Assignment of Absolute Configurations of the Enantiomeric

William L. Neeley , Sarah Delaney , Yuriy O. Alekseyev , Daniel F. Jarosz , James C. Delaney , Graham C. Walker , John M. Essigmann. Journal of Biolog...
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Chem. Res. Toxicol. 2006, 19, 908-913

Assignment of Absolute Configurations of the Enantiomeric Spiroiminodihydantoin Nucleobases by Experimental and Computational Optical Rotatory Dispersion Methods Alexander Durandin,†,‡ Lei Jia,†,‡ Conor Crean,†,‡ Alexander Kolbanovskiy,†,‡ Shuang Ding,† Vladimir Shafirovich,† Suse Broyde,§ and Nicholas E. Geacintov*,† Departments of Chemistry and Biology, New York UniVersity, SilVer Complex, 31 Washington Place, New York, New York 10003-5180 ReceiVed April 17, 2006

The diastereomeric spiroiminodihydantoin (Sp) lesions are oxidation products of guanine and 8-oxo7,8-dihydroguanine (8-oxoG) and have generated considerable interest because of their stereochemistrydependent mutagenic properties in vivo (Henderson, P. T., et al. (2003) Biochemistry 42, 9257-9262). However, the absolute configurations of the two diastereomers have not yet been elucidated, and such information may prove valuable for understanding relationships between biological function and structure at the DNA level (Jia, L., Shafirovich, V., Shapiro, R., Geacintov, N. E., and Broyde, S. (2005) Biochemistry 44, 13342-13353). We have synthesized the two chiral Sp nucleobases by hydrolysis of the nucleosides denoted by dSp1 and dSp2 according to their elution order in HPLC experiments using a Hypercarb column, and determined their absolute configurations using a combination of experimentally measured optical rotatory dispersion (ORD) spectra in aqueous solutions and computed ORD specific rotations using density functional theory (DFT). Recent developments have shown that DFT methods are now sufficiently robust for predicting ORD values of chiral molecules (Polavarapu, P. L. (2002) Chirality 14, 768-781). The nucleobases Sp1 and Sp2 exhibit experimentally measured CD and ORD spectra that are very close to those of the respective precursor nucleosides dSp1 and dSp2 in shape and sign. The first nucleoside stereoisomer (dSp1) to elute from a typical Hypercarb HPLC column has (-)S, while the second (dSp2) has (+)-R absolute configuration. The R and S assignments are applicable to the amino tautomeric forms in each case. Introduction Among the four natural DNA bases, guanine has the lowest ionization potential and is therefore most susceptible to reaction with oxidizing agents (1-6). During the inflammatory response in cellular environments, reactive oxygen species are formed that oxidize guanine in DNA, yielding a number of lesions that include 8-oxo-7, 8-dihydroguanine (8-oxoG)1 (1). Further oxidation of 8-oxoG gives rise to deeper oxidation products such as the diastereomeric spiroiminodihydantoin lesions (Sp, Figure 1) (5-12). Oxidative damage of DNA in vivo has been linked to the initiation of cancer (4, 13-16), aging (17-21), and degenerative diseases such as Alzheimer’s and cardiovascular disease (3, 22). It has been demonstrated in vitro (23, 24) and * Address correspondence to Nicholas E. Geacintov, Chemistry Department, Silver Complex, 31 Washington Place, New York University, New York, NY 10003-5180. Tel., (212) 998 8407; fax, (212) 998 8421; e-mail, [email protected]. † Department of Chemistry, New York University. ‡ The first four authors contributed equally to this work: A. Durandin and A. Kolbanovskiy to the construction of the ORD apparatus and the measurements, L. Jia performed the computational studies, and C. Conor the synthesis of the spiroiminodihydantoin diastereomers. § Department of Biology, New York University. 1 Abbreviations: 8-oxoG, 8-oxo-7, 8-dihydroguanine; Sp, spiroiminodihydantoin; QM, quantum mechanics; BER, base excision repair; Fpg/MutM, formamidopyrimidine glycosylase; Nth, endonuclease III; Nei, endonuclease VIII; yOGG, yeast 8-oxoG glycosylase; hOGG, human 8-oxoG glycosylase; Neil, endonuclease VIII-like; ORD, optical rotatory dispersion; DFT, density functional theory; TDDFT, time-dependent density functional theory; MMFF, MERCK molecular mechanics force field; GIAO, gauge-invariant atomic orbital.

