5.0 (46) with the Cornell et al. force field (47). We find a limited set of allowed structural domains, in which conformers of the R and S isomers are near mirror images; this is governed by the enantiomeric nature of the benzylic ring in the R and S stereoisomers. The benzylic rings are essentially all half-chair-type. Furthermore, a significant possibility exists for cyclohexene-type ring inversion in each isomer, as well as the possibility for inversion of the fjord region twist. Moreover, these two kinds of conformational variabilities are correlated. High-resolution NMR solution structures of fjord region adducts reveal that the modified DNA duplexes select conformers from the allowed families that optimize stacking interactions; this enhances the stability of these intercalated DNA duplex structures (37-40). In turn, this stability could play a significant

DNA Adducts of Benzo[c]phenanthrene Diol Epoxides

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role in the resistance to repair by the human nucleotide excision system observed in 1R(+) and 1S(-)-trans-antiB[c]Ph-N6-dA adducts (48). Finally, based on these investigations and by taking the importance of WatsonCrick base pairing into account, the opposite orientation of the B[c]Ph-dG and B[c]Ph-dA adducts in doublestranded DNA (Scheme 1) can be rationalized. Scheme 1

Figure 2. Half-chair and boat conformations of cyclohexene ring. γ′1, C1-C2-C3-C4; γ′2, C18-C1-C2-C3.

Materials and Methods Creating Starting Conformations. Benzylic Ring. Coordinates of the high-resolution NMR solution structures of the 1R(+) and 1S(-)-trans-anti-B[c]Ph-N6-dA adducts in an 11-mer DNA duplex (37, 38) were employed to construct the corresponding B[c]Ph-N2-G adduct by excising the B[c]Ph-N6-A moiety and replacing the adenine by a guanine (the NMR solution structure of the guanine adduct was not available when this work was initiated). Since we wished to sample the conformation space of the nonplanar cyclohexene type benzylic ring in the present study, we created half-chair and boat starting conformations with symmetric out of plane puckering, each of which consists of a pair of ring-inverted conformers (Figure 2). The γ′1 dihedral angle fully characterizes this cyclohexene-type benzylic ring conformation since it is the only independent dihedral angle not involving the aromatic C5-C18 bond. In our half-chair conformation, this angle adopts values of ∼+60° and ∼-60° in the two conformers. However, in our boat conformation γ′1 adopts a value of 0° in both conformers. Therefore, we employ the dihedral angle γ2′ (Figure 1) as an additional characteristic angle to distinguish the ring-inverted boat conformations. In our boat conformation this angle adopts values of ∼+45.5° for one conformer and ∼-45.5° for its invert. We employed the molecular modeling package SPARTAN from Wavefunction Inc. to create these starting conformers. To produce the two half-chair conformers the dihedral angle γ′1 was constrained to values of +60° or -60°, while still allowing the four atoms, C1-C2-C3-C4 defining γ′1 and their substituents to be flexible. All other atoms in the B[c]Ph-N2-G adduct were held fixed, and the energy was minimized using the MERCK molecular force field (MMFF94) (49) within SPARTAN. Benzylic ring dihedral angles in the resultant structures are given in Supporting Information, Table S1. The boat conformers were constructed similarly using the two half-chair conformers created previously as starting structures, but constraining γ′1 to 0° and minimizing the energy again. Table S1 (Supporting Information) gives resultant benzylic ring dihedral angles. Fjord Region Twist. The conformation of the twist across the fjord region in the aromatic portion of the molecule is another important parameter that must be considered. This twist is characterized by the dihedral angle δ′, C15-C16-C17-C18 (Figure 1). Values of +18° in the 1R(+)-trans-anti-B[c]Ph-N6dA adduct and -16° in the 1S(-)-trans-anti-B[c]Ph-N6-dA

adduct were inferred in the NMR structures (37, 38), and these values were employed in the above boat and chair conformers of the modeled 1R(+) and 1S(-)-trans-anti-B[c]Ph-N2-G adducts, respectively. In addition, a second set of twist conformations for each benzylic ring conformer was created with SPARTAN. This entailed constraining δ′ to -16° in the 1R(+) isomer and to +18° in the 1S(-) isomer and reminimizing with all other atoms fixed. To sample the B[c]Ph-N2-G linkage torsion angles R′ and β′ (Figure 1), we employed a torsion driver program to rotate R′ and β′ at 10° intervals from 0° to 360° in all combinations, creating 1296 different R′, β′ combinations for each γ′ and δ′. Thus, a total of 10 368 different structures (36R′ × 36β′ × 4γ′ × 2δ′) for the 1R(+)-B[c]Ph-N2-G isomer and the same number for the 1S(-)-B[c]Ph-N2-G isomer were constructed. These structures are summarized in Supporting Information, Table S1. Energy Computations. Energy minima of each of the 10 368 starting structures for each adduct were computed with the molecular mechanics program AMBER 5.0 (46), using the Cornell et al. force field (47) with the PARM96.DAT parameter set. Parameters added to the AMBER 5.0 force field for the B[c]Ph-N2-G adducts are listed in Supporting Information, Table S2. These were obtained by analogy with chemically similar atom types already parametrized in PARM96.DAT. To obtain partial charges for the B[c]Ph-N2-G adducts, Hartree-Fock calculations with basis set 6-31G* were used to calculate the electrostatic potential, using GAUSSIAN 94 (50). Four different conformations were used for each adduct (Supporting Information, Table S3). The least-squares fitting algorithm, RESP (51), provided with AMBER 5.0 was then used to fit the charge to each atom center in the molecule. Partial charges were then averaged. Table S4 (Supporting Information) gives partial charges, AMBER atom type, and topology assignments. A sigmoidal distance-dependent dielectric function (52) was employed in the Coulombic term of the force field, to model the dielectric effects of water. Since there are no negatively charged phosphates in the nucleoside, Na+ counterions were not needed. We employed 100 steps of steepest descent minimization to quickly remove collisions, followed by conjugate-gradient minimization with a convergence criterion of 10-4 kcal/(mol Å). All modeling and visualization were carried out with INSIGHT II from MSI Inc, a subsidiary of Pharmacopeia, Inc. Statistical Weights and Thermodynamic Quantities. To analyze the structures after minimization, we classified them into families based on their similarity in the four dihedral angles

