Structures, Ionization Equilibria, and Tautomerism of 6-Oxopurines in

J. Phys. Chem. B 2009, 113, 15101–15118

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Structures, Ionization Equilibria, and Tautomerism of 6-Oxopurines in Solution Spriha Gogia, Ankur Jain, and Mrinalini Puranik* National Centre for Biological Sciences, GKVK Campus, Bellary Road, Bangalore 560065, India ReceiVed: June 19, 2009; ReVised Manuscript ReceiVed: September 17, 2009

6-Oxopurine and its analogues form an important class of biological molecules that include nucleobases and their precursors and are substrates of a wide range of enzymes. Solution structures of purines have been debated in the literature because of the many possible tautomers and protonation states in which they can exist in solution. Substitutions on the pyrimidine and imidazole rings alter tautomerization and protonation equilibria, and as a consequence, the solution compositions and structures of closely related analogues can be significantly different. We have obtained resonance Raman spectra of 6-oxopurines: hypoxanthine, xanthine, their riboside phosphates, guanine monophosphate in the protonated and deprotonated forms with UV excitation at 260 nm. The species present in solution under different pH conditions were identified by isotopic labeling with deuterium as well as by comparison with extensive density functional theoretical calculations. At physiological pH, while N7H and N9H tautomeric forms of hypoxanthine exist in equilibrium, in xanthine, the additional carbonyl group at C2 shifts the equilibrium in favor of the N7H tautomer. The corresponding nucleotide of xanthine, xanthosine monophosphate, on the other hand, is in the anionic form (pKa 5.5). We find that Raman spectra show systematic shifts with change in the protonation state and substitution on the ring. In general, deprotonation of the neutral molecule is marked by a downshift in the observed Raman wavenumbers, and protonation is accompanied by an upshift. Introduction 6-Oxopurines play a variety of roles in cellular function, from storing genetic information as nucleobases and acting as the currency of energy exchange to signaling molecules that drive cell function. In carrying out these activities, the first step is binding to the target protein, induction of conformational changes in the protein, and in the case of nucleic acid enzymes, modification of the nucleic acid. In particular, enzymes constituting the purine metabolic pathway are postulated to involve enzymatic intermediates with purines in varying protonation states. Given their central role in cell function, purine modifying enzymes are implicated in several diseases and also identified as therapeutic targets in some cases, e.g., hypoxanthine guanine phosphoribosyl transferase in malaria and the Lesch-Nyhan Syndrome, xanthine oxidase in gout, etc. The design of inhibitors for these targets depends crucially on knowledge of the exact protonation and tautomeric state of the natural substrate. Purines in solution have a rich repertoire of possible tautomeric structures and ionization states which are potential enzyme substrates. The precise solution structures of the five natural nucleobases and their analogues have been controversial in the literature for a long time. Although many techniques have been applied in the past to resolve these structures, they have met with limited success because of the subtle changes in the physical properties of the tautomers resulting in similar spectral signatures in most commonly used spectroscopic techniques. The current study is aimed at determining the solution structures and vibrational spectra of 6-oxopurines and their anions and protonated states relevant to enzymatic catalysis by using high-resolution resonance Raman spectroscopy. Ultraviolet resonance Raman (UVRR) spectroscopy is ideally suited to study 6-oxopurines because of their large Raman cross-section * Corresponding author. Telephone: +91-80-23666160. Fax: +91-8023636462. E-mail: [email protected].

at ultraviolet wavelengths which gives rise to intense bands from in-plane vibrations.1,2 The ring vibrations of these molecules can be further enhanced with ultraviolet resonant excitation because of the electronic absorption at 260 nm. As shown in this paper, the spectra are also sensitive to the subtle differences between different tautomeric and protonated forms3-7 allowing unequivocal identification of the solution structure. Combined with isotope edited difference spectroscopy and high-level quantum chemical calculations, Raman spectroscopy is a powerful tool to study the differences between structurally closely related molecules. We report ultraviolet resonance Raman (UVRR) spectra of three 6-oxopurines as bases and/or riboside monophosphates important in cell function, hypoxanthine, xanthine, guanosine monophosphate (GMP), inosine monophosphate (IMP), and xanthosine monophosphate (XMP), in various protonation states. While the neutral forms of these bases and nucleotides have been investigated by a range of experimental and computational studies,5,6,8-27 the protonated and deprotonated forms which are directly relevant to enzymatic processes are not well characterized, a fact noted by Shugar and co-workers as well.21 In the following, we have made comprehensive assignments of the spectra of the neutral, protonated, and deprotonated species by deuterium labeling of labile hydrogen atoms. From a detailed comparison of the experimental vibrational spectra with quantum chemical calculations (DFT-B3LYP/6-31G(d,p)), the dominant tautomeric forms and protonation states at various pH values in solution have been identified. Conversely, we have compared experimental and computational spectra to evaluate the accuracy of density functional calculations in the prediction of vibrational spectra and normal mode compositions. These analyses provide a firm basis for further work on the use of vibrational spectra as reporters of interaction between 6-oxopurines and proteins. The most studied among the oxypurines is the guanine mononucleotide guanosine monophosphate (GMP), one of the

10.1021/jp9057753 CCC: $40.75  2009 American Chemical Society Published on Web 10/19/2009

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Figure 1. Structures of guanosine monophosphate: (a) neutral at pH 6.0; (b) anionic at pH 12.0; and (c) protonated at pH 1.0. Hypoxanthine: (d) neutral tautomer with hydrogen on N9 at pH 6.0; (e) neutral tautomer with hydrogen on N7 at pH 6.0; (f) deprotonated at N7/N9 at pH 10.0; (g) dianionic at pH 12.5; (h) protonated form at pH 1.5. Inosine monophosphate: (i) neutral at pH 6.0; (j) anionic (deprotonated) at pH 11; and (k) protonated at pH 1.0. Xanthine: (l) neutral tautomer with hydrogen on N7 at pH 6.0; (m) deprotonated at N3 at pH 10.0; (n) dianionic at pH 13.0. Xanthosine monophosphate: (o) neutral tautomer with hydrogen on N9 at pH 2 and (p) anionic at pH 11. Squares denote hydrogens that have been replaced with deuterium to simulate frequencies of the molecules in D2O.

four nucleotides that make up DNA. The guanine nucleobase is a 6-oxopurine with an exocyclic NH2 at the C2 position (Figure 1(a)). The N1H, amino, and carbonyl groups responsible for base pairing with cytosine in DNA are also crucial in the interaction with proteins for this molecule. Surface-enhanced Raman spectra (SERS) of the neutral, protonated, and deprotonated forms of guanine have been reported previously5,6 in the solid state along with DFT calculations. Solution state studies are confined to Raman spectra of guanosine and GMP due to the insolubility of the nucleobase itself.7,9,10,28-30 In an extensive study with many isotope-substituted analogues of guanosine, Toyama et al.10 have reliably assigned resonance Raman spectra of the neutral form. We recently reported the resonance Raman spectra of neutral and deprotonated GMP.7 In the following, we extend these studies to the protonated state of GMP and discuss the detailed assignments of deprotonated and protonated forms. Hypoxanthine and its nucleotide counterpart inosine monophosphate (IMP) are the molecules from which GMP and adenosine monophosphate (AMP) are synthesized and degraded to in cells. Apart from this, IMP occurs as a minor nucleotide in some tRNAs. Structurally, the only difference in hypoxanthine and guanine is the lack of the exocyclic NH2 in hypoxanthine. Solid state SERS spectra for hypoxanthine in various protonation states have been reported,31 and the only solution state vibrational spectrum available for hypoxanthine base is its UV resonance Raman spectrum at neutral pH.17 Theoretical calculations on hypoxanthine at various levels are available. The objective in many of these reports was to determine the most stable tautomer in solution.13,25,26,32-34 These calculations have suggested that in solution at neutral pH hypoxanthine will exist as a tautomeric mixture of two 6-oxopurine forms, with the hydrogen atom present at either the N9 (Figure 1(d)) or N7 (Figure 1(e)). Calculations reporting the vibrational modes of

