Deoxyribonucleosides: Absolute C - ACS Publications - American

pentofuranosyl)spiroiminodihydantoin (dSp) 1 and 2 (Figure 1), respectively (6, 7). .... be noted that the two protons of the N-2 amino group are none...
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Chem. Res. Toxicol. 2006, 19, 1357-1365

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Nuclear Magnetic Resonance Studies of the 4R and 4S Diastereomers of Spiroiminodihydantoin 2′-Deoxyribonucleosides: Absolute Configuration and Conformational Features Boleslaw Karwowski,† Franc¸ ois Dupeyrat, Michel Bardet,* Jean-Luc Ravanat, Piotr Krajewski, and Jean Cadet* Laboratoire de Chimie Inorganique et Biologique, UMR-E No. 3 (CEA-UJF), De´ partement de Recherche Fondamentale sur la Matie` re Condense´ e, CEA/Grenoble, F-38054 Grenoble Cedex 9, France ReceiVed April 25, 2006

The present study was aimed at gaining further insights into stereochemical and conformational features of the 4R and 4S diastereomers of spiroiminodihydantoin 2′-deoxyribonucleosides that have been shown to be the predominant singlet oxygen oxidation products of 2′-deoxyguanosine in aqueous solutions. It may be added that spiroiminodihydantoin derivatives are efficiently generated by one-electron and singlet oxygen oxidation of the 8-oxo-7,8-dihydroguanine moiety of several nucleic acid components including nucleosides, nucleotides, and oligonucleotides. The reported structural data on the pair of diastereomeric spiroiminodihydantoin 2′-deoxyribonucleosides 1 and 2 are mostly inferred from extensive 1H and 13C NMR analyses including two-dimensional nuclear Overhauser effect measurements performed in both D2O and dimethyl sulfoxide. This approach that has been shown previously to be suitable to assign the stereochemistry of the base moiety of oxidized pyrimidine nucleosides was completed by molecular modeling and quantum mechanics studies. Thus, application of these two complementary approaches together with the consideration of the results of a recent relevant quantum mechanic study has allowed the assignment of the absolute stereoconfiguration of the C-4 carbon of diastereomers 1 and 2. In addition, information is provided on the conformational features of the 2-deoxyribose moiety and the orientation of the base around the N-glycosidic bond of both 2′-deoxyribonucleosides 1 and 2. Introduction The reaction of singlet molecular oxygen (1O2) with the guanine moiety of either free nucleosides or when inserted into short single-stranded oligonucleotides has been the subject of extensive studies during the last two decades (for a recent review, see 1). The two main 1O2 oxidation products of 2′-deoxyguanosine (dGuo)1 initially assigned as a diastereomeric pair of 4-hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine (2-5) were shown to be the (4S*)- and (4R*)-9-(2-deoxy-β-erythropentofuranosyl)spiroiminodihydantoin (dSp) 1 and 2 (Figure 1), respectively (6, 7). The assignment was achieved by comparison of NMR features (6) with those of authentic samples obtained by one-electron oxidation of 8-oxo-7,8-dihydroguanine nucleosides (8) and further confirmed by the determination of carbon connectivities using the SELINQUATE 13C NMR technique (7). Mechanistic insights into the formation of 1 and 2, which is initiated by a [4+2] cycloaddition of 1O2 across the 7,8- and 4,5-ethylenic bonds of dGuo and involves its successive conversion of several instable compounds including the 4,8endoperoxide, 8-hydroperoxide, oxidized quinonoid and 5-hydroxy-8-oxo-7,8-dihydroguanine intermediates, were gained from 18O-labeling isotopic studies (9, 10) and low-temperature * To whom correspondence should be addressed. (M.B.) Tel: +33(0)4 76 88 57 72. Fax: +33(0)4 76 88 50 90. E-mail: [email protected]. (J.C.) Tel: +33(0)4 76 88 49 87. Fax: +33(0)4 76 88 50 90. E-mail: [email protected]. † On leave from the Department of Pharamaceutical Chemistry and Biochemistry, University of Lodz, Lodz, Poland. 1 Abbreviations: dGuo, 2′-deoxyguanosine; 8-oxodGuo, 8-oxo-7,8dihydro-2′-deoxyguanosine; dSp, 9-(2-deoxy-β-D-erythro-pentofuranosyl)spiroiminodihydantoin.