Figure 1. Structures and absolute configurations of R and S spiroiminodihydantoin nucleobase (Sp) enantiomers in the (A) amino tautomeric form with QM geometry optimized models (28) and (B) imino tautomeric form. R and S designations for the chiral centers follow the IUPAC convention as detailed in Prelog and Helmchen (58) and Cahn et al. (59). In this convention, the amino and imino tautomers, although identical in configuration about the asymmetric C4 atom, have different R and S designations; these designations are unrelated to the absolute stereochemistry and the sign of the ORD spectra.

in vivo (25, 26) that Sp is highly mutagenic. Recently, Sp was detected in Nei-deficient Escherichia coli cells following exposure to chromate (27), thus, suggesting that Sp may

10.1021/tx060078e CCC: $33.50 © 2006 American Chemical Society Published on Web 06/30/2006

Absolute Configurations of Sp Lesions

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contribute to oxidative DNA damage in vivo under conditions of oxidative stress (12). The structures of the two Sp stereoisomeric forms have been studied by high-level quantum mechanical (QM) geometry optimization methods (28). These studies have shown that the Sp stereoisomers are enantiomers (in the absence of the furanosyl residue) with two rigid and nearly flat rings in a nonplanar orientation with respect to one another, and with R and S absolute configurations (Figure 1). Because of the unique chemical structure of the Sp stereoisomers, the impact of these lesions on the properties of DNA and their biological functions are of great interest. The Sp lesions cause a severe destabilization of double-stranded oligonucleotides (23, 29). This phenomenon is attributed to the disruption of the base stacking interactions between the nonplanar Sp and the loss of Watson-Crick hydrogen bonding between Sp and cytidine in the complementary strand (29). Like 8-oxoG (reviewed by Neeley et al. (12)), Sp can be repaired by base excision repair (BER) enzymes and is the substrate of Fpg, Nth, and Nei of E. coli, yOGG1 and yOGG2 of yeast, and of mammalian Neil1 (30-33). There is evidence that the two stereoisomeric Sp lesions are processed differently during translesion bypass in vitro. Here, we designate the two lesions by Sp1 and Sp2, according to the order of elution of the corresponding nucleosides dSp1 and dSp2, respectively, in reversed phase HPLC experiments (see below). In E. coli, 72% of the mutations represent G f C and 27% G f T transversions, when one Sp isomer replaces a normal G. In the case of the other Sp isomer, 57% of G f C and 41% G f T transversions were observed (25). These mutations represent a preferential pairing of Sp with the purines G and A, respectively, during translesion synthesis. Our structural and thermodynamic studies with 11-mer duplexes containing Sp at the central base pair suggest that the Sp R stereoisomer favors pairing with G more than the S stereoisomer; these conclusions were based on an analysis of the hydrogen bonding patterns between Sp and the different partner bases (29). However, it is important to distinguish between the absolute configurations of Sp1 and Sp2 for a better understanding of structure-function relationships. In this work, we establish the absolute configurations of Sp1 and Sp2 as (-)-S and (+)-R form, respectively. The R and S designations apply to the tautomeric amino forms of Sp (Figure 1). The identification of the stereochemical characteristics of these isomers was achieved by a combination of experimental optical rotatory dispersion (ORD) measurements using Sp1 and Sp2 nucleobases dissolved in aqueous solutions and calculations of the absolute signs of the ORD values by computational methods using density functional theory (DFT). When linearly polarized light passes through a solution containing asymmetric, optically active molecules, the directions of the electric vector of linearly polarized light is rotated after emerging from the solution. Recently, there has been considerable progress in the development of time-dependent density functional theory (TDDFT) for accurately predicting ORD values of chiral molecules (34-37). These DFT methods are now considered sufficiently robust for the determination of the absolute configurations of asymmetric molecules by comparing calculated and experimentally measured values of the specific rotation, [R]ν, at a frequency ν (38-40). The theoretical expression for [R]ν is (41)

28800π2NAν2 γs,vβ(ν) [R]ν ) c2M

(1)