hypoxanthine in its various protonation states are available,35,36 yet their assignments to experimentally obtained spectra have been carried out only for the neutral state.13 Similarly, the resonance Raman spectra of neutral IMP and inosine have also been reported, although their complete assignment has not been carried out.2,12,14,15,37 There is no experimental Raman report of the deprotonated or protonated forms of IMP. Xanthine and its nucleotide counterpart xanthosine monophosphate (XMP) are important intermediates in the metabolism of purines and their nucleotides in cells. Structurally, xanthine is different from guanine and hypoxanthine with a carbonyl group at the C2 position and containing two hydrogen atoms on the pyrimidine ring at N1H as well as N3H (Figure 1(l)). Shugar and co-workers have reviewed the structure of this nucleobase, corresponding nucleoside, and nucleotides with respect to their relevance to enzymes for which these are substrates.21 They noted many conflicting reports on the nature of the monoanionic species of xanthine and XMP. XMP is indeed different from its analogues in that it is a monoanion at physiological pH. In a later report, Callahan et al. report that xanthine in the solid state is found to be predominantly in the neutral, diketo N7H tautomer from infrared vibrational spectroscopic studies and DFT calculations.19 This difference is crucial in understanding the mechanism of catalysis of several enzymes of which the three 6-oxopurines and their nucleotides are substrates. Computational studies (HF and DFT) on xanthine have been reported recently.18 The only vibrational spectra available for xanthine are its FTIR and FT Raman spectra for which mode assignments have been made on the basis of the Wilson GF method and recently by ab initio Hartree-Fock (HF) and density functional calculations.18,20 This analysis has been carried out only for the neutral form. A large number of tautomers have been predicted for xanthine, and calculations at various levels

6-Oxopurines in Solution have been carried out to predict the most stable solution state tautomer.22,23,38,39 It is predicted that the structure of neutral xanthine is the 2,6-diketo form, while deprotonated xanthine is formed via loss of the N3 proton18-22,38,40-42 (Figure 1(m)). However, the vibrational spectra of xanthine in its various protonation states are undetermined. Experimental and Computational Methods Sample Preparation for UVRR Spectroscopy. Hypoxanthine, xanthine, inosine monophosphate disodium salt, guanosine monophosphate disodium salt, and xanthosine monophosphate disodium salt were purchased from Sigma Chemical Co. and used as supplied. Sample solutions were prepared in 10 mM stock solutions which were subsequently diluted to 500 µM for spectroscopy. For the deuterium labeled samples, the solutions were prepared in D2O and incubated overnight to allow complete H/D exchange. pH of the D2O solutions was adjusted using NaOD or DCl. Ultraviolet Resonance Raman Spectroscopy. Resonance Raman spectroscopy was carried out using excitation light of wavelength 260 nm from a tunable Ti:Sapphire laser (Indigo, Coherent Inc.). Scattered light was collected using back scattering geometry, collimated, and focused onto a monochromator (Jobin Yvon) equipped with a diffraction grating having 3600 grooves/mm and a liquid nitrogen-cooled charge-coupled device (CCD) detector of 1064 × 256 pixels. The detailed configuration has been described before.7 Typically, sample volumes of 200 µL in NMR tubes were illuminated by laser light of power ∼0.6 mW. The tubes were rotated about their axes to minimize sample degradation. The spectra obtained were calibrated using standard solvents: indene, dimethylformamide, cyclohexane, trichloroethylene, acetonitrile, isopropanol, carbon tetrachloride, and chloroform obtained from Ranchem Chemicals and Sigma Co. (HPLC grade) and used without further purification. Positions of observed bands were determined by fitting Lorentzian curves to the observed bands. All data analysis was done using the Synergy software (Jobin Yvon). Computational Methods. Quantum chemical calculations on the 6-oxopurines were carried out and compared with the frequencies obtained from Raman spectra of the nucleobases as well as the nucleotides. All the spectra obtained were in resonance with the nucleobase absorption at 260 nm leading to an enhancement of Raman spectra corresponding to the nucleobase. Thus, most of the observed bands arise from the purine ring in the nucleobases as well as nucleotides. The nucleobase served as a sufficient computational model to predict the vibrational spectra of the nucleotides as well. Density functional theoretical formalism, as implemented in Gaussian 03,43 was used to predict the optimized geometry and vibrational frequencies of all nucleobases. The B3LYP functional was applied since it has been shown previously to predict nucleobase spectra well.5-7,13 B3LYP uses the gradient corrected exchange functional by Becke44 in combination with the correlation functional of Lee, Yang, and Par.45 All the optimized structures obtained are confirmed to be stationary points in the geometry optimization procedure since none showed imaginary wavenumbers. The Gaussian 6-31G(d,p) basis set46 was employed for all the calculations. We note that of all the molecules for which the minimum energy structures were computed two molecules have positive energies for the highest occupied molecular orbitals. These are the hypoxanthine dianion (deprotonated at N7/N9 and N1) and the xanthine dianion (deprotonated at N3 and N7/N9) corresponding to Figures 1(g) and 1(n), respectively.

J. Phys. Chem. B, Vol. 113, No. 45, 2009 15103 Computed vibrational wavenumbers were scaled to improve the correspondence with experimental wavenumbers. Previous studies with this basis set have used 0.96 or 0.98 as scaling factors. We tested scaling factors from 0.96 to 1.00 in increments of 0.01 for each ionization state of each molecule and chose the factor that gave the least mean standard deviation from experimental wavenumbers. The scaling factor used for each species is given at the bottom of the corresponding table of wavenumbers. CdO stretching vibrations were scaled by a factor of 0.93 in all molecules. It is well-known that CdO vibrations are predicted with larger errors due to neglect of solvent environment. Calculations of charged molecules involved the removal or addition of hydrogen atoms from the corresponding neutral molecule according to the structures shown in Figure 1. Vibrational mode descriptions were inferred by visualizing the computed modes using the Molekel47 and Chemcraft software (http://www.chemcraftprog.com). The mode diagrams shown in Figure S1 (Supporting Information) were generated by Chemcraft. The structures shown in Figure 1 were constructed using Symyx Draw version 3.2. Potential energy distribution calculations were carried out using the software VEDA 4.0 (vibrational energy distribution analysis).48 The isotopic shifts on deuterium exchange were computed by changing the mass corresponding to exchangeable hydrogen atoms with the mass of deuterium. Structure and vibrational spectra of the ground state of each molecule were computed for the various possible tautomers of each protonation state. This was followed by a computation of the vibrational spectra at the same level of theory. The tautomer(s) present in solution at each pH were identified by comparison of the UVRR spectra of the molecule (in H2O and D2O) with the computed vibrational spectra and isotopic shifts. Results and Discussion Structures of Guanosine Monophosphate (GMP) in Deprotonated and the Protonated Forms. Several groups have obtained spectra of the neutral form of GMP with UV excitation including us.7-11,30 The guanine nucleobase is not soluble in water, hence most solution studies are on the nucleotide, GMP. UVRR spectra of GMP were obtained with 260 nm excitation at various pH values (Figure 2). At this wavelength of excitation, we expect to observe spectral enhancement of the base vibrations as explained above in the Methods section. Calculations have been carried out on the protonated and deprotonated forms. The final structures corresponding to experimentally observed states are shown in Figure 1, and the structural parameters and Mulliken charge distribution are listed in Tables S1 and S2 in the Supporting Information. The numbering shown for the purine ring of neutral GMP (Figure 1(a)) has been used for all the molecules in the rest of this text. It has been shown earlier that at high pH the proton at the N1 position of GMP is dissociated to form deprotonated GMP (GMP-), while at low pH a proton is attached at the N7 position (GMPH+).5,6 These protonation states of guanine were identified by analyses of UVRR spectra of GMP at various pH values in H2O and D2O as discussed below. The structural parameters (Table S1, Supporting Information) are in good agreement with the previously reported calculations where available.49 Small discrepancies can be accounted for by the different basis sets used. Ionization Products of GMP in Water and D2O. Spectra of GMP at different pH values were recorded and are found to be composed of three distinct species. Figure 2 shows the UVRR spectra of GMP at pH 1, 6, and 12 in water and D2O. As mentioned earlier, the observed bands are expected to be