Figure 1. Structure of the 4S and 4R diastereomers of spiroiminodihydantoin in their preferential amino tautomeric forms. Note that the atom numbering of the modified base that was used is that of guanine as adopted in previous publications. 13C

NMR experiments (11, 12). 1O2 oxidation of 8-oxo-7,8dihydroguanine nucleosides was also found to give rise to spiroiminodihydantoin (Sp) compounds (6, 7, 9, 13-15). This may be rationalized in terms of initial formation of 4,5dioxetanes (16) followed by conversion to 5-hydroperoxides (12) that are reduced to 5-hydroxy-8-oxo-7,8-dihydroguanine nucleosides, the likely precursors of 1 and 2 or related ribonucleosides through a rearrangement involving a 1,2-acyl shift (8). It was also found that 1 and 2 are the predominant oxidation products of 8-oxo-7,8-dihydroguanine either as the free nucleosides,

10.1021/tx060088f CCC: $33.50 © 2006 American Chemical Society Published on Web 09/07/2006

1358 Chem. Res. Toxicol., Vol. 19, No. 10, 2006

nucleotides, or when present in single-stranded oligonucleotides at neutral pH upon exposure to one-electron oxidants including type I photosensitizers (7, 15, 17), peroxynitrite (6, 18, 19), twophoton-excited 2-aminopurine (20, 21), carbonate anion radical (22), high valent IrIV complexes (23-25), photogenerated SO4•radical (26), and guanine (-H)• radical (27). Evidence was recently provided for the formation of dSp in isolated and bacterial DNA upon exposure to chromate (28, 29). Relevant structural and thermodynamic properties of diastereomers 1 and 2 either as isolated 2′-deoxyribonucleosides or when site specifically inserted into a defined sequence 11-mer DNA duplex were recently inferred from theoretical studies involving molecular dynamics simulations and AMBER calculations (30, 31). It may be added that relevant information on the highly mutagenic potential of spiroiminodihydantoin (Sp) was gained from the observed misincorporation by polymerases of adenine and guanine (32) opposite the lesions and further validated in bacterial cell hosts using a suitable restriction endonuclease and postlabeling assay (33, 34). These experiments have necessitated the postsynthesis of suitable DNA probes by selective oneelectron oxidation of site specifically inserted 8-oxodGuo into single-stranded oligonucleotides. It may be noted that spiroiminodihydantoin-containing DNA fragments can be site specifically synthesized using the nucleoside phosphoramidite building blocks of 1 and 2 (35). It was also shown that the spiroiminodihydantoin lesions are excellent substrates for various base excision repair DNA N-glycosylases including bacterial Fpg and Nei (36, 37), yeast yOGG1 and yOGG2 (38), and mammalian NEIL1 and NEIL2 (39). In contrast, it was found that hOGG1 is not able to remove spiroiminodihydantoin from DNA duplexes (38). The main objective of the present work was to determine the absolute configuration of chiral C-4 of spirocyclic nucleosides 1 and 2. The applied approach was based on the consideration of dipolar interactions between the modified base and the sugar moiety after the conformation of the aglycon about the N-glycosidic bond was assessed. For this purpose, extensive one- and two-dimensional (1D and 2D) NMR experiments including correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser enhancement spectroscopy (NOESY) measurements were performed. The assigned C-4 stereochemistry of 1 and 2 received further support from molecular modeling studies. Altogether, the structural and conformational data thus obtained may be of interest to assess biochemical features including mutagenicity and DNA repair enzyme substrate specificity of individual dSp diastereomers.

Materials and Methods Oxidized Nucleosides. The 4R and 4S diastereomers of spiroiminodihydantoin (dSp) were obtained by methylene blue-mediated photosensitization of dGuo as previously described (2). Typically, 1 L of a 2 mM aqueous solution of dGuo that contained 5 × 10-6 M methylene blue was irradiated overnight with visible light generated by a 500 W halogen lamp. During the irradiation, the solution was maintained saturated with oxygen by continuous air bubbling. After irradiation, the solution was concentrated to dryness. Then, the resulting dry residue was dissolved in the HPLC buffer that consisted of a mixture of 20% 25 mM ammonium formate and 80% CH3CN. The two diastereomers of spiroiminodihydantoin 1 and 2 were purified by HPLC using a Hypersil 5 µm (250 mm × 10 mm i.d.) amino silica gel column (Interchim, Montluc¸ on, France) with the above-mentioned HPLC buffer as the eluent (39). The two collected fractions (k′ ) 3.0 and k′ ) 3.5) were found to contain product 1 (4S diastereomer) and product 2 (4R diastere-