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Table 1. Statistical Weight, Thermodynamic Parameters, and Representative Values of Characteristic Angles in Each Family for 1R(+) and 1S(-)-trans-anti-B[c]Ph-N2-G Adductsa,b,c Family Number

Number of Members

W (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

844 775 1112 1132 1209 698 525 825 1167 735 420 245 195 1 1

44.93 28.32 14.41 8.84 1.88 0.53 0.43 0.24 0.20 0.11 0.05 0.02 0.02 0.02 0.00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1081 1116 1573 805 734 1258 673 743 756 465 413 363 148 1 1

44.04 29.82 17.10 6.33 0.91 0.75 0.52 0.19 0.16 0.09 0.05 0.03 0.01 0.00 0.00

R′ (deg)

β′ (deg)

γ′1 (deg)

δ′ (deg)

1R(+)-trans-anti-B[c]Ph-N2-G Adduct -3.90 0.09 3.99 -3.62 0.34 3.96 -3.22 0.90 4.11 -2.93 1.09 4.02 -2.00 2.07 4.08 -1.24 2.49 3.74 -1.13 2.59 3.72 -0.78 2.91 3.69 -0.68 3.35 4.03 -0.30 3.48 3.78 0.23 3.76 3.54 0.64 3.78 3.14 0.67 3.41 2.75 0.80 0.80 0.00 3.93 3.93 0.00

-180.0 1.6 2.6 1.5 -7.8 -1.1 -178.0 174.2 -173.5 174.9 -177.7 178.7 -0.7 -2.0 -173.1

74.2 74.7 -101.1 -77.4 87.6 145.2 -97.9 88.7 -75.0 144.1 -96.7 77.8 80.7 153.2 -149.1

57.4 57.5 59.1 -61.8 -63.1 55.8 58.8 -63.3 -63.4 56.0 60.3 56.9 57.3 -30.1 26.5

-8.8 -9.2 -4.0 14.5 15.9 19.8 -4.2 15.8 16.1 20.3 0.0 20.1 20.5 18.3 -18.4

1S(-)-trans-anti-B[c]Ph-N2-G Adduct -3.97 0.16 4.13 -3.74 0.40 4.14 -3.41 0.93 4.34 -2.82 1.04 3.85 -1.66 2.13 3.80 -1.55 2.61 4.16 -1.32 2.52 3.84 -0.72 2.31 3.03 -0.61 3.33 3.94 -0.30 3.24 3.54 0.11 3.70 3.59 0.35 3.82 3.46 0.74 3.71 2.97 4.30 4.30 0.00 5.16 5.16 0.00

179.8 -1.9 -2.9 -1.7 6.8 177.9 1.0 176.1 -175.1 -176.5 -178.2 0.7 179.4 0.4 174.4

-74.1 -74.5 101.2 77.6 -87.1 97.9 -146.8 74.2 -144.1 -92.7 -77.0 -79.7 94.8 77.3 86.1

-57.4 -57.6 -59.1 61.8 62.3 -58.8 -55.8 58.7 -56.0 64.5 -57.5 -57.5 -62.6 13.3 60.2

8.8 9.2 4.0 -14.6 -15.7 4.2 -19.8 -12.7 -20.3 -16.9 -20.6 -20.9 -3.4 -0.1 10.7

G (kcal/mol)

H (kcal/mol)

TS (kcal/mol)

a G, H, and S are conformational free energy, enthalpy, and entropy, respectively. b Statistical weights, W, are given in percents of the population. c R′, β′, γ′1, and δ′ given in this table are torsion angles values for the structure with lowest AMBER energy in each family.

R′, β′, γ′1, δ′. Structures whose dihedral angles R′, β′, γ′1, and δ′ were all within the following ranges of the lowest energy variant were grouped into one family: R′ ( 15°, β′ ( 15°, γ′1 ( 5°, and δ′ ( 5°. These ranges were chosen based on preliminary evaluations of the data set, which revealed sharp conformational preferences for R′, β′, and γ′1 within these ranges (Supporting Information, Figure S1, parts a and b). The torsion angle δ′ varies continuously in the ∼-20° to ∼+20° range; we selected (5° for grouping this angle based on visual inspection. On this basis, 15 families were obtained for each adduct (see Results). We computed the fractional statistical weights (Wj), together with relative conformational free energies, enthalpies, and entropies for each family, according to standard methods (53). [We retain two decimals in Table S1 (see Results), but six were used for the calculations.]