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Figure 2. Resonance Raman spectra of GMP: (a) neutral in H2O, pH 6.0; (b) neutral in D2O, pH 6.0; (c) deprotonated in H2O, pH 12.0; (d) deprotonated in D2O, pH 12.0; (e) protonated in H2O, pH 1.0; and (f) protonated in D2O, pH 1.0; obtained with 260 nm excitation. The box represents a 10× magnification of the spectrum for a clear depiction of low-intensity modes.

predominantly from the nucleobase at this wavelength of Raman excitation (260 nm). From comparison with data from several groups5,6,9,30 including that from Toyama et al.,28,29 the spectrum at pH 6 is undoubtedly identified as that of neutral GMP. Changes in the spectrum at higher pH indicate the formation of a new species via deprotonation which is identified as deprotonated GMP (GMP-) based on the following arguments. Observed spectra were compared with computed spectra of GMP- (Table 1) formed by removing the proton at N1 and found in agreement. The isotopic shifts in Raman wavenumbers of the experimental spectrum were compared with computed isotope-induced shifts that corroborate the assignment. Table 1 lists the observed UVRR bands in H2O and D2O and their assignment to computed frequencies and vibrational modes of GMP-. The normal modes corresponding to the first few highwavenumber bands are depicted in Figure S1A (Supporting Information). Although we have previously published the spectrum of GMP at high pH,7 a detailed mode analysis for the same was not reported. The spectrum at pH 1 is expected to correspond to the protonated form of GMP (GMPH+). The spectrum of GMP at low pH was compared with computed spectra of GMPH+ with the hydrogen on N7 (Figure 1(c)) and its isotope-labeled analogue (Table 2). The comparison supports the assignment of the observed species as GMPH+. In the following, we discuss the key modes in the three species. Carbonyl Stretch. The band with the highest Raman shift in the spectrum of neutral GMP at 1682 cm-1 in Figure 2(a) is assigned to the C6dO stretch in accordance with previous literature.5-7,10 This band moves down to 1663 cm-1 in D2O

Gogia et al. reflecting the contribution from N1-H bending as reported earlier.5-7,10 The computed vibrational mode for this band correctly reproduces this contribution of N1-H to the predominantly CdO stretching mode. On deprotonation, this mode appears at 1592 cm-1 indicating that the observed weakening in this mode is due to reduction of the double bond character in C6dO. A consequence of the deprotonation is an increase in the negative charge on the oxygen atom. The hydrogen at the N1 position is absent at high pH leaving no exchangeable proton available as evidenced by the absence of an isotopeinduced shift in this band. At low pH, this low-intensity band shows higher Raman shift in comparison to the neutral form and appears at 1707 cm-1. Calculations of the protonated form of GMP show that the partial negative charge on the oxygen atom at the C6 position is reduced leading to a decrease in the C6dO bond length (Table S1 and S2, Supporting Information) by ∼0.01 Å. Triene Stretch. The triene stretch at 1602 cm-1 in GMP involves C2N3-C4C5-N7C8 stretching coupled to the scissoring motion of the NH2 group.6,7,10 The NH2 coupling in this mode leads to a large downshift of 33 cm-1 in this band in D2O. In GMP-, this band appears at 1570 cm-1. Calculations predict a slightly altered mode composition with reduced contribution from N2-H2a (Table 1) and as a consequence a small downshift of 3 cm-1 upon deuterium labeling. However, experiments show a slight upshift to 1575 cm-1 perhaps due to the decoupling of N2-H2a from this mode upon ionization. In GMPH+, this band appears at 1611 cm-1. Unlike in the neutral form, in GMPH+ the N-H bend is decoupled from the triene stretch as seen from the small shift upon H/D exchange. This band has high intensity in all three states, GMP, GMP-, and GMPH+, and can be used as a probe of ring structure and protonation state. We note also that in the neutral form the relative intensity of this band increases in D2O, while in the deprotonated and protonated forms it remains similar in both solvents. In Plane Pyrimidine Ring Distortion. The mode observed at 1576 cm-1 is the second most intense band in the UVRR spectrum of neutral GMP and is assigned to C2N2 and pyrimidine ring stretching coupled to NH2 scissors along with an N1-H bend.7 Deuterium labeling causes a four wavenumber upshift in the position of this band. Computed mode composition indicates that NH2 scissoring motion decouples from the ring motion on H/D exchange to ND2. Removal of a proton at pH 12 is predicted by DFT calculations to lower the Raman shift considerably to 1392 cm-1. The corresponding experimental band is observed at 1359 cm-1. Since the proton on N1 is absent in GMP-, the effect of NH2 decoupling is observed upon H/D exchange as a large upshift of 24 cm-1 in D2O. The larger upshift observed on isotope labeling of GMP- supports the prediction of a contribution from the N1-H bend in the corresponding mode in neutral GMP. In the pH 1.5 spectrum of GMP in water, this mode is not observed, while the band at 1575 cm-1 in the deuterated GMP spectrum at this pH has been assigned to this mode. Coupled Pyrimidine and Imidazole Ring Vibrations. The low-intensity band at 1537 cm-1 is assigned to the pyrimidine and imidazole ring vibrations coupled to the C8-H bending vibration.7 H/D exchange does not affect the position of this band as seen in the spectrum, although a small isotopic shift of 6 cm-1 is expected from DFT calculations. This indicates a very weak coupling to N1-H and NH2. In GMP-, the mode weakens to 1512 cm-1. Calculations predict an isotope labeling induced downshift of 5 cm-1, while the experimental spectrum shows a

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TABLE 1: Experimental Resonance Raman Shifts (λexc ) 260 nm) of Deprotonated GMP at pH ) 12.0 in Water and D2O and Computed (B3LYP/6-31G(d,p)) Vibrational Wavenumbers of Deprotonated Guanine (Figure 1(b)) with Mode Assignmentsa UVRR DFTb in H2O NH2 1592 1570

assignment

PED %

UVRR DFTb in D2O ND2

1603 1592

St C6O+St C4C5 St N3C4-C4C5-N1C2+ Be N9H+N2H2a

78% St OC -10% St NC 28% St NC -17% Be CNC

1592 1575

1603 1589

1562

Sci NH2

67% Be HNH

1270

1180

1512

1508

14% St NC -15% St NC -17% Be NCC

1516

1503

1474

1477

1473

1405

1401

1394

1359

1392

42% St NC -14% Be HCN -10% St NC -21% St NC 21% Be CNC 11% Be CNC -23% St NC -17% St NC 17% St NC