Karwowski et al. omer), respectively. It should be noted that the order of HPLC elution of 1 and 2 was inversed on the Hypercarb column, which was recently used to separate the two latter diastereomers (41). Repeated freeze drying of the collected fractions was required in order to totally remove the ammonium salts. Preparation of Water Free Samples. Nucleosides 1 and 2 strongly absorb water molecules in the solid phase. The H2O molecules are only partially removed by freeze drying under strong vacuum pumping. Therefore, DMSO solutions with low residual water were prepared by leaving the solutions under an argon atmosphere in the presence of lithium aluminum hydride (LiAlH4) for 3-5 days. Sample preparation was performed inside a glove box. Attempts to remove residual water molecules using classical methods were unsuccessful. NMR Spectroscopy. The 1D and 2D NMR spectra were recorded on a VARIAN Unity 500 spectrometer at a constant temperature of 283 K using a 5 mm indirect detection probe. The spectrometer was operated at 500 and 125 MHz for 1H and 13C NMR measurements, respectively. The chemical shifts were referenced to tetramethylsilane (TMS) set at 0 ppm. To avoid water absorption, the preparations were performed inside a glove box using sealed NMR tubes. 1D and 2D experiments were carried out using the standard Varian pulse sequences. The conditions for 1H1H DQF COSY, 1H-13C HMQC, 1H-13C HMBC, and NOESY experiments were identical to those previously reported in details (42-44). In order to delineate the conformational features of the two modified nucleosides 1 and 2, an exhaustive study of long-range coupling constants was performed including both 3JH-H protonproton and proton-carbons 3JC-H. This was achieved by direct measurement of proton-proton coupling constants and protoncarbon indirect coupling constants. Molecular Mechanics Calculations. The energy profiles for the rotation around χ and γ were calculated using the molecular mechanics with universal force field (UFF) (44, 45) with a rotation increment of 5°. The molecular mechanics calculations were performed using GAUSSIAN 03 Revision B.05 program (47). Quantum Mechanics Calculations. The molecular structure and conformers of the diastereomers of spiroiminodihydantoin 2′deoxyribonucleoside 1 and 2 were calculated using the density function theory (DFT) with Becke’s tree-parameter exchange functional and gradient-corrected functional of Lee, Yang, and Parr (B3LYP). For all calculations that were performed using the GAUSSIAN 03 Revision B.05 program, the standard 6-31G** basis set was applied (46).

Results and Discussion Assignments of the Proton and Carbon Resonances of 1 and 2. As a striking feature, it should be mentioned that only exchangeable protons are present within the base moiety of the modified dSp nucleosides (Figure 1). The related 1H NMR signals, which are critical for structural elucidation, collapse when the spectra are recorded in D2O or even if too high content of residual water is present in the deuterated dimethylsulfoxide solution. Therefore, in order to observe these signals, 1H NMR analyses had to be performed in DMSO with residual water content as low as possible to minimize the proton exchange process. Our practical experience on these products has showed that the water, which is found in the solutions prepared for NMR analyses, certainly originated from H2O molecules that remained inside the solid products. Even drastic freeze drying under strong vacuum pumping of the powder was not sufficient to remove traces of water molecules. Therefore, special care was taken to prepare DMSO solutions that contain low levels of residual water. In this study, the determination of the conformation of the 2-deoxyribose moiety and of the orientation of the base about the N-glycosidic bond was achieved by combining NMR and molecular modeling approaches. The latter strategy was applied

Configuration of Spiroiminodihydantoin Nucleosides

Chem. Res. Toxicol., Vol. 19, No. 10, 2006 1359

Figure 2. 1H NMR (500 MHz) spectrum of the 4R diastereomer of spiroiminodihydantoin 2 dissolved in DMSO-d6 and dried by contact with LiAlH4 under argon atmosphere.

Figure 3. Graphic visualization of possible interaction of a N-2 hydrogen with a water molecule (A) or a lone pair of electrons from nitrogen N-1 (B).

to determine in fine the absolute configuration at C-4 of the diastereomers 1 and 2. Water molecules were efficiently removed from the DMSO solution of compound 1 and 2, using the lithium aluminum hydride method. This led to recorded proton spectra, which exhibited all of the expected resonances of the different exchangeable protons. These include OH(5′), OH(3′), NH(7), and NH2(2) and NH(3) signals as illustrated in Figure 2 for compound 2. The proposed proton assignment for both 1 and 2 was mostly based on 2D COSY experiments, which allow correlation of all of the nonexchangeable protons. The assignment was unambiguously achieved starting from H-1′, which is known to resonate at the lowest field among the protons of the 2-deoxyribose moiety (48). In addition, the exchangeable signals at 4.5 and 5.2 ppm were assigned as those of OH-5′ and OH-3′, respectively, on the basis of the observed scalar coupling with the H-5′ and H-3′ protons. The secondary amine, amidine, and NH(7) protons on the other hand were assigned on the basis of their proton exchange rates [kNH(7) > kNH(3) > kNH2], which can be inferred from NOESY spectra recorded with increasing mixing times ranging from 10 to 600 ms. It should be noted that the two protons of the N-2 amino group are nonequivalent. This phenomenon can originate from interactions of one of the two protons with a lone pair of electrons (LP) on the neighboring nitrogen N-1 (Figure 3B). The LP effect of the neighboring N atom is known in coordination chemistry, for example, for 99mTc complexes with 6-hydrazinonicotinamide (HYNIC) (49). Another possibility would involve the formation of a hydrogen bond between the amino protons and a molecule of water, still present in DMSO (Figure 3A). Such interactions were observed in 2D NOESY experiments. It is important to mention here that compounds 1 and 2 exist in several tautomeric