Pi )

The conformational free energy of each family, Gj, was then computed from the relationship nj

Gj ) -RT ln

∑e

-∆Ei/RT

To obtain the conformational entropy of each well, Sj, we first compute pi, the fractional statistical weight of each conformer within its own family.

pi )

e-∆Ei/RT nj

∑e

-∆Ei/RT

i)1

Then,

i)1

nj

where ∆Ei is the relative energy (kcal/mol) of a given conformer with respect to the lowest energy structure; R is the universal gas constant, 1.987 × 10-3 kcal/mol K, T ) 300 K, and N is 10 368, the total number of conformers for each adduct. We computed the combined fractional statistical weight, Wj, for each family by summing the individual statistical weights, Pi, of each structure in the family: nj

Wj )

-∆Ei/RT

i)1

e-∆Ei/RT N

∑e

∑

Pi

i)1

where nj (j ) 1, ..., 15) is the number of conformers in each one of the 15 families.

Sj ) -R

∑p ln p i

i

i)1

Conformational enthalpies for each well, Hj, are obtained from

Gj ) Hj - TSj AMBER Energy of the Boat Conformation. To estimate the AMBER energy of the boat conformation, we used the representative structure in Family 1 (Table 1) of the 1S(-) isomer. We modified the benzylic ring to be in a boat conformation (γ′1 ) 1.76°, γ′2 ) 37.1°), using SPARTAN. Then we used AMBER 5.0 to minimize the energy, constraining the carbon



atoms in the benzylic ring to this conformation while keeping all other atoms flexible.

Results Narrow Conformational Ranges. Our conformational search produced 15 minimum energy structural families, grouped as described in the Materials and Methods, for the 1R(+)-trans-anti-B[c]Ph-N2-G adduct and the same number of minimum energy families for the 1S(-)-trans-anti-B[c]Ph-N2-G stereoisomer. We computed fractional statistical weights, as well as thermodynamic parameters (G, H, and TS) for each family, given in Table 1, together with characteristic torsion angle values, R′, β′, γ′1, and δ′ (Figure 1) for the lowest energy variant in each family. Table S5 (Supporting Information) also gives AMBER energy ranges for each family, as well as ranges in each of the characteristic torsion angles. Table S6 (Supporting Information) gives the number of conformers in 1 kcal/mol AMBER energy shells. The occupied ranges in R′, β′, γ′1, and δ′, irrespective of energy distribution, in the 15 families are shown in Figure 3, panels A and B, for each isomer. Figure S1, panels A and B (Supporting Information) show the occupied domains for R′, β′, γ′1, and δ′ as a function of AMBER energy in each stereoisomer. The R′ and β′ torsion angles govern the relative orientations of the carcinogen and nucleoside residues (54-57). The R′ torsion angle adopts the same regions in the 1R(+) and the 1S(-) adducts. In each case, R′ adopts two domains, one ∼0° and the other ∼180°. The torsion angle β′ falls into three domains for both the 1R(+) isomer and the 1S(-) isomer. In the 1R(+) isomer, β′ is located at ∼+90°, ∼-90°, and ∼+145°, in order of importance according to statistical weights (Table 1). However, in the 1S(-) isomer the β′ domains are ∼-90°, ∼+90°, and ∼-145° in order of importance. These results for the R′ and β′ preference are generally consistent with our previous surveys of the potential energy surface for benzo[a]pyrene diol epoxide adducts to dG and dA, in that 0° and 180° domains are favored for R′, and that the R isomer favors the +90° domain for β′, while the S isomer favors the -90° domain (54-57). The γ′1 torsion angle is characteristic of the benzylic ring pucker. There are two boat ring-inverted conformers, with γ′1 ≈ 0°, distinguished by their γ′2 values of +45.5° and -45.5°, respectively. The +60° and -60° domains are half-chairs. We find that almost all our energyminimized structures adopt the half-chair conformation. There is only one structure near the boat conformation at γ′1 ) 13.3° (Table 1 and Table S5, Supporting Information), although half of the trials were from this family. The half-chair type benzylic ring conformation favored by the 1R(+) isomer has γ′1 +60°, while in the 1S(-) isomer the -60° region is favored. The linkage site is axial in both cases. However, both the +60° and -60° regions are found for each isomer (Figure 3, panels A and B, and Figure S1, panels A and B, in Supporting Information). The torsion angle δ′ characterizes the twist in the fjord region. It adopts a range from ∼-10° to ∼+21° in the 1R(+) isomer and from ∼+10° to ∼-21° in the 1S(-) isomer (Table 1 and Table S5, Supporting Information). Our results show that when the twist in the fjord region is toward the linkage site, it is smaller than when it is in the direction opposite to the C1-N2 linkage site, and this applies to both isomers.

Figure 3. Occupancies for characteristic torsion angles R′, β′, γ′1, and δ′ in (A) 1R(+)-trans-anti-B[c]Ph-N2-G and (B) 1S(-)trans-anti-B[c]Ph-N2-G.

