Methylation Stabilizes the Imino Tautomer of dAMP and Amino

ARTICLE pubs.acs.org/JPCB

Methylation Stabilizes the Imino Tautomer of dAMP and Amino Tautomer of dCMP in Solution Namrata Jayanth and Mrinalini Puranik* National Centre for Biological Sciences, TIFR, GKVK Campus, Bellary Road, Bangalore 560065, India

bS Supporting Information ABSTRACT: Alkylating agents cause methylation of adenosine and cytidine in DNA to generate 1-methyladenosine and 3-methylcytidine. These modified nucleosides can serve as regulators of cells or can act as agents of mutagenesis depending on the context and the partner enzymes. Solution structures and the chemical interactions with enzymes that lead to their recognition are of inherent interest. At physiological pH, 1-methyladenosine and 3-methylcytidine are presumed to be in the protonated amino forms in the literature. We report the structures, ionization states, and UV resonance Raman spectra of both substrates over a range of pH (2.511.0). The Raman excitation wavelength was tuned to selectively enhance Raman scattering from the nucleobase (260 nm) and further specifically from the imino form (210 nm) of 1-medAMP. We find that contrary to the general assumption, 1-me-dAMP is present in its neutral imino form at physiological pH and 3-me-dCMP is in the amino form.

’ INTRODUCTION DNA methylation involves covalent modification of nucleobases by the addition of methyl groups at specific positions. Depending on the site of base methylation, the modification can be desirable or disruptive to cell function. While 5-methylcytosine is pivotal for cell regulation, 1-methyladenosine (Figure 1) and 7-methylguanosine, present in tRNA, are essential for maintenance of tertiary structure of tRNA.1,2 Methylation of nucleobases is brought about by both endogenous and exogenous alkylating agents. There are 12 different sites on DNA bases, including the exocyclic oxygens and ring nitrogens that can react with methylating agents.3 The second-order nucleophilic substitution (SN2) alkylating agents predominantly generate 1-methyl-deoxyadenosine-50 -monophosphate (1-me-dAMP) and 3-methyl-deoxycytidine-50 -monophosphate (3-me-dCMP). These modified bases when present in DNA disrupt the normal basepairing process hindering the process of replication4 and hence prove lethal to cells. Efficient repair enzymes such as AlkB in Escherichia coli and ABH2 and ABH3 in humans exist, which counteract the deleterious effects of these lesions. These enzymes recognize methylated nucleosides present within DNA or RNA strands and demethylate the base restoring it to its natural form. AlkB is known to be promiscuous in its substrate recognition and is able to accommodate small (3-me-dCMP) as well as large substrates, such as ethenoadenosine (εA), into its active-site pocket. Thus, the solution structures of the substrates and their interactions with AlkB that lead to substrate recognition are of inherent interest. The structural tautomers and ionization states of 1-methyladenine (1-meA) have been extensively studied by theoretical r 2011 American Chemical Society

and experimental methods to deduce structurefunction relationships.1,510 Three stable forms of 1-meA are possible: imino-N7H, imino-N9H, and amino form.10 Quantum chemical calculations of various forms of solvated and unsolvated 1-meA have deduced that the imino form, protonated at N9 position,7 is the stable ground state. On the basis of the pKa, it has been proposed that 1-methyladenine exists as a mixture of amino and imino forms at physiological pH.11 NMR studies of 1-methyladenosine carried out in context to the tRNA structure have revealed the presence of the amino form at pH below 6, and the imino form is dominant at physiological pH.1 Theoretical analysis of the relative stabilities of the various forms using SCF MO LCAO method predict the imino-N9H to be the most stable followed by the imino-N7H form.10,12 The IR spectra of 1-meA in argon matrix and in solution interpreted using normalcoordinate analysis show the presence of imino form.10 To our knowledge, the solution-state structures of 1-me-dAMP, the minimal substrate of the repair enzyme, at different pH values have not been previously characterized. We examined the vibrational spectra of 1-me-dAMP at various pH values using ultraviolet resonance Raman spectroscopy (UVRR), and we report the dominant protonation state and its structure in this study. Purrello et al. have reported the resonance Raman spectrum of 3-methylcytidine at pH 5 and 11.0, but a detailed vibrational assignment has not been made. Their work demonstrates the presence of the imino form of 3-methylcytidine at higher pH.13 Received: January 7, 2011 Revised: March 21, 2011 Published: April 15, 2011 6234

dx.doi.org/10.1021/jp200185k | J. Phys. Chem. B 2011, 115, 6234–6242

The Journal of Physical Chemistry B

Figure 1. Structure and numbering used in computational calculations for (A) protonated 1-methyladenine, (B) neutral 1-methyladenine, (C) protonated 3-methylcytosine, and (D) neutral 3-methylcytosine. Hydrogen atoms replaced with deuterium for simulation of H/D exchange effects on the vibrational spectra are marked with squares.