1468

1405

St N1C2-C4N9-C5C6+ +Be C8H+ N9H+N2H2b St N7C8-N3C4-N1C2+ Be C8H St C4C5+N1C2+N7C8+ rock NH2

1383

1407

1343

1326

-26% St NC -11% Be CNC 32% Be HNC

1340

1306

1277

12% St NC 31% Be HCN

1258 1209

1257

1188

1166 1124

a

St C2N2+C6N1+ C8N9+Be N9H+Sci NH2 Be N9H+C8H+St C8N9+C2N2+C2N2N3C4 Be C8H+Im Bre+St C5C6 N9-C1′ Be C8H+N9H+St C5N7-N3C4 rock NH2+St N1C6+C2N2 Be N9H+C8H+St C4N9-C5N7+ C6N1+C2N2

-12% St NC -15% St NC 40% St NC -15% Be HCN -10% St NC 22% St NC -31% Be HNC 12% St NC -10% St NC -14% Be CNC 20% Be HCN

assignment

PED %

St C6O St N3C4-C4C5-N1C2+Be N9H rock NH2+St N1C6-C2N2 St N1C2+C4C5-N7C8+ Be C8H+N9H

78% St OC 29% St NC -18% Be CNC

St N7C8+N3C4-N1C2+ Be C8H St C4C5-N1C2+N7C8+ Be N9H St C2N2+N1C2-C2N3+ Be N9H

31% St NC 12% St NC -11% Be HCN -21% St NC -20% St NC 16% Be CNC 13% Be CNC -14% St NC -24% St NC 28% St NC

1329

Be N9H+St C2N2+C8N9-C4C5

-26% St NC 36% Be HNC

1300

1277


1258 1191

1258

Be C8H+Im Bre+St C5C6+C2N2 N9-C1′ Be C8H+St C5N7-N3C4

1165

1098 1124

rock NH2+St N1C6+C2N2 Be N9H+C8H+St C4N9-N3C4-C5N7

-17% St NC 37% Be DND -21% St NC 17% Be NCC

-10% St NC -14% St NC 46% St NC -10% Be HCN 33% St NC 21% Be DND -16% Be CNC 18% Be HCN 10% Be CNC

Abbreviations: St, stretch; Be, bending; Sci, Scissors; Im, imidazole; Bre, breathe. b Scaling factor used: 0.97.

shift in the opposite direction to 1516 cm-1. Since the calculations are carried out on the guanine molecule, the computed mode contains contribution from the N9-H bend which is absent in GMP-. This explains the observed reversal in shift direction as compared with theory. On protonation, the mode up shifts to 1561 cm-1 reflecting an overall strengthening of the C-C and C-N bonds in the ring. As observed for other ring vibrations in GMPH+, the band has much lower intensity. Imidazole N7-C8 Stretching Mode. The spectra of GMP and GMP- have their highest intensity bands at 1486 and 1474 cm-1, respectively. Following the preceding assignment of neutral guanosine10 and our computational results, these bands are assigned to N7C8 and pyrimidine ring stretching coupled to C8-H.7 The computed PED shows 41% contribution from N-C stretching coordinates. In accordance with the same report, a downshift of 7 units is observed in this mode in D2O. A comparable downshift of 7 and 6 cm-1 is observed in GMP and GMP-, respectively, suggesting that this mode has a larger contribution from the exocyclic NH2 rather than N-H. In the GMPH+ spectrum, the mode appears as a low-intensity band at 1538 cm-1 that downshifts to appear at 1514 cm-1 on deuteration. A detailed assignment of the remaining bands of lower intensities of deprotonated and protonated GMP is provided in Tables 1 and 2. Hypoxanthine (Hx) and Inosine Monophosphate (IMP). Protonated states of hypoxanthine and IMP observed in the experimental UVRR spectra are shown in Figure 1(d)-(k). Computed bond distances and Mullikan atomic charges corresponding to these structures are listed in Tables S3 and S4

(Supporting Information), respectively. Ultraviolet resonance Raman spectra of hypoxanthine and IMP, in H2O and D2O, are shown in Figures 3 and 4, respectively. The species contributing to the observed spectra at each pH were identified based on the following arguments. Neutral Forms of Hypoxanthine and Inosine Monophosphate. Hx has been reported to exist as a mixture of two tautomers, one with H7 (Figure 2(e)) and another with H9 (Figure 2(d)) at pH 7.13 At first glance, it can be observed that the UVRR spectrum of neutral hypoxanthine has many more bands than expected for a single tautomer (Figure 3(a) and Table 3). Since IMP has a sugar attached at the N9 site, it correlates with the tautomer of hypoxanthine with the hydrogen on N9 (Figure 1(i)). By comparing the observed hypoxanthine spectrum with that of IMP and computed vibrational spectra of the tautomers, we have identified the bands corresponding to the N7H and/or N9H tautomers as summarized in Table 3. These assignments have been found to agree with previous assignments made by Fernandez et al.13 for vibrational spectra of neutral hypoxanthine (N7H/N9H) tautomers. We note that several bands are common to the two tautomers. Deprotonated IMP (IMPs). On raising pH, IMP must necessarily lose the only available proton on N1 (Figure 1(i)). Thus, IMP- must have the structure given in Figure 2(j). The spectrum of IMP at pH 11.0 in H2O and D2O is identical (Figures 4(c) and 4(d)) lending further proof that at this pH the observed Hx species does not contain any protons available for exchange with deuterium. The normal mode assignments of the observed IMP- bands are given in Table 4.

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TABLE 2: Experimental Resonance Raman Shifts (λexc ) 260 nm) of GMP at pH ) 1.5 in Water and D2O and Computed (B3LYP/6-31G(d,p)) Vibrational Wavenumbers of Protonated Guanine (Figure 1(c)) with Mode Assignmentsa UVRR DFTb in H2O N1H, N7H, NH2 1707

1611

assignment

PED %

assignment

1717

St C6O+Be N1H

79% St OC

1689

1710

St C6O

1659

Sci NH2+St C2N2+Be N1H St N3C4-C4C5+C8N9+ Be N9H+N7H Sci NH2+St C2N3-C4C5-C6N1+Be N1H+N7H Sci NH2+Be N1H+N7H+C8H+St N1C2+C4N9-C5N7 St N7C8+Be C8H+N7H

-10% St NC 33% St NC 41% Be HNH -38% St CC 29% St NC

1235

1234

1609

1636

22% St NC 29% Be HNH

1575

1602

Sci NH2+Be N1H+ C2N2-C6N1 St N3C4-C4C5+ Be N9H St C2N2+C2N3-C5C6+ Sci NH2+Be N9H

-21% Be HNH 12% Be NCC

1552

1551

42% St NC -15% St NC -20% Be HCN

1514

1524

1480 1421

1414

1389

1430

1343

1358

1638 1585

1561

1558

1538

1537

1487 1470

1468

1411

1422

1357

1365 1346

from neutral St C8N9+N7C8+N1C2+ Be N7H+N9H+N1H+ C8H St C4C5-C8N9+N1C2+ Be N9H+N2H2b St C5N7-C4N9+ Be N7H+C8H+N9H Be N1H+N2H2b+St C2N2 Be C8H+N9H+N7H+ N1H+St C5C6-N1C2