forms, which may facilitate the formation of different hydrogen bonds with water molecules. The reliability of the assignments of the nonexchangeable protons was checked by spin simulation of the experimental 1D proton spectra with PERCH software (University of Kuopio, Finland), using as the initial data the measured chemical shifts and coupling constants (Tables 1 and 2). The 1H NMR spectra of 1 and 2 exhibit similar features as expected for a pair of diastereomers with, however, a major exception that concerns the H-2′ resonance that is strongly sensitive to the orientation of the base about the N-glycosidic bond for β-anomers of 2′-deoxyribonucleosides. Thus, H-2′ was found to resonate at δ ) 2.45 and 1.98 ppm for 1 and 2, respectively. In 2, the electronegative oxygen atom in the C-6 carbonyl group through overlapping with H-2′ forces a shift of its NMR signal toward higher fields. In addition, the H-5′ and H-5′′ signals appear as the characteristic A and B patterns of ABMX systems in nucleoside 2 whereas they are isochronous in diastereomer 1. The magnetic nonequivalence of the latter protons is likely to be caused by hydrogen bond interactions between the 5′-OH group acting as the proton donor and the spiroiminodihydantoin moiety as the acceptor. Starting from the previously assigned proton resonances, the corresponding protonated carbons were inferred from HMQC experiments. The C-4 and C-8 carbons of the base were assigned from HMBC analyses. This attribution is in agreement with the observation of 1J (C-C) scalar coupling involving C-4 and C-8 on one hand and C-4 on the other hand as inferred from dedicated SELINQUATE experiments (7). The assignments of 1H and 13C chemical shifts for both compounds 1 and 2 are reported in Table 1. Conformational Features of 2′-Deoxyribonucleosides. The conformation of the sugar moiety of nucleosides is rationalized in terms of a dynamic equilibrium between different conformers involving mostly the puckered forms C-2′ endo (S type) and C-3′ endo (N type) (50). Other relevant features include the staggered rotamers of the 4′-hydroxymethyl group (g+, t, and g-) and the orientation of the base with respect to the sugar moiety described by the torsion angle χ as defined by O-4′, C-1′, N-9, and C-4 for purine bases (51). The typical χ values, namely, -120 to -180 and 0 to 90°, correspond to anti and syn conformations, respectively. Syn and anti glycosidic conformations are depicted in Figure 4. Assessment of sugar conformational properties mainly relies on the determination of the 3JH-H coupling constants. Thus, the conformational features of the sugar moiety and of the 4′hydroxymethyl group for nucleosides 1 and 2 can be inferred

1360 Chem. Res. Toxicol., Vol. 19, No. 10, 2006 Table 1. Chemical Shifts of 1H and

13C

Karwowski et al.

NMR in DMSO-d6 at 303 K and Partial Charges Taken from Ref 30 for Diastereomers 1 and 2a

4S diastereomer 1

4R diastereomer 2

position

proton δ(ppm)

carbon δ(ppm)

partial charge P

∆SH - RH δ(ppm)

1′ 2′ 2′′ 3′ 3′OH 4′ 5′ 5′′ 5′OH

5.10 2.45 1.77 4.07 5.02 3.58 3.32 3.32 4.46

82.6 36.3

0.163843 0.064315 0.064315 0.097122 0.426801 0.09243 0.077426 0.077426 0.44813

-0.46 0.47 0.07 0.05 -0.02 0.02 -0.01 0.11 0.1

70.7 86.4 61.9

∆SC - RC δ(ppm) sugar part

proton δ(ppm)

carbon δ(ppm)

partial charge P

81.15 35.0

0.021568

5.56 1.98 1.70 4.02 5.04 3.56 3.33 3.21 4.36

0.201288 0.045338 0.045338 0.090867 0.430971 0.094296 0.097851 0.097851 0.426562

0.003345 0.003345 0.05573

7.87 8.15 8.39

∆SP - RP

1.45 1.30

-0.037445 0.018977

0.00

0.006255 -0.00417 -0.001866 -0.020425

0.4 -0.3

70.7 86.0 62.2

base part C2 NH2′ NH2′′ N3 C4 C5 C6 N7 C8

172.1 7.96 8.24 8.50

0.5 0.430557 0.430557 0.43356

79.0 169.2 182.0 11.24

0.38307

0.09 0.09 0.11

-0.16

-0.3 -0.2 0.9 -1.4

155.0

171.6

-0.014359

0.427212 0.427212 0.37783 79.3 169.4 181.1

11.40

0.397429 156.4

a ∆S - R ; ∆S - R difference between chemical shifts of suitable protons or carbons for diastereomers R and S. ∆S - R difference between partial H H C C P P charges of atoms calculated from literature data (30) for R and S diastereomers.