NA is Avogadro’s number, M is the molar mass, and γs,v is a

factor that depends on solvent and vibrational effects. The value of β(ν) can be calculated by DFT methods and is defined as

β(ν) ) 1/3Tr[βRβ(ν)]

(2)

βRβ(ν) are the elements of the frequency-dependent electric dipole-magnetic dipole polarizability tensor:

βRβ(ν) )

c

[

Im

3πh

∑ k*0

]

〈0|(µeel)R|k〉〈k|(µemag)β|0〉 ν2k0 - ν2

(3)

µel and µmag are the electric and magnetic dipole operators, respectively, |0〉 is the ground state of the molecule, and |k〉 runs over all excited states whose excitation frequencies are νk0 (40). The values of β(ν) can be calculated quantum-mechanically for any molecular configuration. In this work, we calculated the wavelength-dependent ORD values for both the amino and imino forms and determined the absolute configurations of the stereoisomeric Sp1 and Sp2 nucleobases by comparison of the experimentally measured ORD values with those computed according to eq 1.

Materials and Methods Calculation of the ORD of the Sp Nucleobases. The amino and imino forms of the nucleobase Sp R and S enantiomers were built with SPARTAN from Wavefunction, Inc. As described previously (28), these structures were first geometry-optimized with the MERCK molecular mechanics force field (MMFF94) (42) in SPARTAN. In the next stage, the quantum-mechanical density functional theory (DFT) method (B3LYP/6-31G*) (43, 44) in Gaussian 98 (45) from Gaussian, Inc. was used to obtain the final geometry-optimized structures for our ORD calculations. The specific optical rotations were calculated using ab initio timedependent DFT (34-36, 46, 47) and Gauge-Invariant Atomic Orbital (GIAO) (34, 48, 49) methods utilizing the B3LYP/6311++G (2d, 2p) basis set in Gaussian 03 (50). B3LYP (43, 44) is a widely used, state-of-the-art hybrid functional. The 6-311++G (2d, 2p) basis set (51-55) contains diffuse functions, which have been shown to significantly reduce basis set errors in the calculated ORD values (36). Although the ORD values are mostly calculated at a single wavelength, usually the Sodium D line at 589 nm, we chose to perform the calculations at multiple wavelengths in the 300-589 nm range. We did not take into account solvent and vibrational effects (γs,v ) 1), partially because there are questions about achieving accurate estimates of solvent effects in the calculated ORD values (40). Our major objectives were to determine the absolute sign of the optical rotation and to mimic the overall wavelength dependence of the specific rotation, rather than to obtain exact matches of the calculated and experimentally measured ORD values (56). The DFT method B3LYP, coupled with the high-level basis set 6-311++G (2d,2p), is widely used for the determination of absolute configurations (35, 36). The presently available computational methods are still not considered to be sufficiently adequate for accurately reproducing the experimentally measured ORD values, especially the wavelength dependence where the minima and maxima in the ORD values occur (56). Nevertheless, the computation of the wavelength dependence of ORD is considered more robust than single-wavelength calculations, since this avoids potential errors associated with the calculation of specific rotation values in the wavelength region where the ORD signal is weak (e.g., at the Na D line of 589 nm (56)). Therefore, the multiple-wavelength approach provides more reliable results for the assignments of the absolute configurations by comparing the computed and experimentally measured wavelength dependence of the absolute sign of [R]λ. Synthesis of the Spiroiminodihydantoin Nucleosides and Nucleobases. We utilized the procedure published by Ravanat and

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Durandin et al.

Scheme 1. Oxidation of 2′-Deoxyguanosine to Spiroiminodihydantoin, and Its Subsequent Hydrolysis to the Base Form