Because both 1-me-dAMP and 3-me-dCMP bind at the same active site, we studied the structure of 3-me-dCMP as well to compare the two substrates. In the following, we report the UVRR spectra over a range of pH and the corresponding density functional theory (DFT) calculations of 3-meCMP. The dominant protonation and tautomeric states present in solution are identified.

’ EXPERIMENTAL AND COMPUTATIONAL METHODS

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analyzed with a single-grating (3600 grooves/mm) monochromator (Jobin-Yvon). The detector used was a 1024 256 pixel, back-illuminated CCD camera (Jobin-Yvon). Calibration was carried out using spectra of standard solvents: dimethyl formamide, acetonitrile, trichloroethylene, isopropanol, indene, and cyclohexane of HPLC grade from RANCHEM chemicals and SIGMA and were used without further purification. In experiments where the relative shift was measured, the spectra were recorded without interruption and without changing the spectrometer position. We estimate that they are accurate to (3 cm1. All spectral processing was carried out using the software Synergy (Jobin-Yvon). Band positions were determined by fitting Lorentzian line shapes to the bands in the observed spectra. Conversion of Raman Intensities into Concentrations. Sodium nitrate (1049 cm1) was used as an internal standard to determine the relative cross sections and concentrations of the two forms, amino and imino, of 1-me-dAMP in solution. Raman intensities of all bands were determined by calculating the area under the band after fitting a Lorentzian line shape. As a first step, cross sections of the two forms of 1-me-dAMP were determined using the known cross section of NaNO3. The Raman cross section of the 1049 cm1 NaNO3 (σ1049) has been reported by Billinghurst and Loppnow16 at 257 nm as 6.35 1011 cm2/ molecule 3 sr. We have taken this value as it is the value closest to the wavelength of excitation that we have used (260 nm). We also assumed that at extreme limits of the pH titration range all the 1-me-dAMP in solution will be present in either the amino (pH 2.5) or imino (pH 10.0) forms. Thus, the spectrum at pH 2.5 was used to determine the cross section of amino 1-medAMP and that at pH 10.0 for the cross section of the imino form using eq 1. σðaminoÞ ¼ ½σ 1049 I ðaminoÞ CNaNO3 =I 1049 CðaminoÞ

Sample Preparation. Deoxyadenosine-50 -monophosphate

(dAMP), deoxycytidine-50 -monophosphate (dCMP), 1-me-dAMP, and 3-me-dCMP, purchased from Chemgenes, United States, were of chromatographic grade and were used without further purification. Samples for Raman experiments were of 0.5 mM concentration prepared by dissolving appropriate quantities of the corresponding nucleotides in buffers (pH 2.5: 0.1 M potassium phosphate buffer; pH 5.5: 0.1 M sodium acetate buffer; pH 6.5, 9.5, 10.0, and 11.0: 0.1 M sodium carbonate buffer; pH 7.08.5: 20 mM Tris buffer). For the hydrogen/deuterium (H/D) exchange experiments, the buffers were prepared in D2O. Samples were dissolved in the appropriate buffers and were left overnight before recording the spectrum to ensure complete H/D exchange. The pH of the samples was adjusted by the addition of required amounts of acid or base (NaOD for samples in D2O). Acetonitrile, trichloroethylene, isopropanol, indene, and cyclohexane used for calibration were HPLC grade purchased from RANCHEM chemicals or SIGMA and were used without further purification. UVVisible Absorption Spectroscopy. Ultraviolet spectra were recorded with Ultraspec 4000 UV/visible spectrophotometer from Pharmacia Biotech. Ultraviolet Resonance Raman Spectroscopy. The detailed ultraviolet resonance Raman setup has been described in earlier publications.14,15 Resonance Raman spectra were obtained by excitation with 260 nm light. This is the third-harmonic output of a nanosecond-pulsed Nd:YLF laser pumped TiS laser (Indigo, Coherent Inc.) operated at 1 kHz. Typical average power at the sample was less than ∼600 μW. Reabsorption of the scattered light was minimized by using backscattering collection configuration. Light was collected using home-built optics and was

ð1Þ

where C(amino) and CNaNO3 are the concentrations of amino 1-me-dAMP and NaNO3. σ1049 and σ(amino) are cross sections of the internal standard, NaNO3 and 1-me-dAMP, respectively. I(amino) corresponds to the area under the Raman peak at 1503 cm1, a band assigned to the amino form of 1-me-dAMP. I1049 is the Raman intensity of the 1049 cm1 band of NaNO3. A similar equation was used to determine the concentration of the imino form using the Raman intensity of the band at 1193 cm1 corresponding to the imino form of 1-me-dAMP. The concentration of the internal standard, CNaNO3, used in the experiments was 30 mM. Once the cross sections were determined, these values were used to determine the concentrations of the amino and imino forms of 1-me-dAMP at other pH values from the measured Raman intensities using eq 1. Computational Methods. Quantum mechanical calculations were carried out on the nucleobases corresponding to the nucleosides and nucleotides used in the experiments. Since the Raman excitation wavelength used is in resonance with the nucleobase, the vibrational spectrum is dominated by vibrational bands from the nucleobases. Density functional theoretical calculations were performed with the Gaussian0317 program using the B3LYP parametrization.18,19 The basis set used is Gaussian 6-31G** which has additional polarization functions on the hydrogen atoms. The geometry was optimized taking solvent effect into account by using polarizable continuum model (PCM). The potential energy distributions (PEDs) were computed using Vibrational Energy Distribution Analysis (VEDA) 4.0 program.20 6235