Table 2. Coupling Constants (J/Hz) for Diastereomers 1 (S) and 2 (R) in DMSO-d6 Obtained from 1H NMR Spectra (*)a and after Simulation (**) Jij /Hz ij

1 (S)*

1 (S)**

2 (R)*

2 (R)**

1′2′ 1′2′′ 2′2′′ 2′3′ 2′′3′ 3′4′ 4′5′ 4′5′′ 5′5′′

8.3 6.2 -13.1 5.2 2.6 ∼0.9 5.6 5.6

8.3 6.2 -13.1 5.2 2.4 1.0 5.8 5.8

8.8 6. -13.1 5.3 2.5 ∼0.5 5.0 7.0 -11.4

8.6 5.9 -13.1 5.3 2.3 0.6 4.9 7.1 -11.1

a These values were taken for the calculation of rotamer and conformer populations.

from the consideration of dedicated 3JH-H values. The respective contribution of the three staggered rotamers g+, t, and g- of the exocyclic 4′-hydroxymethyl group was determined with the following equations:

% g+ ) {1.46 - [3J4′,5′ + 3J4,5′′]/8.9}100

(1)

% t ) {(3J4′,5′′/8.9) - 0.23}100

(2)

% g- ) {(3J4′,5′/8.9) - 0.23}100

(3)

The percentage of C-2′ endo conformation can be estimated using the following equation:

% C-2′ endo ) 100(J1′2′/9.3)

(4)

The results that were obtained in DMSO and D2O solutions are given in Table 3. For comparison, the values concerning dGuo and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) are also reported. It was inferred that the furanose ring of the two oxidized nucleosides 1 and 2 exists predominantly in the C-2′ endo C-3′exo conformation (S conformation). Moreover, the contribution of the C-2′ endo conformer is slightly higher for nucleoside 2 than for nucleoside 1. However, we may note a slight shift in the dynamic equilibrium toward the C-3′ endo

conformer when the analyses are performed in water with respect to DMSO. The trans rotamer of the exocyclic 4′-hydroxymethyl group, in either DMSO or D2O, is predominant for both diastereomers 1 and 2. Nevertheless, there are some notable differences between populations according to the nature of the solvent. Moreover, it is worth noting that the distribution of the three staggered conformers (g+, t, and g-) for both compounds was not affected in a similar way in DMSO and D2O. This clearly indicates that according to the C-4 stereochemistry the conformations adopted by 1 and 2 lead to some specific intramolecular interactions. Proton-carbon 3JC-H values can be used to define the orientation of the base about the N-glycosidic bond. In this respect, Davies (48) proposed a method to assess the relative distribution of syn type and anti type conformers by considering the value of the difference ∆(J) ) (3JC8-H1′ - 3JC4-H1′). By using this criterion, a preferential syn conformation is proposed for the diastereomer 2 since ∆(J) was equal to -0.80 Hz. On the other hand, the conformational features of the spiroiminodihydantoin base about the N-glycosidic bond of 1 were accounted for by the presence of both syn and anti conformers as inferred from a ∆(J) value equal to 0.49. Additional support for a syn orientation was provided by the observation of weak dipolar interactions between amino protons from N-2 and sugar protons H-3′ and H-4′ in the 2D NOESY NMR spectra of both diastereomers 1 and 2 (Figure 5). Absolute Configuration. The NOESY analyses recorded in water free DMSO solutions appear to be the key experiments to determine the absolute configuration of the chiral C-4 atom of the modified base of 1 and 2. Indeed, it was possible to record 2D spectra in which the exchangeable protons are correlated with other protons not only as the result of exchange processes but also through dipolar interactions. It should be noted that the correlation peaks from exchange and dipolar interactions could be differentiated since they have opposite signs. Enlargements of the NOESY spectra of the two diastereomers 1 and 2 were recorded and plotted under similar conditions. It may be remembered that the assignment of exchangeable protons of the spiroiminodihydantoin moiety was a critical prerequisite. This