Near-Mirror Image Symmetry. Table 1 and Table S5 (Supporting Information) reveal that the 1R(+)-transanti-B[c]Ph-N2-G and 1S(-)-trans-anti-B[c]Ph-N2-G adducts are distinctly different in families 1-4, which take up more than 96% of the fractional statistical weight. Specifically, each characteristic torsion angle in a given family of the 1R(+) isomer is the inverse in the 1S(-) isomer (for R′ the 0° and 180° domains are the same as their sign inverted values), which is a hallmark of mirror image structures. Figure 4 shows color views of representative structures of the lowest energy variants for families 1-4. Figure 5, panels A and B, show these structures in stereo. Figure 4 shows that for each of the first four families, the 1R(+)-trans-anti-B[c]Ph-N2-G adduct structure is essentially a mirror image of the 1S(-)-trans-anti-B[c]Ph-N2-G adduct. Benzylic Ring Inversion and Relation to Fjord Region Twist. The benzylic ring conformation and its relation to the fjord region twist are of particular interest.

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Figure 4. Color views of representative structures for 1R(+) and 1S(-)-trans-anti-B[c]Ph-N2-G base adducts. I-IV denote the 4 families with the highest statistical weights (Table 1). In each pair the left structure is the 1R(+)-trans-anti-B[c]Ph-N2-G adduct and the right is the 1S(-) adduct. R′, β′, γ′1, and δ′ values and AMBER energies, ∆E, relative to the global minimum are the following. Family 1: 1R(+) adduct -180.0°, 74.2°, 57.4°, -8.8°; 1S(-) adduct 179.8°, -74.1°, -57.4°, 8.8°; ∆E 1R(+) adduct 0.06 kcal/mol, 1S(-) adduct 0.06 kcal/mol. Family 2: 1R(+) adduct 1.6°, 74.7°, 57.5°, -9.2°; 1S(-) adduct -1.9°, -74.5°, -57.6°, 9.2°; ∆E 1R(+) adduct 0.32 kcal/mol, 1S(-) adduct 0.32 kcal/mol. Family 3: 1R(+) adduct 2.6°, -101.1°, 59.1°, -4.0°; 1S(-) adduct -2.9°, 101.2°, -59.1°, 4.0°; ∆E 1R(+) adduct 0.81 kcal/mol, 1S(-) adduct 0.81 kcal/mol. Family 4: 1R(+) adduct 1.5°, -77.4°, -61.8°, 14.5°; 1S(-) adduct -1.7°, 77.6°, 61.8°, -14.6°; ∆E 1R(+) adduct 0.93 kcal/mol, 1S(-) adduct 0.93 kcal/mol.

In families 1-3, the benzylic ring conformation contains H1 equatorial, H2 equatorial, H3 axial, and H4 axial (correspondingly, the linkage to G is axial, O2 is axial, O3 is equatorial, and O4 is equatorial) in both isomers. Thus, the linkage site prefers an axial position in both isomers, with the γ′1 of the 1R(+) isomer in the +60° region and the 1S(-) isomer in the -60° region. We also see that this benzylic ring conformation is correlated with the twist of the fjord region. When γ′1 is in its favored domain, the twist in the fjord region prefers to be toward the linkage site with a value of ∼-10° in the 1R(+) isomer and ∼+10° in the 1S(-) isomer. The R′ and β′ angles of the first three families are in their favored regions where R′ is either in the 0° region or 180° region for each isomer and β′ is in the +90° region for the R isomer and the -90° region for the S isomer (54-57). In family 4, the benzylic ring conformation contains H1 axial, H2 axial, H3 equatorial, and H4 equatorial. In this case, the linkage site is in the equatorial position, which we observe to be more crowded than the favored axial position. In this family, γ′1 is in the -60° region for the 1R(+) isomer and +60° for the 1S(-) isomer, with the linkage site in the equatorial position. That is, the cyclohexene-type benzylic ring is inverted from its more favored conformation of families 1-3. Again, the twist is opposite in sign for each isomer, but in this case, away from the linkage site and somewhat steeper, ∼+15° in the 1R(+) isomer and ∼-15° in the 1S(-) isomer. Furthermore, in this family β′ adopts the less favored region of ∼-90° for the 1R(+) isomer and ∼+90° for the 1S(-) isomer. The origin and possible importance of these structures is further considered in the discussion.

Discussion Benzylic Ring Pucker and Fjord Region Nonplanarity: Torsion Angles γ′1 and δ′ and Their Correlation. Benzylic Ring Pucker, γ′1: The Axial Conformation for the Linkage Site Bond Is Preferred. The characteristic angle of the benzylic ring pucker γ′1 adopts the +60° region in 1R(+)-trans-anti-B[c]Ph-N2-G and the -60° region in 1S(-)-trans-anti-B[c]Ph-N2-G, corresponding to an axial conformation at the linkage site, in families 1-3 (Table 1, Figure 4), whose combined statistical weight is ∼90%. However, in family 4 with statistical weight ∼8-9%, γ′1 of the 1R(+)-trans-antiB[c]Ph-N2-G is in the -60° region and γ′1 of the 1S(-)trans-anti-B[c]Ph-N2-G is in the +60° region, with the linkage site conformation equatorial (Table 1, Figure 4). When the linkage site is equatorial, the cyclohexene ring is inverted compared with the benzylic ring conformations of families 1-3. The benzylic ring prefers to adopt a conformation in which the linkage is axial rather than equatorial, because there is less crowding (Figure 4), since the guanine plane is more distant from the phenanthrenyl ring system. Fjord Region Twist, δ′: Minimal Twist Is Achieved by the Axial Linkage Site. The aromatic phenanthrenyl ring has an intrinsic preference to keep all of the aromatic rings in the same plane; this achieves maximum parallelism among the aromatic π bonds and maximum stability. However, the steric hindrance across the fjord region at C1 to C15 must be alleviated, and it costs energy to deform the adjacent aromatic ring. There is an energy balance between the release of the steric hin-