Cadet (57), with some modifications, to synthesize the Sp 2′deoxynucleosides. The formation of Sp via the oxidation of 2′deoxyguanosine is outlined in Scheme 1 and requires pathways that are equivalent to the removal of four electrons and the addition of two oxygen atoms; in this work, the Sp nucleosides were hydrolyzed to the nucleobases as indicated in this scheme. Briefly, 100 mL of an aqueous solution of 0.5 mM Methylene Blue and 1.0 mM of 2′-deoxyguanosine was irradiated with light from a 100 W xenon lamp passing through a 500 nm cutoff filter, for 60 min. After irradiation, the solution was concentrated to dryness and dissolved in 10 mL of H2O; 500 µL portions of this solution containing the reaction products were purified by HPLC methods using a Microsorb Amino HPLC column, Varian Corp., 250 mm × 4.6 mm (the mobile phase was 80% methanol and 20% of a 25 mM ammonium formate solution). The diastereomeric spiroiminodihydantoin 2′-deoxynucleosides (denoted by dSp1 and dSp2) were first collected as a mixture (eluting between 12 and 15 min.). Following solvent removal, the dSp1/dSp2 mixture was dissolved in water, and the diastereomers were separated from one another using HPLC and a Hypercarb column from Thermo Electron Corp., 100 mm × 3 mm (mobile phase, 98% H2O:2% acetonitrile with each solvent containing 0.5% formic acid). The identities of the two dSp products were determined utilizing MS/MS methods as described by Luo et al. (7) using an LC/MSDtrap XCT spectrometer (Agilent Technologies, Palo Alto, CA) with an Agilent 1100 Series Capillary LC System. The mass spectra (MS/MS) were recorded in the positive mode. The nebulizer gas pressure was 15 psi, the dry gas flow rate was 4.0 L/min, and the dry temperature was 325 °C. Samples in aqueous solution were infused using a Kd Scientific pump at a flow rate of 600 µL/h. The procedure for obtaining the nucleobases Sp1 and Sp2 by hydrolysis of the 2′-deoxynucleosides dSp1 and dSp2, respectively, was also adapted from Ravanat and Cadet (57), but with some minor modifications. Typically, 5-10 µmoles of either of the pure anhydrous nucleosides was treated with 80 µL of an HF/pyridine solution at 37 °C for 90 min. To this solution, 1 mL of an aqueous solution containing 160 mg of CaCO3 was added, and the mixture was agitated until the pH reached the value of ∼6. The insoluble salts were removed by centrifugation, and the supernatant was evaporated to dryness. The residue was then dissolved in 100 µL of H2O, and the residual solids were removed by centrifugation. The Sp1 and Sp2 products were then purified using HPLC methods. Measurement of the CD and ORD Spectra. The circular dichroism spectra of the Sp nucleobases were recorded in aqueous solution using an AVIV model 202SF CD spectrometer. A commercially available polarimeter was not suitable for our purposes because the large volume of the optical cell required more of the optically active material than was convenient to synthesize. Therefore, we built our own, highly sensitive apparatus that was suitable for making measurements utilizing a reduced-volume 4 mm × 10 mm optical cell. The home-built ORD instrument was equipped with a 150 W Cermax Xenon arc lamp (Perkin-Elmer, Inc., Wellesley, MA), and an H-10UV monochromator (JobinYvonHoriba, Longjumeau, France) with the wavelength drive controlled by a microprocessor unit. The light was passed first through a quarter-wave Fresnel romb retarder (Karl Lambrecht Co., Chicago, IL) that is transparent from 250 to 600 nm, then a photoelastic modulator operating at a frequency of 50 kHz (model PM3, Hinds International, Inc., Portland, OR). The light passed through the 4 mm × 10 mm quartz sample cell (10 mm optical path length), and finally through a crystal polarizer before reaching the detector. The

latter was a Hamamatsu model R960 photomultiplier whose output was coupled directly to a model 5209 lock-in amplifier (Princeton Applied Research, Princeton, NJ). The output current of the photomultiplier was kept constant as the wavelength was varied by a home-built feedback circuit that adjusted the voltage applied to the photomutiplier in order to compensate for the differences in the incident light intensities at the different wavelengths. The photoelastic modulator provided an analogue Voltage output that was proportional to the wavelength setting of the monochromator which, in turn, provided for easy recording of the ORD signal output as a function of wavelength on a computer. The output of the lockin amplifier was transmitted to a computer through a GPIB interface (National Instruments Co., Austin, TX). The output signal was calibrated using (20 g/100 mL) aqueous solutions of L-(+) and D-(-)-Tartaric acid (Aldrich Chemical Co., Inc., St. Louis, MO) that yield standard rotations of (12° (1 dm path length), respectively (Sodium D-line, 589 nm). The calibration coefficients determined separately with either L-(+) and D-(-)-Tartaric acid solutions were the same within 3%. The (1R)-(+) and (1S)-(-)-camphor samples were purchased from Fluka Chemie Gmbh, Buchs, Switzerland (standard rotations of +44.1° and -43.0° (1 dm path length, 10 g/100 mL in ethanol), respectively, at 589 nm).