dx.doi.org/10.1021/jp200185k |J. Phys. Chem. B 2011, 115, 6234–6242


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Figure 2. (A) UV absorption spectra of aqueous solutions (50 μM) of dAMP at pH 7.5 (dashed line), 1-me-dAMPHþ at pH 2.5 (solid line), and 1-me-dAMP at pH 10.0 (dotted line). (B) UV absorption spectra of aqueous solutions (50 μM) of dCMP at pH 2.5 (solid line), dCMP at pH 7.5 (dashed line), 3-me-dCMP at pH 7.5 (dotted line), and 3-me-dCMP at pH 11.0 (dasheddotted line). The excitation wavelengths used in the UVRR experiments are shown as arrows at 210 nm and 260 nm.

Figure 3. UVRR spectra of 1-me-dAMP in buffer at different pH values using Raman excitation of 260 nm: (A) pH 2.5, (B) pH 7.5, and (C) pH 10.0 in water; (D) pD 2.5, (E) pD 7.5, and (F) pD 10.0 in D2O.

’ RESULTS AND DISCUSSION pH Induced Changes Monitored by Absorption Spectroscopy. The spectra of 1-me-dAMP and 3-me-dCMP are markedly

different from their parent compounds as expected because of the addition of the methyl group (Figure 2). In general, the extinction coefficient of 1-me-dAMP is lower than that of dAMP. Changes in the UVvisible absorption spectrum of 1-me-dAMP with pH titration indicate a change of protonation state at ∼pH 5.0. At pH 2.5, there are two bands corresponding to πfπ* transition at 204 and 260 nm. The UV absorption spectra are essentially unchanged from pH 2.5 to 5.5. With further elevation of the pH from 6.5 to 10.0, the two bands red shift to final positions of 225 and 269 nm at pH 10.0. The pKa of 1-me-dAMP has not been established so far. The pKa for 1-methyladenine is reported at 7.211 and that of 1-methyladenosine is 8.25.21,22 By extrapolating these pKa's to 1-me-dAMP, the amino form of 1-me-dAMP is expected to be the primary species at pH 7.0. In the following, UVRR spectra provide unequivocal identity of the species present in solution under different pH conditions.

3-Me-dCMP shows two intense absorption bands at 212 and 277 nm, whereas dCMP contains three absorption bands at 206, 232, and 271 nm.

’ ULTRAVIOLET RESONANCE RAMAN SPECTRA OF 1-ME-DAMP 1-Me-dAMP Exists in Equilibrium between the Protonated Amino and Neutral Imino Forms at Physiological pH. Figure 3 shows the UVRR spectra of 1-me-dAMP obtained

with 260 nm excitation at three pH conditions: 2.5, 7.5, and 10.0. Spectra at intermediate values of pH are shown in Figure S1 of theSupporting Information. In comparison with the corresponding spectrum on dAMP, the spectrum at pH 7.5 of 1-me-dAMP clearly shows many more bands. The spectra at pH 2.5 and pH 10.0 contain fewer bands. The systematic change in the band intensities with pH likely indicates the presence of more than one species in solution. Two features stand out: the band at 1503 cm1 seen in the spectrum at pH 2.5 gradually loses 6236



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Table 1. Experimental (pH 2.5) and Computed Wavenumbers (cm1) of 1-Methyl-deoxyadenosine-50 -monophosphate in Water and D2O

a

DFTa (H2O)

exptl (H2O) λexc 210 nm


1624

1644

1644

1604

1591

1593

1569

1565

1566

C2H be, N6H6 be, C6N6 str, C1H1be, C2N3 str

1460

1503

1503

1339

1331

1330

C1H1 be, C2N3 str, C2H be, N6H6 be, C4N9 str, C5N7 str, C5C6 str, N1C6 str N1C6 str, C5N7 str, N1C2 str, C8H be, N9H be, C1H1 be, N6H6 be

assignments N6H6 be, C2N3 str, C2H be, N1C6 str, C5C6 str, N7C8 str, C4N9 str, C8H be be N6H6 be, N3C4 str, C8H be, C4C5 str, N9H be, C1H1 be

PED (%)

DFTa (D2O)

exptl (D2O) λexc 210 nm


1607 (þ3)

1582

1585 (8)

1578 (þ9)

1555

1556 (10)

1459 (1)