Figure 5. (A) Stereoviews of 1R(+)-trans-anti-B[c]Ph-N2-G adduct. Representative structures of Figure 4. I-IV denote the four families with the highest statistical weights (Table 1). (B) Stereoviews of 1S(-)-trans-anti-B[c]Ph-N2-G adduct. Representative structures of Figure 4. I-IV denote the four families with the highest statistical weights (Table 1).

drance in the fjord region and the deformation of the aromatic phenanthrenyl ring. The fjord region tends to adopt a minimum energy conformation in which there is no collision between the hydrogens on C1 and C15


(minimum distance between them of ∼1.92-1.97 Å) and the twist of the phenanthrenyl ring is as small as possible. The fjord region twist angle δ′ is negative in the 1R(+) isomer and positive in the 1S(-) isomer, with magnitude less than 10°, in families 1-3. In these families, the linkage site (N2-C1) bond is axial. The small twist angles suffice to relieve the steric hindrance across the fjord region when the aromatic ring system is twisted toward the axial linkage site (Figure 6), because H1 and H15 are on opposite sides of the benzylic ring in this conformation. In this way, it is possible to achieve H1 to H15 distances in the fjord region of 1.92-1.97 Å with the least nonplanarity. In family 4, the twist angle δ′ is larger, ∼15°, and positive in the 1R(+) isomer, while negative in the 1S(-) isomer. In this family the linkage site (N2-C1) bond is equatorial, producing crowding (nonbonded repulsions) between the guanine and the phenanthrenyl ring. Consequently, there is no room for aromatic ring twisting toward the linkage site, and hence the aromatic rings must twist away from the linkage. However, a larger twist angle is needed to release the H1 to H15 crowding, because in this case, H1 and H15 are on the same side of the benzylic ring (Figure 6). Thus, for a given twist angle magnitude, maximal distance between H1 and H15 is achieved when the N2C1 linkage site is axial. This is true because the axial linkage site conformation places H1 and H15 on opposite sides of the benzylic ring. On the other hand, when the linkage site is equatorial H1 and H15 are brought to the same side of the benzylic ring and hence are intrinsically closer. In addition, we note higher energy structures in Table 1 with combined statistical weights less than 1%, in which γ′1 and δ′ are the same sign. In these structures the twist angle δ′ is also large, ∼20° in both isomers. The larger twist angle is required to relieve the steric hindrance in this case because H1 and H15 are again on the same side of the benzylic ring (Figure 6). Structural Preferences for r′ and β′ Torsion Angles. R′: 0° and 180° Are Preferred. The R′ torsion angle prefers the 0° and 180° regions for both isomers. When R′ ) ∼0° or ∼180°, the nonbonding orbital on guanine N2 and the guanine ring π orbitals are nearly parallel to one another (56). The R′ ) ∼0° region is less favorable than the R′ ) ∼180° region in the case of B[c]Ph-N2-G; here, the N1-H1 edge of guanine is directed toward the benzylic ring, causing the conformational space to be constrained. When R′ ) ∼180°, the guanine N1-H1 edge is directed away from the benzylic ring, producing a more favorable open structure. The R′ ) ∼180° region is also favored in DNA duplexes as it is required to form the Watson-Crick hydrogen bonds (58). β′: R Isomers Favor ∼+90° While S Isomers Prefer ∼-90°. The torsion angle β′ plays a key role in the conformations of the two stereoisomers due to primary steric hindrance between base and benzylic ring (58). Our previous work on the nucleoside level for adducts of antibenzo[a]pyrene diol epoxide has revealed that the β′ torsion angle of R isomers favors the +90° domain, while S isomers favor the -90° domain: the mirror image nature of the benzylic rings in the two isomers causes crowding when an R isomer adopts the β′ domain favored by the S isomer and vice versa (54-57).

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Figure 6. Stereoviews of representative conformational combinations of B[c]Ph moiety in (A) 1R(+)-trans-anti-B[c]Ph-N2-G and (B) 1S(-)-trans-anti-B[c]Ph-N2-G adduct. I, the aromatic ring system is twisted toward the axial linkage site to G, with H1 and H15 on opposite sides of the benzylic ring. I is found in families 1, 2, 3, and 7 of the 1R(+) isomer and families 1, 2, 3, and 6 of the 1S(-) isomer. II, the aromatic ring system is twisted away from the equatorial linkage site to G, with H1 and H15 on the same side of the benzylic ring. II is found in families 4, 5, 8, and 9 of the 1R(+) isomer and families 4, 5, 8 and 10 of the 1S(-) isomer. III, the aromatic ring system is twisted away from the axial linkage site to G, with H1 and H15 on the same side of the benzylic ring. III is found in families 6, 10, 12, and 13 of the 1R(+) isomer and families 7, 9, 11, 12, and 13 of the 1S(-) isomer. Table 1 gives family numbers and their characteristics.