Configuration of Spiroiminodihydantoin Nucleosides

Chem. Res. Toxicol., Vol. 19, No. 10, 2006 1361

Figure 4. Schematic representation and corresponding nomenclatures of the main conformational domains for nucleosides 1 and 2. Table 3. Conformational Features of the Sugar Ring and Hydroxymethyl Groups of Diastereomers 1 and 2 dGuo and 8-oxodGuo in D2O and DMSO-d6 CH2OH conformation C-2 endo (%) in molecule dGuo 8-oxodGuo 1 (S) 2 (R) a

gauche+

(%) in

gauche- (%) in

trans (%) in

DMSO-d6

D2O

DMSO-d6

D2O

DMSO-d6

D2O

DMSO-d6

D2O

38.0 35.7 20.1 10.9

50.2

32.7 36.1 40.0a 56.1

32.0

29.3 28.2 40.0a 31.0

17.8

89.7 94.6

74 71 66 74.4

23.0 27.0

49.0 45.0

28.0 28.0

Only the sum of g- and t rotamers around C-4′-C-5′ is accessible since H5′ and H5 are isochronous for 1.

Figure 5. Enlargements of 500 MHz 1H 2D NOESY spectra of the 4R and 4S diastereomers of spiroiminodihydantoin 2′-deoxyribonucleosides.

concerns in particular the N-3 proton, which is likely to play a central role for the C-4 stereoconfiguration assignment since it is located ortho to the N-glycosidic bond. It may be added that recent quantum mechanic geometry optimization calculations indicated that among the three possible tautomers for the hydantoin ring B of the base, the amino form exhibits the lowest

energy (30, 31). Interestingly, dipolar interactions were observed between the NH-3 proton on one hand and H-2′ and H-2′′ on the other hand for nucleoside 1. In contrast, these different correlations are not detected in the 2D NOESY spectrum of nucleoside 2. These different pieces of information once considered together are strongly indicative of a S stereochemistry

1362 Chem. Res. Toxicol., Vol. 19, No. 10, 2006

Karwowski et al.

Table 4. Torsion Angles in Degrees and Energy in Hartree of the Refined Structures of 4R Diastereomer 2 Calculated Using DFT 6-31G** from the Two Puckered Forms of the Sugar glycosidic conformation anti

syn

conformation

χ′ angle

γ′ angle

energy

χ′ angle

γ′ angle

energy

g+ t g-

-108.31 -95.12 -86.10

45.60 -173.42 -69.51

C- 3′ endo -1114.0397567 -1114.0313093 -1114.0359953

69.05 57.84 56.18

66.01 179.40 -68.47

-1114.0451228 -1114.0342441 -1114.0397306

G+ t g-

-82.45 -63.53 -93.26

48.43 177.82 -64.49

C-2′ endo -1114.0289445 -1114.0392675 -1114.0350063

67.99 64.25 75.96

64.52 157.80 -64.53

-1114.0422173 -1114.0435369 -1114.0360033

Table 5. Torsion Angles in Degrees and Energy in Hartree of the Refined Structures of 4S Diastereomer 1 Calculated Using DFT 6-31G** from the Two Puckered Forms of the Sugar glycosidic conformation anti conformation

χ′ angle

syn

γ′ angle

energy

χ′ angle

γ′ angle

energy (kcal mol)