1501

1501 (2)

C1H1 be, C2H be

1343 (þ4)

1332

1335 (þ5)

C1H1 be, N9H be, C8H be, N1C2 str, N1C6 str, C5N7 str, N6H6 be

assignments

HNH be (48), NC str (14) HNH be (13), NC str (22), CNC be (10) HNH be (17), NC str (16), HCN be (12) NC str (16), HCH be (12) NC str (18), NC str (18)

N3C4 str, N1C6 str, N9H be, C8H be, C1H1 be, N7C8 str C2H be, C1H1 be, N9H be, C6N6 str, N3C4 str

Scaling factor used 0.99.

intensity as the pH is raised while the triplet of bands at 1423, 1443, and 1472 cm1 seen clearly in the spectrum at pH 10.0 gradually loses intensity as pH is lowered and disappears at pH below 7.0 (Figure 3). The intense band at 1330 cm1 in the low pH spectrum shifts to 1324 cm1 at high pH and also decreases in intensity (Figure S4 of the Supporting Information). These features point clearly to the presence of at least two species in solution. We find that the spectrum obtained at each pH can be reproduced by combining the spectra at pH 2.5 and pH 10 in different ratios. On the basis of these observations, we conclude that only two species are present in solution. These changes qualitatively follow the pH dependence of UVvisible absorption spectra. Figure 5 shows the titration curves obtained by plotting the concentration of the respective tautomer that was determined from the integrated intensity of the Raman bands that are exclusive to each of the observed species. As discussed below, the band at 1503 cm1 serves as the marker of the protonated amino form (1-me-dAMPHþ), and the band at 1193 cm1 is a characteristic band of the neutral imino (1-me-dAMP) form. From these titration curves, the pKa of 1-me-dAMP is measured as 7.2. This value is lower than that reported for the 1-methyladenosine21,22 and is closer to that observed for the free nucleobase. To determine the protonation states and structures of 1-medAMP involved in this ionization equilibrium, we compared the experimental spectra with those computed using density functional theory. Structures and vibrational spectra were computed for the nucleobases in the neutral imino form of 1-meA (Figure 1B) and in the protonated, amino form of 1-meA (Figure 1A). A detailed normal-mode analysis was carried out for both forms (Tables S1 and S2 of the Supporting Information). Similar calculations were carried out on corresponding isotopically labeled (HfD) molecules (Figure 1) in order to analyze spectra obtained in D2O. From a comparison with the computed band positions, spectrum at low pH was identified as that of protonated, amino 1-me-dAMP indicating that at low pH the nucleobase in 1-me-dAMP is protonated. The spectrum at high pH corresponded to the computed spectrum of the neutral, imino 1-me-dAMP. Table 1 (1-me-dAMPHþ) and Table 2 (1-me-dAMP) list the computed and experimental Raman shifts

for the two species. As expected from the red shift in the absorption spectrum maximum with changing pH, Raman scattering from the amino and imino forms will be differentially resonance enhanced as the excitation wavelength is tuned. We carried out experiments with two excitation wavelengths: 260 nm (both forms in resonance) and 210 nm (primarily 1-medAMPHþ in resonance). In the following, a detailed analysis of the bands observed with both wavelengths is presented. 1-Me-dAMP Exists in Its Amino Form in Single-Stranded Oligomer. The physiologically relevant form of methylated adenine is the nucleotide incorporated into a single- or doublestranded DNA. It has been shown that the ssDNA strand with 1-me-dAMP is a substrate of the demethylating repair enzymes. Hence, we investigated the protonation state of a 1-me-dAMP incorporated into an ssDNA strand. The pKa of 1-me-dAMP is altered when present in a single-stranded DNA. The amino form of 1-me-dAMP predominates at physiological pH when present in an oligomer (Figure 4). The characteristic bands of the amino form of 1-me-dAMP are seen distinctly in the spectrum of a pentanucleotide containing 1-me-dAMP. The representative modes of NH2 group are seen at 1581 cm1 and 1554 cm1. The 1510 cm1 mode and a single band at 1426 cm1 further support the presence of the amino form in the oligomer. Protonated Amino Form 1-me-dAMPHþ is Present at Low pH. In the spectrum in Figure 3A, obtained with Raman excitation at 260 nm, two bands are observed in the 1600 cm1 region. These bands are in the typical positions for an amino group vibration as also predicted by DFT calculations of the vibrational spectra of the amino form. They are relatively weak bands which shift to lower frequencies at high pH. The three bands seen in the 1500 cm1 region at 1593, 1566, and 1503 cm1 are unique to the amino form. They contain significant contribution from the ring modes, mainly the pyrimidine ring and the amino group. The band at 1593 cm1 is comparable to the 1581 cm1 band of dAMP. It is assigned to modes involving the NH2 group and the pyrimidine ring. This mode disappears at high pH as deprotonation at the N6 position will cause significant change in the C6N6 stretch and NH2 bending vibrations. The 1565 cm1 band is composed of ring vibrations, namely, the pyrimidine C4C5 and N3C4 6237