The results here show that in the first two families, comprising more than 70% of the statistical weight, the favored β′ region is adopted: the ∼+90° region in the 1R(+)-trans-anti-B[c]Ph-N2-G isomer, and the ∼-90° region in the 1S(-)-trans-anti-B[c]Ph-N2-G isomer. However, in families 3 and 4, comprising ∼30% of the statistical weight, the β′ torsion angle is in the -90° region for 1R(+)-trans-anti-B[c]Ph-N2-G and the +90°

region for 1S(-)-trans-anti-B[c]Ph-N2-G, i.e., the less favored β′ region for each isomer. We find that weak hydrogen bonds or favorable electrostatic interactions provide the stability for families 3 and 4. The interaction is between the benzylic ring O4 and N1-H1 of guanine. In family 3, the N1 to O4 distance is 3.24 Å and the N1-H1-O4 angle is 126.6°; in family 4, the N1 to O4 distance is 3.60 Å and the N1-



Table 2. r′, β′, γ′1, δ′ Values in NMR Solution Structures Structure

R′ (deg)

β′ (deg)

1R(+)-trans-anti-B[c]Ph-N2-dGa 1S(-)-trans-anti-B[c]Ph-N2-dGa 1R(+)-trans-anti-B[c]Ph-N6-dAb 1S(-)-trans-anti-B[c]Ph-N6-dAc 14R(+)-trans-anti-B[g]C-N6-dAd

Fjord Region Adducts 142 101 -156 -109 154 104 -131 -111 144 ( 2 118 ( 1

10S(+)-trans-anti-B[a]P-N2-dGe 10R(-)-trans-anti-B[a]P-N2-dGf 10S(+)-trans-anti-B[a]P-N6-dAg 10S(+)-trans-anti-B[a]P-N6-dAh 10R(-)-trans-anti-B[a]P-N6-dAi 10R(-)-trans-anti-B[a]P-N6-dAj 10R(-)-trans-anti-B[a]P-N6-dAk

137 152 25 -134 141 108 126

Bay Region Adducts -102 78 -90 -118 97 178 148

γ′1 (deg)

δ′ (deg)

-69 65 -66 -53 -75 ( 1

16 -26 18 -16 10 ( 0.3

-66 66 -63 -58 60 57 -47

∼0 ∼0 ∼0 ∼0 ∼0 32 15

a Ref 39. b Ref 37. c Ref 38. d Ref 40 (torsion angle values are ensemble average with standard deviation). e Normal partner (59). f Normal partner (60). g A-G mismatch major conformer (61). h A-G mismatch minor conformer (62). i A-T normal partner (63). j A-G mismatch conformer (65). k A-T normal partner (66).

H1-O4 angle is 152.3°. Nonetheless, families 3 and 4 remain less favored because of the inherent crowding due to primary steric hindrance (58), which occurs between the N1 and O6 containing edge of the guanine and the C2-C3 region of the benzylic ring in these two structural types with R′ ) ∼0°. Other structural types adopting the less favored β′ domain for each isomer with R′ ) ∼180° are also seen in our Results (Table 1), but with combined statistical weights of less than 1%. These are disfavored due to crowding between the N2 and N3 containing edge of guanine and the C2-C3 region of the benzylic ring. Relevance to DNA Duplexes. The sets of R′, β′, γ′1, and δ′ values adopted by the fjord region adducts [1R(+)/ 1S(-)-trans-anti-B[c]Ph-N6-dA, N2-dG, and 14R(+)-transanti-B[g]C-N6-dA] in DNA duplexes, obtained from NMR solution studies, are listed in Table 2 (37-40). We also give similar information for bay region trans-anti-B[a]P adducts containing full duplexes with normal WatsonCrick partner in Table 2 (59-66), to permit comparison of benzylic ring conformations. Linkage Site, R′ and β′. The R′ and β′ angles in the NMR structures of the modified DNA duplexes are generally in the preferred regions for each isomer found in our computational results. In DNA duplexes, the β′ of the R isomer is generally in the ∼+90° region and the S isomer is in the ∼-90° region; this governs the opposite orientation phenomenon in the R and S DNA adducts (37-39, 59-63, 65-78). The B[c]Ph-N6-dA adducts intercalatively insert the PAH residues on the 5′ side of the modified adenine in the 1R(+) isomer and on the 3′ side in the 1S(-) isomer. In the case of the B[c]Ph-N2dG adducts, the intercalation of B[c]Ph is to the 3′-side of the modified guanine in the 1R(+) isomer and to the 5′-side in the 1S(-) isomer (see Scheme 1 and Figure 7). The explanation for this phenomenon is as follows: in both cases, Watson-Crick base pairing is maintained, which requires the ∼180° region of R′ (since base pairing is distorted, R′ is ∼(150°, as shown in Table 2). However, when R′ is near 180°, the 1R(+)-trans-anti-B[c]Ph-N6-dA adduct must intercalate to the 5′ side of the modified adenine to maintain the Watson-Crick hydrogen bond, while the 1R(+)-trans-anti-B[c]Ph-N2-dG adduct must intercalate on the 3′ side. Similarly, with R′ ) ∼180°, the 1S(-)-trans-anti-B[c]Ph-N6-dA adduct must be 3′ intercalated and the 1R(+)-trans-anti-B[c]Ph-N2-dG 5′ intercalated (37-39). To reverse the direction of intercalation in each case requires rotating R′ to the less favored 0°