38.10 43.91 39.89

53.04 -170.61 -68.06

-1114.0431104 -1114.0294287 -1114.0341116

24.82 64.61 36.82

43.40 172.03 -65.86

-1114.0351594 -1114.0378893 -1114.0346553

g+ T g-

-136.21 -125.73 -122.47

46.34 -175.02 -68.90

C-3′ endo -1114.0431037 -1114.0360193 -1114.0391316

g+ T g-

-97.92 -136.77 -121.68

52.21 159.39 -64.24

C-2′ endo -1114.0323198 -1114.0400942 -1114.0378457

at C-4 for 1, whereas diastereomer 2 would exhibit a 4R stereoconfiguration. This applies when 1 and 2 are in the preferential amino tautomeric forms, whereas the absolute configuration of the spirocyclic carbon has to be inversed for the related imino tautomers. It is worth noting that the preferential syn conformation for the R diastereomer 2 would favor, on the basis of model consideration, hydrogen bond occurrence between the nitrogen of the amino group at C-2 and the protons of the 4′-hydroxymethyl group. Such interactions are expected to decrease the equilibrium rates between the different conformational domains of the 4′-hydroxymethyl group for 2. This is fully consistent with the fact that in nucleoside 2, the NMR signals of proton H-5′ and H-5′′ are nonequivalent, whereas they are isochronous in nucleoside 1. As a result, a doublet is observed for the H-5′, H-5′′ pattern for the S diastereomer 1, whereas a complex multiplet is observed for 2. Molecular Modeling and Quantum Mechanics Calculations. Because of the flexibility of the sugar moiety and the possibility of free rotation of the nucleobase and the 4′hydroxymethyl group around the N-glycosidic and C-4′-C-5′ bonds, respectively, 12 possible conformers are expected for each of the two diastereomers 1 and 2. The strategy that was applied to obtain the most probable structures exhibiting the lowest energy has involved a two-step process. First, the conformer populations were assessed by molecular mechanics calculations. Then, further information on the potential energy and the geometry of the obtained structures was gained from quantum mechanics calculations. For both molecules, one can build correlation graphs, which give the rms deviations for the backbone atoms of the generated structures from dynamic studies. These involve the atoms of the 2-deoxyribose unit with the exception of those of the 4-hydroxymethyl group. In the graphs of each compound, two conformational families corresponding to the 3E (C-3′ endo) and 2E (C-2′ endo) puckered conformers can be distinguished. The two (C-3′ endo and C-2′ endo) conformations found for the sugar moiety were further used as starting structures to assess the preferential orientation of the base with respect to the

2-deoxyribose ring. This was achieved by calculating the energy of the minimized structures obtained by varying the torsion angle χ (O-4-C-1′-N-9-C-4) around the N-glycosidic bond by increments of 5°. The respective resulting energy profiles were analyzed. The calculated energies are not absolute. However, the data for the individual structures are comparable to each other. For each diastereomer, two energy minima were found, indicating two preferential conformational domains. These conformations correspond to the expected syn and anti orientation domains, which are known to represent preferential conformations for such nucleosides. A similar approach was used to determine the rotameric population of the 4′-hydroxymethyl group around the C-4′-C5′ bond. This was achieved upon energy minimization of the low-energy structures obtained by varying the torsional angle (C-3′-C-4′-C-5′-O-5′) by increments of 5°. Three preferential conformational domains that correspond to the staggered g+, t, and g- rotamers were found for each of the two diastereomers 1 and 2. The 2 × 12 structures that were thus obtained were subsequently optimized by quantum mechanics calculations. The respective potential energies and γ angle values for 1 and 2 are reported in Tables 4 and 5. One of the striking results deals with the fact that the syn conformation is the preferential domain for both diastereomers 1 and 2. The energy difference between the syn and anti conformers is significantly larger (3.37 kcal mol-1) for the 4R diastereomer 2 than for the 4S nucleoside 1 (0.063 kcal mol-1). Another relevant structural piece of information concerns the interproton distances between the amino proton at N-3 and each of the two sugar methylenic protons at C-2′ that were assessed by considering the most stable forms after the structures were minimized. The results thus obtained for diastereomers 1 and 2 are reported in Table 6. The estimated distances between NH-3 and H-2′ on the one hand and H-2′′ on the other hand are 4.76 and 5.74 Å for the most stable structure of the 4R diastereomer 2 that exhibits a syn orientation and a C-3′ endo puckered form. Interestingly, shorter distances (3.20 and 4.88 Å, respectively) were found for the 4S

Configuration of Spiroiminodihydantoin Nucleosides

Chem. Res. Toxicol., Vol. 19, No. 10, 2006 1363

Table 6. H2′/H2′′ NH3 Interproton Distances for the Lowest Energy Conformers of 4S and 4R Diastereomers 1 and 2 Calculated by DFT 6-31G** interproton distances (Å) glycosidic conformation χ′ angle (°)

sugar pucker

average

energy (Hartree)

H2′-NH3R

38.10 (syn) 64.61 (syn) -136.21 (anti) -136.77 (anti)

C-3′ endo C-2′ endo C- 3′ endo C-2′ endo

-1114.0431104 -1114.0378893 -1114.0431037 -1114.0400942

diastereomer S 3.20 3.31 5.52 5.61

69.05 (syn) 64.25 (syn) -108.31 (anti) -63.53 (anti)