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Table 2. Experimental (pH 10.0) and Computed Wavenumbers (cm1) of 1-Methyl-deoxyadenosine-50 -monophosphate in Water and D2O

DFTa (H2O)


1695

a

exptl (H2O) λexc 260 nm 1681

1424

1421

1423

1409

1440

1443

1313

1321

1324

1076

1192

1193

assignments

PED (%)

DFTa (D2O)

C6N6 str, C5C6 str, C2C3 str, C2H be, N6H be C2H be, N9H be, C1H1 be, C8N9 str

NC str (45), CNC be (18), NC str (10) HCN be (39), HNC be (16), NC str (12), NC str (11) NC str (22), HNC be (16), HNC be (11), NC str (11), NC str (11) NC str (33), HNC be (19)

1692 (3)

N6H be, N1C6 str, C5N7 str, N9H be, C4N9 str N6H be, C5N7 str, N1C1 str, C4N9 str, N9H be, C8H be C1H1 be, N6H be

HCNC tors (21), HCNC tors (21), HCH be (14), NC str (14)



assignments C6N6 str, N6H be, C4C5 str, C2H be

1424 (0)

1421

1423 (0)

C2H be, N9H be, C1H1 be

1393 (16)

1437

1439 (4)

1294 (19)

1326

1328 (þ4)

C2H be, N9H be, C5N7 str, C1H1 be, N1C6 str, N6H be C8H be, N9H be, C5N7 str, N1C1 str

1059 (17)

1186

1187 (6)

C1H1 be, N6H be, C2H be, C8H be

No scaling factor used.

Figure 5. pH titration plot of concentrations determined from Raman intensities of 1503 cm1 band of the protonated amino form (1-medAMPHþ) and 1193 cm1 band of neutral, imino form of 1-me-dAMP.

Figure 4. UVRR spectra of a pentamer, 50 -dC-dT-(1-me-dAMP)-dTdC-30 , obtained using Raman excitation at 260 nm in (A) water and (B) D2O, pH 8.0.

stretch along with the imidazole N7C8 stretch. This band shows 10 cm1 downshift on deuterium exchange owing to the contribution of NH2 bending along with the ring vibrations. The 1503 cm1 band appears as a prominent band in 1-me-dAMPHþ spectrum at low pH and can be used as a diagnostic band for the protonated form of 1-me-dAMP. The dominant feature in the spectrum of 1-me-dAMPHþ at pH 2.5 is at 1330 cm1. On the basis of the previous mode assignment of dAMP23 at 1339 cm1, we assign this band to a predominantly imidazole ring vibration, in particular, to the C5 —N7 stretch. Upon H/D exchange, an upshift of 5 cm1 is observed, which correlates well with that predicted by DFT (4 cm1). Detailed assignments, PED, and isotopic shifts are provided in the tables (Table S1 of the Supporting Information). Deprotonated Neutral Imino Form of 1-me-dAMP is Present at High pH. The observed vibrational spectrum correlates

well with the computed spectrum of the neutral imino form. The 1681 cm1 band has been assigned to C6N6 stretch, pyrimidine ring stretch, and N6H bending vibrations. The 1637 cm1 band is composed of pyrimidine ring vibrations and imino nitrogen supported by a large upshift to 1656 cm1 in D2O because of decoupling of the NH bend. The 1472 cm1 band is unique to the imino tautomer. This band has been assigned mainly to the C2H group and methyl modes with significant contribution from imidazole and pyrimidine ring modes. As compared to the amino tautomer, where only one band is observed at 1428 cm1 in water, the imino tautomer shows two additional bands in this region at 1443 cm1 and 1423 cm1. The 1443 cm1 band is assigned to N6H bend, coupled to C5N7 and N1C6, and shows a downshift to 1439 cm1 in D2O. The 1423 cm1 mode comprises mainly C2H bending vibrations along with methyl group bending motions and is insensitive to H/D exchange. Another marker of the imino form is the band at 1393 cm1 which is absent at lower pH values. It has been assigned to C2H stretch, N1C2 stretch, and methyl group bending vibrations. The sharp band at 1356 cm1 is a highly delocalized mode 6238



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Figure 6. UVRR spectra of 1-me-dAMP in buffer at different pH values using Raman excitation at 210 nm: (A) pH 5.5, (B) pH 6.5, (C) pH 7.5, and (D) pH 8.5 in water; (E) pD 5.5, (F) pD 6.5, (G) pD 7.5, and (H) pD 8.5 in D2O.