Figure 7. NMR solution structures: (A) 1R(+)-trans-anti-B[c]Ph-N2-dG adduct. (B) 1S(-)-trans-anti-B[c]Ph-N2-dG adduct. (C) 1R(+)-trans-anti-B[c]Ph-N6-dA adduct. (D) 1S(-)-trans-anti-B[c]Ph-N6-dA adduct. The 5′-ends of the modified strands are on the top, and the views are into the minor groove in dG adducts and into the major groove in the dA adducts. The central fivebase-pair segments of the duplex 11-mers are shown.

domain where one of the Watson-Crick base pairs is disrupted. Figure 8 illustrates these concepts. Adaptability in Fjord Region Twist and Benzylic Ring Conformation Help Stabilize Intercalated Structures. Benzylic Ring. The geometry of the benzylic ring in these adducts is closely related to that of cyclohexene, which can adopt either a half-chair or a boat conformation. We note that the observed benzylic rings of the NMR structures are half-chairs (γ′1 ≈ (60°), and no boats (γ′1 ≈ 0°) are observed, in accord with our computed preference. Indeed, in our 10 396 energy minimization trials for both isomers, only a single boatlike conformer resulted (Table 1, Figure 3, panels A and

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Figure 8. Comparison of adenine and guanine B[c]Ph adduct conformations, showing 5′ versus 3′-orientations of the B[c]Ph. The left panel shows NMR solution structures (37-39) at the lesion site, with R′ in the 180° region (exact values given in Table 2). The right panel shows the same structures with R′ rotated to 0°. Hydrogen bonds are indicated with dashed lines. Views are from the major groove in the adenine adducts and from the minor groove in the guanine adducts. The arrows designate the R′ torsion angle.

B). Boats are higher energy than half-chairs in cyclohexene type compounds (41, 45). MM3 molecular mechanics and ab initio calculations (44) have indicated that the energy for the half-chair conformation of cyclohexene (Figure 2) is a minimum and the transition state for halfchair ring inversion is the boat form. The barrier for interconversion is about 5-6 kcal/mol (44), close to the NMR experimental values of 5.2-5.3 kcal/mol (41, 45). Other molecular mechanics data have also indicated that the two half-chair conformations in substituted cyclohexenes are interconverted through a symmetrical boat conformation transition state and that the calculated energies of the transition states are in the range of 5.27.3 kcal/mol, in reasonable agreement with experimental

values (42, 79-86). NMR and ESR experimental data on substituted cyclohexenes also give ring inversion energy barriers in the range of 5.3-8.4 kcal/mol (42). We estimated the AMBER energy (an approximate enthalpy) of the half boat conformation as described in Materials and Methods, to be about 7.0 kcal/mol in our adduct, which is in line with the barrier values for ring inversion in substituted cyclohexenes. We also noted from computer graphics modeling that the transition between the two half-chairs through the boat intermediate, via linkage site flipping between the axial and equatorial positions, can take place without collision. Therefore, energetic and structural considerations reveal the possibility of interchange between half-chair conformations


in DNA. The NMR results have revealed this possibility; according to Table 2, the observed γ′1 in the B[a]P modified dG/dA adducts and the 1S(-)-trans-anti-B[c]PhN6-dA are in the calculated favored γ′1 region of B[c]PhG for each isomer, with the S isomers in the ∼-60° region and the R isomers in the ∼+60°. However, the γ′1 of 1R(+)-trans-anti-B[c]Ph-N6-dA and 14R(+)-trans-antiB[g]C-N6-dA are also in or near the ∼-60° region, although the ring-inverted ∼+60° domain is favored more in our calculations for the R isomer (Table 1). Thus, in DNA duplexes, the cyclohexene type benzylic ring pucker (γ′1) can adopt the less preferred half-chair conformation for a given isomer since favorable interactions with the DNA compensate through enhanced stacking interactions. This is not surprising in light of the experimentally observed possibility for cyclohexene ring inversion discussed above. An interesting question concerns the γ′1 value of 14R(+)-B[g]C-N6-dA, around 75°, while the values are at ∼60° for 1R(+)-B[c]Ph-G (Tables 1 and 2). B[g]C contains a chrysene ring which has one more aromatic ring than the phenanthrene ring in B[c]Ph, and therefore the π bond delocalization energies in the aromatic ring system of 14R(+)-B[g]C-N6-dA are stronger than for the 1R(+)B[c]Ph-N6-dA adduct. Thus, it costs more energy to deform the chrysene ring than the phenanthrene ring, and the aromatic chrysene ring has a stronger intrinsic preference for keeping the aromatic rings in a plane. Consequently, the fjord region twist (δ′ ) 10.2°) of the 14R(+)-B[g]C-N6-dA, inferred from the NMR solution structures, is smaller than the twist observed in the B[c]Ph-N6-dA adducts (∼15-18°). This smaller positive twist would bring the fjord region H1 and H14 atoms too close for this R isomer; however, the distortion in the γ′1 benzylic ring pucker from the favored ∼60° region alleviates the close contact. Still, the H1 to H14 distance in this structure is ∼1.84 Å which is smaller than the 1.92-1.97 Å we obtained. Fjord Region Twist. The NMR structures of the 1R(+)/ 1S(-)-trans-anti-B[c]Ph-N6-dA and N2-dG adducts and the 14R(+)-B[g]C-N6-dA adduct also reveal that the fjord region twist angle δ′ can be adaptable. We note that the δ′ of the two B[c]Ph-N6-dA and N2-dG isomers is opposite in sign, revealing this adaptability itself. Also, both the 14R(+)-B[g]C-N6-dA and the two B[c]Ph-N6-dA and N2dG adducts have δ′ values opposite in sign to those most favored in our computations for R and S B[c]Ph-G isomers (see Table 1). This δ′ variability is likely to be coupled with the possibility for benzylic ring inversion. Inversion of δ′ from positive to negative (or vice versa) through the 0° region would entail collisions across the fjord region hydrogens, which would present a very high barrier. However, models show that the benzylic ring can adjust during the motion through the δ′ ) 0° region to prevent collision. In the DNA duplexes the twist is adjusted to optimize stacking interactions between the intercalated carcinogen and the DNA in the 1R(+)/ 1S(-)-B[c]Ph-N6-dA, 14R(+)-B[g]C-N6-dA, and 1R(+)/ 1S(-)-B[c]Ph-N2-dG adducts (37-40). Comparing the γ′1 and δ′ combinations observed in the NMR solution structures of the fjord region adducts (Table 2) with our computed regions (Table 1), we note that the observed combinations are in family 4 and in the higher energy families 6, 10, 12, and 13 in the 1R(+) isomer and families 7, 9, 11, and 12 in the 1S(-) isomer (Table 1). Again, favorable interactions between the carcinogen and