14%St NC -11% St NC -14% St NC 18% Be HNC -17%St CC -18% St NC 22% Be HNC -13%St CC -23% St NC 32% St NC -19% St NC 52% Be HNC 13% St NC -12% Be HNC 17% Be HNC 13% Be HCN 24% St NC 28% Be HNC 10% Be CNC

1295

1300

1259

1208

Be N7H+C8H+N9H+ purine ring torsion

1188

1134

1173

1118

Be N9H+N7H+C8H+Im 13% St NC 26% St NC Bre+St C5C6+rock NH2 -12% St NC 15% Be HNC Be C8H+N9H+St C6N1 -19% St NC 34% Be HCN

a

UVRR DFTb in D2O N1D, N7D, ND2

1318 1065 1271

1283

1215

1165

1165

1133

1119

1111

St N1C2+C5N7-C4N9+Be C8H St N7C8+Be C8H+ N7H+N9H from neutral St C8N9-C4C5-N7C8+ Be N9H+C8H St C4C5-N7C8+N1C2+ Be N9H+C8H St C5N7-C4N9+ Be C8H+N7H From neutral Be N1H+Sci NH2+St C2N2 Be C8H+N9H+St C5C6

PED %

80% St OC -10% Be CNC 11% St NC 41% Be DND -18% Be DNC -35% St CC 31% St NC -28% St NC 42% St NC -11% St CC -11% Be CNC 21% Be NCC 38% St NC -13% St NC 21% Be HCN -12%St CC -12%St NC 21% St NC 13% Be CCN -31% St NC 29% Be HNC -13%St CC -20% St NC 38% St NC 20% Be DND 10% Be DNC 14% Be OCN -25% St NC -11% Be HNC 20% Be HCN

-12% St NC 19% Be NCN -22% Be HNC 11% Be CNC Be C8H+N9H+Im Bre+ 12% St NC -19% St NC St C5C6+Sci NH2 14% Be HCN Be N9H+purine ring torsion

Be C8H+N9H+St C6N1+ Sci NH2


Abbreviations: St: stretch; Be: bending; Sci: Scissors; Im: imidazole; Bre: breathe. b Scaling factor used: 0.98.

Deprotonated Hypoxanthine (Hx-). The structure of neutral hypoxanthine contains exchangeable protons at the N1 and N9/ N7 positions. If hypoxanthine were to form Hx- via deprotonation at the N1 site, the resulting spectrum is expected to be similar to that of IMP- at pH 11.0. A preliminary comparison of the spectra of Hx- and IMP- suggests that this is not the case (Figures 3(c) and 4(c)). The observed spectrum implies that the structure of Hx- does not correspond to the one formed via loss of the N1H proton. Further, the number of bands observed in this spectrum tally only if a single tautomer is present in solution, while a loss of the proton on N1 could result in two tautomers of Hx-. We conclude that the structure of hypoxanthine at high pH is an anion formed via the loss of the protons on N7/N9 (Figure 1(f)) from the corresponding tautomers of neutral Hx. The experimental and computed isotopic shifts upon H/D exchange of the N1 proton in the Hx- tautomer are found to be in agreement (Table 5) confirming the assignment of the structure of hypoxanthine at high pH to that shown in Figure 1(f). Double Deprotonated Hypoxanthine (Hx2-). The UVRR spectra of hypoxanthine obtained at pH 12.5 are identical in H2O and D2O (Figures 3(e) and 3(f)) thus implying that hypoxanthine does not contain any exchangeable protons at this pH. This is only possible if both the protons viz. at N1 and

N7/N9 positions are abstracted. Thus, the structure of double deprotonated hypoxanthine is the one shown in Figure 1(g). The assignment of the observed spectrum is given in Table 6. Protonated Hypoxanthine (HxH+) and Inosine Monophosphate (IMPH+). HxH+ and the purine ring in IMPH+ are in the same protonation state as evidenced from the similarity in the UVRR spectra obtained for the two molecules (Figures 3(g) and 4(e)). The species of Hx and IMP present at low pH, viz., HxH+ and IMPH+, were identified via comparison of the shifts obtained from DFT calculations with those obtained experimentally in water and D2O (Table 7). On the basis of this analysis, the HxH+ and IMPH+ are ascribed to structures with protons on the N1, N7, and N9 nitrogen atoms in hypoxanthine and at the N1 and N7 nitrogen atoms in IMP. Previous theoretical studies have predicted the formation of this tautomer on protonation.35 As has already been stated, the spectrum of neutral hypoxanthine corresponds to a mixture of the N9H and N7H tautomers, with the N9H tautomer being dominant in solution. The observed bands have been assigned to the N9H or N7H tautomers based on the presence or absence of a corresponding band in the IMP spectrum and theoretically predicted spectrum of the tautomer. The assignments for hypoxanthine and IMP and their different protonation states are given in Tables 3-7,



Figure 3. Resonance Raman spectra of hypoxanthine: (a) neutral in H2O, pH 6.0; (b) neutral in D2O, pD 6.0; (c) deprotonated in H2O, pH 10.0; (d) deprotonated in D2O, pD 10.0; (e) double deprotonated in H2O, pH 12.5; (f) double deprotonated in D2O, pD 12.5; (g) protonated in H2O, pH 1.5; and (h) protonated, in D2O, pD 1.5; obtained with 260 nm excitation.

Figure 4. Resonance Raman spectra of IMP (a) neutral in H2O, pH 6.0; (b) neutral in D2O, pD 6.0; (c) deprotonated in H2O, pH 11.0; (d) deprotonated in D2O, pD 11.0; (e) protonated in H2O, pH 1.0; and (f) protonated in D2O, pD 1.0; obtained with 260 nm excitation. Inset shows fitting and 10× enlarged view of low-intensity IMP deprotonated bands in the region 1200-1300 cm-1.

and a few representative normal modes are depicted in Figures S1B and S1C (Supporting Information). Assignments of the intense bands that are diagnostic of the ring structure and protonation states are discussed below. Carbonyl Stretch. The CdO stretch mode at 1694 cm-1 in Hx has low intensity and is weakly coupled to the N1-H bend and C5C6 stretch as predicted by DFT (Table 3). This band downshifts by 17 cm-1 in D2O confirming that N1-H is coupled to this mode. In IMP, the band appears at 1690 cm-1 and downshifts by 17 cm-1 in D2O indicating a normal mode composition similar to that of the nucleobase. In Hx-, electron density on the oxygen atom at the C6 position (Table S4, Supporting Information) increases resulting in lesser double bond character for the carbonyl bond, weakening its stretching frequency. Thus, in the UVRR spectrum of hypoxanthine at pH 10, this band appears at 1585 and 1566 cm-1 in water and D2O, respectively (Table 5). The C6dO stretching mode in the IMP- spectrum appears at 1594 cm-1, much lower than in Hx and IMP. Although IMP- is deprotonated at N1 and Hx- at N7/N9 (Figures 1(j) and 1(f), respectively), DFT calculations predict a similar change on the charge and bond length of C6dO in the two cases (Tables S3 and S4, Supporting Information). Second deprotonation of hypoxanthine to Hx2- is predicted to exhibit further weakening in the C6dO stretch mode due to higher negative charge on this molecule as compared to Hx- and appears at 1580 cm-1 (Table 6). The CdO stretch/N1-H bend does not show isotopic shift in the dianion because no exchangeable protons remain in the molecule. Thus, while in Hx and Hx- there is a downshift