assigned to the C8H bend, the C2H bend, and the pyrimidine ring vibrations coupled to the imino group and methyl group vibrations. The dominant band in the spectrum of 1-medAMP at pH 10.0 at 1324 cm1 contains contribution from the imino group and ring vibrations, mostly from the imidazole ring. It upshifts to 1328 cm1 in D2O, although DFT predicts a downshift. The band undoubtedly corresponds to the 1330 cm1 band seen in the amino form which downshifts to 1324 cm1. Deprotonation has been shown to cause delocalization of π electrons, which in turn reduces the double-bond nature of the ring leading to a decrease in the vibrational frequency.24 A band sensitive to H/D exchange in the lower frequency region is at 1299 cm1 (N6H bend). It shifts to 1287 cm1 in D2O, which is a downshift that is well-reproduced by DFT calculations (16 cm1). The band at 1193 cm1 observed as a faint band at pH 6.5 gains intensity with increase in pH and is characteristic of the imino tautomer. This mode has been assigned to vibrations from the N6H stretching vibrations coupled to methyl group. It downshifts by 6 cm1 upon deuteration. This assignment is also confirmed on comparison with the observed IR frequency at 1176 cm1 for the imino tautomer in argon matrix.10 Spectra with 210 nm Raman Excitation. The spectra obtained with 210 nm excitation are shown in Figure 6. Since 210 nm is in resonance with a higher electronic state than 260 nm, the spectrum obtained shows intensity enhancements complementary to the spectra at 260 nm excitation. Several of the bands observed at 260 nm lose intensity while several additional bands appear in the spectrum. The NH2 vibration of the amino form appears at 1644 cm1 and is comparable to the corresponding dAMP mode at 1642 cm1.23 The pyrimidine ring mode at 1591 cm1 (1593 cm1 at 260 nm) gains intensity in this spectrum. Similar intensity patterns were observed in dAMP in the band at 1581 cm1 which gains intensity at 218 nm excitation.21 At 260 nm, a band was observed in D2O at 1415 cm1 which was absent in the water spectrum. This mode appears as an intense band in the spectrum obtained with 210 nm excitation in both water and D2O. Modes with ring contributions are predicted to gain intensity at lower excitation wavelength.23 Fodor et al. have

observed selective enhancement of the NH2 modes of dAMP and dCMP on going from 260 to 200 nm laser excitation. This has been attributed to the promotion of NH2 p electrons.23 This same trend is observed for the three modes of 1-me-dAMP at 1644, 1591, and 1565 cm1 corroborating assignment of the protonated amino form of 1-me-dAMP at lower pH. Excitation with 210 nm leads to change in the intensity pattern of imino 1-me-dAMP as well. Weak bands in the spectrum at 260 nm excitation gain intensity, for example, 1565 cm1 (weak band at 260 nm) is seen as a sharp, intense band at 1560 cm1. The 1391 cm1 (1394 cm1 at 260 nm) band is unique to the imino tautomer. It gains in intensity at 210 nm excitation. The mode that is characteristic of the imino form is the NH bend at 1192 cm1, a well-defined, intense band. 3-Me-dCMP Is in the Protonated Amino Form at Physiological pH. Fourier transform infrared (FTIR) and FT-Raman of CMP25 and UVRR spectra of dCMP and 3-me-dCMP at various pH values have been previously reported.13,23,2527 Purrello et al. have made assignments in the high wavenumber region between 1500 and 1800 cm-1 for neutral and protonated 3-methylcytidine by comparison with various analogous structures.13 At pH 11.0, 3-methylcytidine (pKa = 8.7)28 exists in the imino form. It undergoes protonation at low pH and is present as the amino form at pH 7 and below. We obtained spectra of 3-me-dCMP at a range of pH values to study both the neutral, imino form (3-medCMP) and the protonated, amino form (3-me-dCMPHþ). As with 1-me-dAMP, spectra were recorded with two Raman excitation wavelengths: 260 nm in resonance with both protonation states (Figure 7) and 210 nm to enhance spectra from 3-me-dCMPHþ (Figure 8). Spectra in D2O were also obtained to aid assignment of the observed bands to vibrational normal modes. Consistent with previous reports, we find 3-medCMPHþ present at low pH and the 3-me-dCMP at high pH. The spectrum at pH 7.5 is dominated by vibrational features of 3-me-dCMPHþ. The computed and experimental bands are listed in Table 3 (3-me-dCMPHþ) and Table 4 (3-me-dCMP) along with the normal-mode assignment and PED composition. An interesting feature in the UVRR spectrum of protonated dCMP is the presence of a single band at 1730 cm1 which is attributed to the rare iminol tautomer.13 A similar band is 6239



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Figure 7. UVRR spectra of 3-me-dCMP in buffer at different pH values obtained using Raman excitation of 260 nm: (A) pH 7.5, (B) pH 8.5, and (C) pH 11.0 in water; (D) pD 7.5, (E) pD 8.5, and (F) pD 11.0 in D2O.