C-3′ endo C-2′ endo C- 3′ endo C-2′ endo

-1114.0451228 -1114.0435369 -1114.0397567 -1114.0392675

diastereomer R 4.76 4.26 4.23 5.36

diastereomer 1 in the structure of the lowest potential energy that shows a syn orientation together with a C-3′ endo conformation. The distance between NH-3 and either H-2′ (3.31 Å) or H-2′′ (4.08 Å) becomes even shorter for the structure displaying a syn orientation and a C-2′endo puckered form (Table 6). It is important to mention that the distances between protons of interest, namely, NH-3 on the one hand and H-2′ and H-2′′ on the other hand may fluctuate due to the conformational flexibility of the N-3 atom. Therefore, it may be pointed out that in Table 6 are reported the averaged internucleus distances between the mentioned protons, which have been calculated for all optimized structures of conformers presented in Tables 4 and 5. Comparison of Quantum Mechanics and Experimental Data. Quantum mechanics calculations aimed at providing insights into structural and conformational features on both 4S and 4R diastereomers of dSp 1 and 2 recently became available. The study has focused on the determination of the statistical weights for the torsion angle around the N-glycosidic bond and the rotameric distribution around the C-4′-C-5′ bond. Additionally, information was gained on the sugar pseudorotation conformers. According to the proposed model, which predicts free rotation of modified nucleobases around the C-1′-N-9 bond, the distribution of the syn and anti conformers significantly differs. When 1 and 2 appear in syn and anti or anti and syn conformation, respectively, the effect of geometry gives rise to the same chemical shifts of protons and the unequivocal assignment of the configuration is impossible (see Figure 4). It was shown by quantum chemical analysis that both syn and anti conformers are possible; however, the syn orientation is favored over the anti conformation for the 4R diastereomer 2. In contrast, both syn and anti conformation are permitted for the 4S diastereomer 1. This is in a good agreement with the C-1′-N-9 bond orientation study based on the consideration of 1H-13C NMR measurements involving the anomeric proton and either C-4 or C-8 of the base. Thus, the observed differences ∆(J) ) (JC8-H1′ - JC4-H1′) are consistent with a higher abundance of the syn form for the 4R nucleoside with ∆(J) ) -0.80, whereas both anti and syn forms are present in the 4S diastereomer as inferred from ∆(J) ) 0.49. The energy difference between the syn and the anti conformers of the two diastereomers calculated by quantum mechanics (DFT, B3LYP/ 6-31G**) is 0.063 kcal/mol for the 4S diastereomer 1 and 3.37 kcal/mol for the 4R diastereomer 2. The syn orientation can be stabilized by hydrogen bonding formed between the 4′-hydroxymethyl group of the sugar from one side and the amino group from the other. This is in agreement with the data inferred from 1H NMR experiments showing that the trans rotamers are the most frequent (Table 3). Using eq 4, which is based on the magnitude of trans coupling constant between H-1′ and H-2′,

H2′′-NH3

H2′-NH3

H2′′-NH3

4.88 4.08 5.77 5.40

3.2

4.6

5.5

5.6

5.74 5.51 4.82 5.16

4.2

5.2

4.9

4.9

it is possible to estimate the % of S conformer population in the system. It has been found that 1 and 2 have a preference for the C-2 endo conformation. It was clearly shown by considering trans J1′,2′ and J3′,4′ coupling constant values that the 2-deoxy-β-D-erythro-pentofuranose ring of both 1 and 2 when dissolved in either D2O or DMSO adopts a preferential S puckered form. Finally, a comparison of partial charges of protons in 1 and 2 seems instructive. In Table 1, the measured chemical shift is correlated with the calculated charges (30). It follows that the same protons of two diastereomers 1 and 2 show a clear correlation between the charge and the chemical shift in all but one case; the higher positive charge is, the lower value of δ. The largest difference between the δ values has been found for the H-2′ protons; this is consistent with a significant difference in their partial positive charge.

Conclusion The absolute configuration of the two main singlet oxygen oxidation products of dGuo, namely, the 4S and 4R diastereomers of spiroiminodihydantoin 2′-deoxyribonucleoides 1 and 2 has been assessed on the basis of detailed NMR analyses that have involved the determination of dipolar interactions between the base moieties and the 2-deoxyribose unit. The proposed assignment received confirmation from the results of both molecular modeling and quantum mechanics investigations. This should now allow making correlations between structural parameters of the two modified nucleosides and eventual differences in biochemical end points such as the rate of excision by DNA repair glycosylases and the mutagenic potential. Finally, an excellent correlation between our experimental NMR data and the quantum mechanics calculations recently reported by Broyde et al. (30, 31) should be pointed out. During the preparation of the manuscript, we have been informed of the determination of the absolute configuration of 1 and 2 that was achieved using a different strategy (41). However, this, which is based on theoretical calculations of ORD features, has led to opposite conclusions and it is not clear at the present stage what is at the origin of the observed discrepancies in the assignment of the stereochemistry of the spirocyclic carbon of 1 and 2. The use of dipolar interactions that constitutes a well-established approach to delineate absolute configuration cannot, however, be considered as most of the methods aimed at delineating absolute configuration with the exception of X-ray diffraction to be unambiguously accurate. Acknowledgment. We thank the EU Marie Curie Training and Mobility Project MRTN-CT2003 “CLUSTOXDNA” for partial support.

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