Results and Discussion Synthesis. The purified spiroiminodihydantoin 2′-deoxynucleosides were synthesized as described above. Typical HPLC elution profiles of these two nucleoside products denoted by dSp1 and dSp2 according to their elution order using the Hypercarb column (3.2 and 4.6 min, respectively) are depicted in Figure 2A. The two products were identified as the Sp nucleosides by comparison of their mass spectra and MS/MS spectra with the results published by Luo et al. (7) (see Figure S1 in Supporting Information). The characteristic fragmentation pattern gives m/z ) 184 representing the Sp nucleobase followed by further fragmentation to m/z ) 156 (loss of CO) and m/z ) 141 (loss of NHCO). We note that the dSp1 and dSp2 nucleosides can also be separated using a normal phase amino Hypersil column as described in the original work of Ravanat and Cadet (57). However, we found that the order of elution using our Hypercarb column is reversed compared to that of ref 57 in which a Hypersil column was used. We confirmed this conclusion by performing HPLC experiments with both types of columns in our laboratory and by comparing the shapes and signs of CD spectra with those of Ravanat and Cadet. Accordingly, we find that the nucleosides eluted first and second from an amino Hypersil column, denoted in ref 57 as products 2 and 3, have the same spectral characteristics as our dSp2 and dSp1, respectively. After acid hydrolysis (with HF/pyridine) of the individual dSp nucleosides, the corresponding Sp nucleobases were purified using the Hypercarb column, and their structures were identified by mass spectrometry (Figure S2, Supporting Information). The CD spectra of the enantiomeric nucleobases are depicted in Figure 2B and are symmetric, as expected, within experimental error. The CD spectra of the individual precursor dSp1 and dSp2 nucleosides are very similar in sign and wavelength dependence to the Sp1 and Sp2 spectra, respectively, shown in Figure 2B (Figure S3, Supporting Information).

Absolute Configurations of Sp Lesions

Figure 2. (A) HPLC elution profile of a mixture of the spiroiminodihydantoin nucleosides dSp1 and dSp2 using a Hypercarb column (100 mm × 3 mm). The composition of the mobile phase was 98% H2O, 2% acetonitrile with each solvent containing 0.5% formic acid. (B) CD spectra of Sp1 (blue) and Sp2 (red) nucleobases in aqueous solution (0.0083 g/mL). The vertical axis is expressed in degrees divided by the absorbance of the sample solutions measured at 230 nm (1 cm path length).

ORD Spectra. To validate our methods for computing and measuring the specific rotation of the Sp enantiomers, we calculated and measured the wavelength dependence of the ORD spectrum of the two enantiomers of the well-studied chiral compound, camphor (56). The computed and measured ORD spectra of (1R)-(+)- and (1S)-(-)-camphor are compared in Figure 3A. The agreement is satisfactory even in the wavelength region where the ORD signal rises sharply. Our measured ORD spectrum of (1R)-(+)-camphor is also in good agreement with the ORD spectrum published most recently (56). The ORD spectra of the two nucleobases, Sp1 and Sp2, dissolved in water, are shown in Figure 3B. The difficulty of synthesizing and working with greater amounts of the purified and enantiomeric Sp bases, as well as the limitations of our apparatus, limited the wavelength range accessible to measurement in our experiments to greater than ∼280 nm. The computed ORD values are also shown in Figure 3B. While there are differences in the absolute computed and measured values of the specific rotation values of the Sp enantiomers, the wavelength dependence of the measured and computed ORD values resemble one another. These differences may be inherent in the computational methods used (56), and/or to the neglect of the solvent-dependent factor γs,v (eq 1). The signs of the experimentally measured and computed ORD values indicate that Sp1 has (-)-S absolute configuration, while Sp2 has (+)-R absolute configuration for the amino tautomer,