Figure 8. UVRR spectra of 3-me-dCMP in buffer at different pH values obtained using laser excitation of 210 nm: (A) pH 7.5, (B) pH 8.5, and (C) pH 11.0 in water; (D) pD 7.5, (E) pD 8.5, and (F) pD 11.0 in D2O.

observed in 3-methylcytidine at 1723 cm1 which is also predicted to arise from the iminol tautomer.13 We observe this band at 1718 cm1 in 3-me-dCMPHþ which downshifts only slightly to 1717 cm1 in D2O. The carbonyl stretching mode is a prominent feature at 1654 cm1 in the 3-me-dCMPHþ spectrum at low pH. Deprotonation causes an upshift (1670 cm1) and a gain in intensity to make it the most intense band of 3-me-dCMPHþ. The ring vibration is another intense band at 1549 cm1 comprising N3C4 stretching vibration in 3-me-dCMPHþ. DFT predicts significant contribution from the NH2 mode which is manifested as a large downshift in the D2O spectrum. Methyl group vibrations are represented by two weak bands at 1457 cm1 and 1427 cm1. DFT predicts coupling of the former mode with N1H, but since the experimental data corresponds to the mononucleotide, the contribution from N1H can be excluded. The C6H bending vibration at 1388 cm1 is an important mode that decouples from NH2 upon H/D exchange to show an upshift of 5 cm1 in D2O. A corresponding vibration with similar composition is seen in dCMP at 1374 cm1 which also upshifts by 19 cm1 in D2O.23 The dominant feature of the

3-me-dCMPHþ spectrum at 260 nm is the band at 1268 cm1. Similar to dCMP which also contains prominent bands in the 12001300 cm1 region, we assign it to vibrations from C2N3 and C4N4 stretching. In addition to the ring-stretching vibration, significant contribution from the methyl group is also present. Spectra in Resonance with Higher Excited State (Raman Excitation λexc = 210 nm). The most prominent band of the amino form at 210 nm excitation is at 1553 cm1 followed by the carbonyl stretching mode at 1656 cm1. Previously reported UVRR spectra of 3-methylcytosine obtained at different excitation wavelengths21 show a similar trend wherein the modes containing pyrimidine ring vibrations, namely, N3C4 in the 1500 cm1 region, are enhanced in the 218 nm excitation. With 260 nm excitation, a weak band at 1585 cm1 attributed to C5C6 stretching occurs only in D2O. This mode appears as a sharp band at 1581 cm1 with 210 nm excitation. The Neutral Imino Form of 3-Me-dCMP. As stated before, the most intense band in the imino form of 3-me-dCMP is at 1670 cm1 comprising the carbonyl mode, which downshifts to 6240



ARTICLE

Table 3. Experimental (pH 7.5) and Computed Wavenumbers (cm1) of 3-Methyl-deoxycytidine-50 -monophosphate in Water and D2O

DFTa (H2O)

exptl

exptl

exptl

exptl

(H2O)

(H2O)

(D2O)

(D2O)

λexc

λexc

210 nm

260 nm

assignments

PED (%)

λexc

λexc

DFTa(D2O)

210 nm

260 nm

assignments

1719

1718

iminol str

1716

1717

iminol str

1778

1656

1654

C2O str, N1H be

OC str (75)

1777 (1)

1661

1659 (þ5)

C2O str, N1H be

1542

1553

1549

N4H4 be, N3C4 str,

NC str (32),

1541 (1)

1539

1537 (12)

N1H be, N3C4 str,

C3H3 be, C4N4 str,

NC str (19)

N1C2 str, C3H3 be

N1H be, C6H be 1243

1299

1299

N1H be, C5H be, C6H be, N4H4 be

HCC be (24),

1239 (4)

1302

1302 (þ3)

HNC be (18), HCC be (18),

N1H be, C6H be, C5H be

HCC str (15) 1265

1264

1268

C3H3 be, C2N3 str,

HCNC tors (17),

1270 (þ5)

1265

1275 (þ7)

C3H3 be, C2N3 str,

N1H be, N4H4 be,

HCNC tors (17),

N4H4 be, C6H be,

C4N4 str

HCH be (11), NC str

C4N4 str

(17), NC str (10) a


Table 4. Experimental (pH 11.0) and Computed Wavenumbers (cm1) of 3-Methyl-deoxycytidine-50 -monophosphate in Water and D2O exptl

exptl

exptl

exptl

(H2O)

(H2O)

(D2O)

(D2O)

λexc

λexc

DFTa (H2O)

210 nm

260 nm

1749

1674

1670

C2O be, N1H be, C3H3 be

OC str (66)

1629

1585

1574

C4N4 str, C5C6 str,

NC str (68)

assignments

λexc

λexc

210 nm

260 nm

assignments

1749 (0)

1673

1669 (1)

C2O str, N1H be, C3H3 be, C4N4 str

1624 (þ5)

1585

1578 (þ4)

C4N4 str, N4H4 be,

DFTa(D2O)

PED (%)

C6H be, C5H be,

C6H be, N1H be,

N1H be 1502

1490

1492

N1H be, C3H3 be,

C5C6 str, C5H be HNC be (18), NC str

N4H be, C6H be,

(13), NC str (11),

C5H be

HCH be (10)