in the band position in D2O with respect to that in water, the band remains at 1580 cm-1 in Hx2- in both solutions. Protonation of hypoxanthine and IMP leads to opposite effects on the C6dO stretching mode as compared to the neutral forms since the partial negative charge on carbon is reduced in this case (Table S4, Supporting Information). The bands observed at 1715 and 1717 cm-1 in protonated hypoxanthine and IMP UVRR spectra are assigned to this mode (Table 7). Thus, the carbonyl mode is an excellent marker of the protonation state of both the nucleobase and the nucleotide of hypoxanthine. Pyrimidine Ring Stretching Mode. The band at 1596 cm-1 has been assigned to pyrimidine ring stretching coupled to C8-H, N1-H, and N9-H/N7-H bending corroborated by the observed downshift of 18 cm-1 in D2O. In Hx- and Hx2- this band appears at 1551 and 1526 cm-1, respectively (Tables 5 and 6). No downshift is observed in this mode on deuterium labeling in Hx- in contrast with the computed mode composition which contains contribution from N1-H. In neutral and deprotonated IMP, this mode appears at 1593 and 1561 cm-1, respectively (Tables 3 and 4), and is one of the three most intense bands in the spectrum. In protonated hypoxanthine and IMP, the band is present at 1619 and 1614 cm-1, respectively, as given in Table 7. This band can be used as a marker for protonation/deprotonation on hypoxanthine and IMP since it has high intensity and undergoes large downshifts as the pH is increased. Triene Stretching Mode. This band at 1575 cm-1 is assigned to N3C4-C4C5-N7C8 stretching coupled to N9-H and C8-H bending. In D2O, this band downshifts to appear at 1552 cm-1 in accordance with DFT calculations. The effect of having a

DFTb

1593

1554

1596

1575

1468

1464

1381

1396

1259

1270

a

1182

1290

1348

1367

1397

1426

1471

1521

1585

1624

1705

Be C8H+N7H+St C4N9-C5C6 sugar Be C8H+N1H+N9H/ N7H+St N1C6-C5N7C4N9

Be C8H+N9H+C2H+ N1H+St N3C4-C5N7

Be C2H+N7H+C8H+Im Bre

Be C2H+C8H+N1H+St C4N7-N7C8 Be C8H+N9H+C2H+Im Bre

Be N1H+C2H+N7H+ C8H+St N1C2+ N7C8-C4C5 Be N9H+C2H+N1H+ C8H+St C8N9-C4C5-C4N7 Be C2H+N1H+St N3C4-C5N7 St C4N9-N7C8-C5C6+Be N7H+C8H+N1H

Be N1H+C2H+St C2N3-N3C4-C4C5

St C4C5-N7C8+C2N3+Be N1H+C8H Be N1H+C8H+N9H+St N7C8-C4C5+N1C2 St C5N7-C8N9-N9C4+Be C8H+N1H

St C4C5-N7C8+C2N3+Be C8H+C2H

St C2N3-C4C5-C5C6+Be C2H+N1H+N9H/N7H St C4C5-N3C4+N7C8+Be N9H+C8H St C2N3-C4C5-N7C8+ Be N7H+C8H+N1H

St C6dO+Be N1H

1495

15% St NC -12% St NC -16% St NC 10% Be CNC 17% Be CNC 30% St NC 21% Be CNC

sugar 11% St NC 19% Be HCN


15% St NC 15% St NC -16% St NC -29% Be HCN 16% St NC -10% St NC 33% Be HCN

-10% St NC 10% Be HCN -13% Be HCN 10% Be CNC


-17% St NC -10% Be CNC 20% Be HNC -17% Be HCN 13% St NC -10% St NC 42% Be HCN -11% St NC -21% St NC 21% St NC 11% Be HNC

1167

1268

1256

1319

1352

1388

1372

1332

1458

1427

1552

1578

1677

1536

-15% St NC -11% St NC -23% Be HNC 24% St NC 19% St NC -12% Be CNC -16% Be HCN 17% St NC 10% St NC -10% Be CNC -17% Be HNC 18% Be HCN -15% St NC 15% Be HNC 39% Be HNC

DFTb

1193

1309

1363

1383

1334

1428

1506

1549

1580

1673

1163

1274

1367

1329

1403

1303

1452

1535

1525

1624

1677

1158

1246

1322

1365

1384

1335

1451

1514

1566

1617

1698

Hyp IMP in D2O N1D,N7D N1D,N9D

UVRR

76% St OC -12% Be CNC 55% St NC -12% Be HCN -22% St NC 12% St NC 15% Be HNC 13% St NC 15% Be HNC

PED %

Abbreviations: St: stretch; Be: Bending; Im: imidazole; Py: pyrimidine; Bre: breathe. b Scaling factor used: 0.99.

1196

1288

1341

1393

1405

1426

1461

1536

1547

1634

1684

assignment

Be C8H+St C5N7-C2N3C6N1+C4N9

Be C8H+N7H+St N7C8N9C4-C5C6

Be C8H+N9H+C2H+Py ring+St N7C8

Be C8H+C2H+Im Bre+St N1C6-C2N3 St N1C2+Im Bre+Be N1H+C8H+C2H

St C5N7-C4N9+C5C6+Be C2H+N9H St C2H+C8H+N9H+Im Bre

Be C2H+C8H+St C2N3-C4C5-C5N7 St C5N7-C4N9-C8N9+Be C2H+C8H

Be C2H+N9H+C8H+St C8N9+C2N3

Be C2H+C8H+N1H+ N9H+St N1C2+N7C8C8N9 Be C8H+C2H+N1H+St N1C2+Im Bre

St C4C5-C5N7+C2N3+Be C8H St C4C5-N7C8-C8N9+Be C2H+C8H St C5N7-C8N9-N9C4+Be C8H

St C2N3-C4C5-C5C6C6N1+Be C2H(+N1H) St C4C5-N3C4-N7C8+Be C8H St C4C5-C5N7-C8N9 +C2N3+Be C8H+C2H +N7H St C4C5-N3C4+Be C2H+ C8H

St C6dO+C6C5+Be N1H

assignment

-11% St NC 26% Be HCN 17% Be CNC

-11% St NC 18% St NC -25% Be HCN -10% St NC -18% St NC 30% St NC -13% Be HCN -11% St NC 12% St NC 28% St NC -12% Be HCN -15% St NC 22% St NC -23% Be HCN

10% St NC 45% Be HCN -11% Be CNC -12% St NC -11% St NC -16% Be HCN 12% Be HCN 23% St NC 27% St NC -15% Be HCN -13% St NC -10% St NC 28% Be HCN

14% St NC 36% Be HCN -25% Be HNC

35% St NC -11% St NC -16% Be DNC

14% St NC 25% St NC 14% St NC 13% Be HCN

78% St OC -10% Be CNC 55% St NC -11% Be HCN 26% St NC -10% St NC -12% Be CNC -12% St NC 14% St NC -11% St NC 12% Be HCN 11%St NC -19% St NC 16% Be CNC 20% Be CNC 40% St NC -10% Be HCN 19% Be CNC -24% St NC 20% Be CNC -11% Be NCC 21% St NC 25% St NC 13% Be CNC

PED %


1219

1244 1217

1322

1338

1287

1350

1367

1382

1421

1424

1452

1515

1512

1541

1690

1694

Hyp IMP in H2O N1H,N7H N1H,N9H

UVRR

TABLE 3: Experimental Resonance Raman Shifts (λexc ) 260 nm) at pH ) 6.0 in Neutral Hypoxanthine and IMP in Water and D2O and Computed (B3LYP/6-31G(d,p)) Vibrational Wavenumbers of Neutral Hypoxanthine (H7/H9 Tautomers) (Figures 2(a) and 2(b)) with Mode Assignmentsa