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Figure 3. (A) Measured ORD spectra (smooth lines) of (1R)-(+)- and (1S)-(-)-camphor (0.1 g/100 mL) in ethanol. The computed values are indicated by the red and blue circles, respectively. The vertical axis is expressed in units of specific rotation, [R]λ, at 20 °C. (B) Measured ORD spectra (smooth lines) of the spiroiminodihydantoin Sp1 and Sp2 nucleobases dissolved in water (0.0083 g/mL), expressed in units of specific rotation (20 °C). Red and blue circles: computed ORD values for the Sp2 and Sp1 nucleobase enantiomers, respectively. The computed specific rotation values at the sodium D-line (589 nm) are [R]D ) (6.1°[dm (g/cm3)]-1; the experimentally measured values are somewhat larger (see Figure 4).

as indicated in Figure 3B. We note here that the absolute configuration descriptors R and S are based on accepted sequence rules (58, 59) and apply to the amino tautomers (Figure 1). The computed ORD values shown in Figure 3B were obtained for the amino Sp tautomeric forms. However, Sp might partially exist also in the imino form (Figure 1), as discussed in detail in our earlier publication (28). The tautomer with the exocyclic amino group is usually favored over the imino tautomer in a variety of heterocyclic ring systems (60, 61). The amino tautomer permits conjugation of the amino group with the carbonyl group in the same ring, which may contribute to its stability, and has been favored as the dominant form in the past (28, 32, 62). To date, the equilibrium constant of the aminoimino tautomerism is unknown. On the basis of our QM geometry optimization calculations, the amino form is lower in energy by only ∼1 kcal/mol than the imino form (28). Experimental measurements of glycocyamidines, a group of molecules that mimic the Sp B-ring, show that the amino form is also preferred (63). Nevertheless, the contributions of the imino form to the specific rotation cannot be neglected a priori. We therefore calculated the specific rotation of the Sp imino form as well from 400 to 589 nm (Figure 4). The results show that the ORD values of the imino forms do not differ significantly from those of the amino forms, and thus, ORD cannot distinguish between the two tautomers. Note that the R

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Figure 4. Computed specific rotation ORD values in the wavelength range of 400-600 nm for the amino (open red and blue circles) and imino forms (filled red and blue circles) of the nucleobases Sp1 (blue) and Sp2 (red). The smooth lines are the experimentally measured ORD values.

and S designations in the tautomers are inverted according to the IUPAC convention (58, 59) (Figure 1). This does not, however, impact the sign of the ORD spectra. The aminoimino tautomerism therefore does not influence the assignment of the absolute configurations, nor does it account for the differences between the computed and measured specific rotation values (Figure 3B). During the preparation of this manuscript, we learned that J. Cadet and co-workers (Karwowski, B., Dupeyrat, F., Bardet, M., Ravanat, J.-L., Krajewski, P., and Cadet, J., submitted for publication) have studied the absolute configurations of the two spiroiminodihydantoin 2′-deoxynucleosides (dSp) by considering the dipolar interactions between the base and the sugar moieties using NMR methods. In summary, the absolute configurations of the two Sp enantiomers were determined by measuring the ORD spectra and by comparing the wavelength dependence and signs of the ORD values calculated by DFT methods for each nucleobase enantiomer. The signs of the ORD spectra at wavelengths beyond ∼280 nm are the same as the signs of the longest wavelength CD band with maxima or minima at 258-259 nm. Neither the signs nor the shapes of the ORD and CD bands are significantly different in the dSp nucleosides and the Sp nucleobases derived from each of the diastereomeric nucleosides. On the basis of the results of this ORD/DFT method, the dSp1 nucleoside that elutes first in Hypercarb column HPLC experiments has (-)-S while dSp2 has (+)-R absolute configuration. Acknowledgment. This work was supported, in part, by NIH Grant 1R01 ES11589 (V.S.), and by NIH 2R01 CA75449 (S.B.). Components of this work were conducted in a Shared Instrumentation Facility constructed with support from Research Facilities Improvement Grant C06 RR-16572 from the National Center for Research Resources, NIH. We thank Professor Robert Shapiro for careful reading of the manuscript and helpful discussions. Supporting Information Available: MS/MS fragmentation spectra of dSp, mass spectrum of Sp, and CD spectra of dSp1 and dSp2. This material is available free of charge via the Internet at http://pubs.acs.org.

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