1400

1397

1395

C6H be, C5H be, C3H3 be, N4H4 be,

1325

1318

1317

C3H3 be, N4H4 be,

1495 (7)

1473

1473 (19)

C3H3 be, N1H be, C5H be, C6H be

HCC be (35), HCC be (25)

1395 (5)

1390

1393 (2)

C6H be, C5H be, C3H3 be, N1C6 str

NC str (22),

1315 (10)

1314

1312 (5)

C3H3 be, N3C4 str,

N1C2 str, C2N3 str, N3C4 str,

NC str (16)

C5H be, N1H be

C5H be a


1669 cm1 upon deuteration. This is undoubtedly valuable as a marker of hydrogen-bonding interaction with the protein hosts for these substrates. The bands at 1574 cm1 and 1492 cm1 mark the pyrimidine C5C6 stretch coupled to the C4N4 stretch. The band at 1492 cm1 is essentially a C4N4 stretching vibration coupled to N4H bending and is a characteristic band of the imino form. The assignment is also supported by DFT calculations in which a large downshift (19 cm1) upon deuteration of H4 is predicted. Another band unique to the imino form is at 1317 cm1 assigned to vibrations of pyrimidine ring, namely, N3C4 and C2N3 stretch, coupled to the methyl group vibrations. At 210 nm excitation (Figure 8), the carbonyl mode is significantly reduced in intensity. Bands at 1585, 1490,

and 1397 cm1 are enhanced. The most prominent mode is observed at 1490 cm1. We have demonstrated the remarkable differences in protonation equilibrium between the amino and imino forms of methylated adenine in its two forms: as a nucleotide and as a nucleic acid. While 1-me-dAMP exists as a mixture of both amino and imino forms at physiological pH, 3-me-dCMP exists in its amino form at pH 7.5. Deprotonation occurs because of the loss of the amino proton at N6 and N4 positions of 1-me-dAMP and 3-me-dCMP, respectively. Both convert to their respective imino forms at pH higher than physiological range. The presence of the methyl group alters the electronic properties and the base-pairing ability of adenine and cytosine. 6241


The Journal of Physical Chemistry B Methylation at the N1 and N3 positions of dAMP and dCMP prevents the formation of normal WatsonCrick base pairing and leads to the formation of Hoogsteen interactions in doublestranded DNA. 1-Me-dAMP and 3-me-dCMP when present in DNA block the replication process acting as inhibitors of DNA polymerase I.27 They are repaired by demethylating repair enzymes such as AlkB, ABH2, and ABH3. Knowledge of the dominant protonation states of the bases in mononucleotide and DNA is important to understand the mechanism of recognition of these by polymerases and repair enzymes. Our finding that 1-me-dAMP is present in equilibrium between the amino and imino forms raises the question of which form is the true substrate of these enzymes. The presence of the methyl group renders the bases to be more basic, and they bear a positive charge. The positive charge of the bases is implicated to have an advantage over neutral substrates in stabilizing the nucleic acidrepair enzyme complex on the basis of the active site geometry.4 Hydrogen bonding with amino acids of the enzymes plays an important role in sequence-specific recognition of nucleic acids. Since the hydrogen-bonding abilities of the amino and imino forms of these bases are quite different, we predict that only one of the forms is recognized by the enzyme. The vibrational spectra reported here and the accompanying detailed normal-mode analysis open up possibilities of elucidation of the finer structural changes occurring in these nucleotides upon interaction with repair enzymes. The marker bands of amino and imino forms are uniquely identified as are the markers of methyl group vibrations. Further work on the repair enzymemethylated base complex is in progress.

’ ASSOCIATED CONTENT

bS

Supporting Information. UVRR spectra of 1-me-dAMP in (a) water and (b) D2O at various pH values using Raman excitation of 260 nm. Computed bond lengths of adenine, amino form; 1-methyladenine, amino form; 1-methyladenine, imino form; cytosine, amino form; 3-methylcytosine, amino form; and 3-methylcytosine, imino form. Spectra of the various buffers used in these experiments. Relative intensities of the 1330 cm1 (1-me-dAMPHþ) and 1324 cm1 (1-me-dAMP) normalized to an internal standard NaNO3. Detailed vibrational assignments for each of the forms of 1-me-dAMP and 3-medCMP. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Telephone: þ91-80-23666160. Fax: þ91-80-23636462. E-mail: [email protected].

’ ACKNOWLEDGMENT This research was supported by grants from Tata Institute of Fundamental Research and the Department of Biotechnology, India. N.J. was supported by the Ph. D. fellowship of the Council for Scientific and Industrial Research (CSIR) India. M.J.P. is a recipient of the Innovative Young Biotechnologist Award of the Department of Biotechnology, India.

ARTICLE

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Methylation Stabilizes the Imino Tautomer of dAMP and Amino

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