15108 Gogia et al.



TABLE 4: Experimental Resonance Raman Shifts (λexc ) 260 nm) of IMP at pH ) 11.0 in Water and D2O and Computed (B3LYP/6-31G(d,p)) Vibrational Wavenumbers of Deprotonated Hypoxanthine (Figure 2(g)) with Mode Assignmentsa UVRR in H2O /D2O

DFTb

assignment

1594 1561 1512

1602 1593 1521

1473 1424 1372 1336

1484 1423 1372 1360

St C6O St C4C5-C2N3+Be N9H+C2H St C4C5-N7C8-C2N3-N1C2-C5C6+Be C8H+C2H+N9H St C2N3-C4C5-N7C8+Be C8H+C2H Be C2H+St C4C5-C4N9-C5N7+N1C2 Be C2H+N9H+St C8N9-C4N3-C2N1-N1C6 Be C2H+N9H+St N1C2+C4C5-C4N9

1302 1270 1247

1312 1267 1230

Be C8H+C2H+Im Bre+St N1C6-C2N1 St C5N7-N3C4-C2N3+Be C8H St N1C2-N3C4+N7C8+Be C8H+N9H+C2H

a

PED % 77% St OC -11% Be CNC 25% St NC -16% Be CNC -24% St NC 12% St NC 10% Be NCC 29% St NC 13% St NC -10% Be HCN 14% St NC -19% Be CNC 28% Be HCN 39% St NC -11% St NC -10% Be HNC -25% Be HCN -11% St NC -16% St NC 10% St NC 24% Be HNC -14% Be HCN -15% St NC -25% Be HCN -15% St NC 52% St NC 24% St NC 28% Be HCN

Abbreviations: St: stretch; Be: Bending; Im: imidazole; Bre: breathe. b Scaling factor used: 0.98.

TABLE 5: Experimental Resonance Raman Shifts (λexc ) 260 nm) at pH ) 10.0 in Water and D2O and Computed (B3LYP/ 6-31G(d,p)) Vibrational Wavenumbers of Deprotonated Hypoxanthine (Figure 2(c)) with Mode Assignmentsa UVRR DFTb in H2O N1H

assignment

1585

1646

St C6O+Be N1H

1551

1618

St C2N3+Be C2H+N1H

1511

1515

from dianion St C4C5-N7C8-N9C4+ Be C8H+C2H

1469

1466

1444 1430

1417

1397

1385

St C4N9-C5N7-C5C6+ Be C2H+N1H

1379

1365

1334

1347

1271

1274

1248 1233

1236

1178

1170

Be C2H+C8H+N1H+ St C4N9-C5C6 St C4C5-C6N1+N7C8+ Be C8H+C2H+N1H St N7C8+Be C8H+ N1H from dianion St C8N9-C5N7+Be C8H+N1H+C2H Be C8H+N1H+St C5N7-C4N9-C6N1

a

St C2N3-C4C5-N1C2+ C6O+Be N1H+C2H from dianion Be N1H+C2H+C8H+St N1C2-N3C4-C5N7

PED % 64% St OC -13% Be CNC 63% St NC -12% Be HCN -14% St NC 24% St NC 12% St NC -13% St NC -18% Be HCN 11% St OC 21% St NC -14% Be CNC

UVRR DFTb in D2O N1D 1566

1639

St C6O+C5C6

1551

1609

St C2N3+Be C2H

1526 1508

1515

from dianion St C4C5-N7C8-N9C4+ Be C8H+C2H

1467

1459

1444 12% St NC 12% St NC -31% Be HNC -12% Be HCN 38% St NC -15% Be CNC 11% Be NCN 45% Be HCN -15% Be CNC 23% St NC 26% Be HCN -13% Be CNC

49% St NC -11% Be NCN 10% St NC 30% Be HCN 19% Be CNC

assignment

1313

St C4C5-C2N3+Be C2H+C8H from dianion St N1C2-C5C6-N7C8+Be C8H+C2H+N1H Be C2H+C8H+St C4C5-N3C4-N7C8-N9C4

1380

1389

1364

1354

Be C2H+C8H+St C4N9

1333

1379

1259

1265

1249 1207

1220

1150

1166

St C5N7-C4N9-C5C6+ Be C2H+C8H St N7C8-N7C8+Be C8H+N1H+C2H from dianion St C8N9-C5N7+Be C8H+N1H Be C8H+St C5N7-C4N9-C6N1N3C4

PED % 64% St OC -14% Be CNC 63% St NC -11% Be HCN -14% St NC 24% St NC 11% St NC -12% St NC -19% Be HCN -27% St NC 14% Be CNC 30% St NC -14% St NC 12% Be HCN -12% Be CNC 18% St NC 16% St NC 13% Be HCN -16% Be CNC -20% St NC 36% Be HCN -12% Be HCN 30% St NC -16% Be HCN -13% Be HCN 44% St NC 15% St NC -18% Be NCN 42% St NC 13% St NC 12% Be HCN 27% Be HCN 21% Be CNC

Abbreviations: St: stretch; Be: Bending. b Scaling factor used: 0.99.

ribose moeity attached at the N9 position would encumber any vibration involving this atom. The shift at which this mode occurs should be comparable to the vibrational frequency obtained for Hx deuterated at N9. For this reason, the band observed at 1554 cm-1 in neutral IMP has been assigned to this mode. This is the most intense band observed in the UVRR spectrum of neutral IMP. Since ring modes are expected to be maximally resonance enhanced at this Raman excitation wavelength, the high intensity in this mode corroborates its assignment to a ring mode. In Hx- and HxH+, the mode appears at 1511 and 1589 cm-1, respectively. Spectra of corresponding deprotonated and protonated forms of IMP contain this mode at 1512 and 1576 cm-1, respectively. This mode is observed to be high intensity in neutral and protonated forms of both IMP and hypoxanthine, while it loses intensity upon loss of a proton.

C-H Bending and Imidazole Ring Stretch. Spectra of neutral hypoxanthine and IMP contain intense bands at 1464 (Hx) and 1468 cm-1 (IMP) arising from a vibrational mode comprised of N1-H and C8-H bending coupled to N7C8-C4C5 and N1C2. Both downshift in D2O to appear at 1427 and 1428 cm-1, respectively. This is the second most intense band in both these spectra and a potential diagnostic mode of interaction of the ring hydrogen atoms with their solvent or protein environment through interaction of the N1 hydrogen atom or basepairing. Corresponding bands in protonated and deprotonated spectra of hypoxanthine appear at 1503 and 1430 cm-1, while in protonated IMP the mode is at 1492 cm-1. The mode is absent in IMP- and Hx2-. The band at 1452 cm-1 has no counterpart in the UVRR spectrum of neutral IMP which suggests that it arises from the

15110


Gogia et al.