the DNA can provide the needed energy stabilization of only at most 5 kcal/mol. Studies utilizing human nucleotide excision cell extracts have revealed that B[c]Ph-N6-dA adducts, which are intercalated without disruption of Watson-Crick base pairing, are not repaired by the nucleotide excision repair enzyme system (48). The adaptability in fjord region twist, and correlated benzylic ring flexibility, stabilizes the intercalated structures by optimizing stacking with the adjacent base pair. This stability is manifested in the similar thermal transition midpoints (Tm) for the unmodified duplex (Tm ) 43.8 ( 0.5 °C) and for both (+) and (-)-trans-anti-B[c]Ph-N6-dA 11-mer duplexes (Tm ) 43.3 ( 0.5 °C) (87). Importantly, the stability of the modified duplexes also plays a key role in the resistance to repair, which may be relevant to the high mutagenicity and tumorigenicity of these substances (88-90). By contrast, the intercalated bay region B[a]PN6-dA adducts are repaired to some extent in the human nucleotide excision system (48). Moreover, their stacking interactions with neighboring base pairs are much less optimal in the NMR solution structures (61-66), because stacking cannot be optimized via twist distortion. Consequently, the greater genotoxicity of fjord, versus bay region PAHs, mentioned earlier, may stem in part from the enhanced stability of the fjord region DNA adducts achieved through distortability in the aromatic twist. In turn, the enhanced stability may help convey resistance to removal by DNA nucleotide excision repair enzymes.

Conclusions For the base adduct, B[c]Ph-N2-G, we find that the benzylic ring favors the axial conformation at the linkage site. However, the equatorial conformation is also possible, though less favored, in agreement with the wellknown ring inversion of cyclohexene-type compounds. In addition, the twist of the aromatic ring in the fjord region has a preferential orientation toward the linkage site; this permits a smaller distortion while still alleviating the steric hindrance across the fjord region. Moreover, the benzylic ring conformation is correlated with twist. Fjord region adducts in DNA duplexes (37-40) select conformers from our computed allowed families, with energies up to ∼5 kcal/mol, that permit enhanced stacking interactions between carcinogen and the adjacent base pair; this is effectively an induced fit. Conformational multiplicity in twist and benzylic ring conformation observed in solution structures and computed here, provides a stabilizing feature in the fjord region adducts, which may be a contributing factor to their resistance to DNA repair, and thus to their genotoxicities.

Acknowledgment. This research is supported by NIH Grants CA 28038 and RR-06258, DOE Grant DEFG0290ER60931 to S.B., NIH Grant CA-20851 to N.E.G., and NIH Grant CA-46533 to D.J.P. We thank Professor Robert Shapiro, Chemistry Department, New York University, for very helpful discussions. Computations were carried out at the National Science Foundation San Diego Supercomputer Center, the Department of Energy National Energy Research Scientific Computing Center, and on our own SGI machines. Supporting Information Available: Table S1 lists conformational features of the phenanthrenyl residue of modified bases in the starting structures. Table S2 gives additional

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molecular mechanical parameters. Table S3 lists conformational features used in partial charge calculation. Table S4 gives partial charges, topology and atom type assignments for the 1R(+) and 1S(-)-trans-anti-B[c]Ph-N2-G base adducts. Table S5 gives AMBER energy ranges for each family, as well as ranges in each of the characteristic torsion angles (R′, β′, γ′1, and δ′). Table S6 gives number of conformers in 1 kcal/mol AMBER energy shells. Figures S1a and S1b show the distribution of the characteristic torsion angles (R′, β′, γ′1, and δ′) versus AMBER energy.

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TX010152N

Cyclohexene Ring and Fjord Region Twist Inversion in Stereoisomeric

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