TABLE 6: Experimental Resonance Raman Shifts (λexc ) 260 nm) at pH ) 12.5 in Water and D2O and Computed (B3LYP/ 6-31G(d,p)) Vibrational Wavenumbers of Double Deprotonated Hypoxanthine (Figure 2(d)) with Mode Assignmentsa UVRR in H2O /D2O

DFTb

assignment

PED %

1580 1526 1444 1390 1338 1328 1309 1248

1585 1538 1505 1443 1397 1351 1313 1303 1237

St C6O St C2N3-C4C5-C6N1+C6O+Be C2H+C8H St N3C4-C5N7-C8N9+Be C8H+C2H St C4C5-N7C8-C4N9+Be C8H+C2H Be C2H+C8H+St C4C5-C5N7-C8N9 Be C2H+St N1C2+C4C5-C4N9 Be C8H+C2H+St C4C5-C6N1 St N1C2-C2N3-C4N9-C5N7+Be C8H+C2H Be C8H+St N7C8+N1C2

1203 1156

1209 1140

Be C8H+St C8N9 St N3C4-C5N7-C4N9-C6N1+Be C8H

65% St OC -12% Be CNC 26% St NC -13% St NC -21% Be HCN 11% St NC -14% St NC -18% St NC 12% St NC 15% Be HCN 12% St NC 11% St NC -15% St NC 24% Be CNC 12% Be HCN 42% Be HCN -20% Be CNC 17% St NC 22% St NC -18% Be HCN -17% Be CNC -11% St NC 27% St NC 10% St NC -11% Be CNC 17% St NC 20% St NC 20% St NC 39% St NC 11% St NC -13% St NC -12% Be HCN -12% Be NCN 56% St NC 26% Be HCN -15% St NC 17% Be HCN -10% Be CNC 25% Be CNC

a

Abbreviations: St: stretch; Be: Bending. b Scaling factor used: 0.99.

N7H tautomer and is found to upshift in D2O by 6 cm-1. Analogous to the 1464 cm-1 mode in Hx (N9H), the internal coordinates involved in this normal mode are expected to be imidazole ring stretching. While a downshift is predicted in this mode on deuterium labeling, a slight upshift is observed. This upshift points to an uncoupling of N1-H from this mode in D2O which is not reproduced well by DFT calculations. The loss in intensity observed in this mode on going from H2O to D2O is intriguing and suggests a reorganization of the normal mode composition of the deuterium labeled molecule. C-H Bending Mode. The Hx band at 1396 cm-1 has a large isotope-dependent downshift of 24 cm-1 in D2O and is assigned to N9-H, C2-H, and N1-H bending coupled to imidazole ring stretching. In neutral IMP, this band appears at 1381 cm-1 in H2O and upshifts by 2 cm-1 in D2O. The considerably smaller isotopic shift in IMP compared to Hx indicates a reorganization of the normal mode due to the presence of ribose at N9 in IMP. The bands observed at 1379, 1372, and 1338 cm-1 in deprotonated hypoxanthine, deprotonated IMP, and double deprotonated hypoxanthine, respectively, are assigned to this mode. In protonated IMP and hypoxanthine, this mode appears at 1369 and 1361 cm-1. C-H Bending Coupled to N9-H Bend and Imidazole Ring Breath. This band appears at 1338 cm-1 in native neutral hypoxanthine in water, and on deuterium labeling of the N9-H, it moves down by 19 cm-1. In IMP, this mode appears at 1322 cm-1 in H2O and at a lower isotopic shift of 13 cm-1 in D2O. In Hx- and Hx2-, this band is observed at 1334 and 1309 cm-1, while in IMP- this mode is ascribed to the band at 1302 cm-1. In the protonated forms of IMP and hypoxanthine, this mode is observed at 1350 and 1335 cm-1, respectively. Xanthine and Xanthosine Monophosphate. The solution structures of xanthine and XMP and their ionization states under various pH conditions have been debated extensively.21-23,38,39 The xanthine nucleobases as well as the monophosphate are known to exist in the diketone forms in solution.18-21 Figure 1(l)-(p) shows the energy minimized structures of xanthine and XMP in various ionization states. The bond distances and Mullikan atomic charge distribution of neutral xanthine obtained from DFT calculations have been listed in Tables S5 and S6 (Supporting Information), respectively. The solution tautomers at each pH were identified by comparing the observed Raman spectra with the computed vibrational spectra of all possible tautomers of a protonation state. Neutral Xanthine and Xanthosine Monophosphate. Xanthine at neutral pH is known to exist as a 2,6-diketone tautomer. Of the two possible tautomers, with the hydrogen atom on the

Figure 5. Resonance Raman spectra of xanthine in the region 1100-1750 cm-1: (a) neutral, in H2O, pH 6; (b) neutral, in D2O, pD 6; (c) deprotonated, in H2O, pH 10.0; (d) deprotonated, in D2O, pH 10.0; (e) double deprotonated in H2O, pH 13; and (f) double deprotonated in D2O, pD 13; obtained with 260 nm excitation.

imidazole ring on either N7 or N9 nitrogen, the tautomer with hydrogen on N7 is the most stable (Figure 1(l)) in agreement with previous work.22,39 Computed spectra of this structure agree with the Raman spectrum of xanthine at neutral pH in H2O and D2O at 260 nm (Figure 5(a), S2(a), and Table 8). Due to the presence of the N9-C1′ bond in XMP, its spectrum (Figure 6(a), S3(a)) is expected to resemble that of the xanthine tautomer with the hydrogen atom on N9. The excellent agreement found between the computed spectrum of the xanthine tautomer with N9H and XMP (Table 9) corroborates this. Isotopic labeling of XMP with deuterium and corresponding calculations of the isotope effect on vibrational frequencies indicate that the normal mode composition is not significantly altered in the nucleotide

1131

1503

1456

1394

1361

1335

1267

1257

1195

1519 1492

1469 1427

1385

1369

1350

1319 1296

1265

a

1204

1538 1522

1554 1534

Be C8H+N9H+N1H+ C2H+St N1C2+C8N9

from neutral Be N9H+C8H+N7H+ C2H+St C6C5 purine ring torsion

Be C2H+N7H+C8H+ N1H+St C5N7+C2N3 Im Bre+Be C2H+N1H

St C6O+Be N1H St C4C5-C2N3+Be N9H+C2H+N7H from neutral Py ring torsion+Be C2H+N9H+N1H+C8H from neutral St N7C8-C8N9-C4C5+ C2N3+Be C8H+C2H+N1H from neutral St C4C5-N7C8+Be N7H+N1H+C2H from neutral St N1C2+N7C8-C8N9+Be N1H+C2H+N7H+N9H Be N1H+N9H+St N1C2+C5N7-C2N3

assignment

-13% St NC 30% St NC 17% Be HNC -18% Be HCN

14% Be HNC 20% Be HNC -17% Be HCN -21% St NC 24% Be HNC

12% St NC -12% St NC 11% St NC 42% Be HCN -14% St NC 36% St NC -18% Be HCN

-17% St NC 14% Be HNC 22% Be HNC 20% St CC -12% St NC 23% Be HNC

20% St NC 21% Be HNC 18% Be CCN

18% St NC -20% St NC 27% Be HCN -10% Be CNC

39% St NC -12% St NC

79% St OC -12% St NC 40% St CC

PED %

1150

1265

1323

1352 1341

1363 1339 1326

1382

1429 1404

1495 1463

1513

1568

1709 1585

1386

1432 1417

1505 1470

1549 1530

1565

1700 1604

IMP Hyp in D2O

UVRR

Abbreviations: St: stretch; Be: Bending; Im: imidazole; Py: pyrimidine; Bre: breathe. b Scaling factor used: 0.97.

1281

1309

1368

1408

1450

1501

1516

1574

1589

1593 1576

1722 1607

1715 1619

N1H, N7H, N9H

DFTb

1717 1614

IMP Hyp in H2O

UVRR

1122

1042

1245

1278

1319

1369

1381

1465

1502

1563

1717 1589

N1D, N7D, N9D

DFTb

Be C8H+St N3C4-C5N7

Be C8H+C2H+St C6C5-N7C8 purine ring torsion

from neutral St C4C5-N7C8+Be C2H+N7H from neutral St N1C2+N7C8-C8N9+Be C2H Be C2H+C8H+St C5N7-C2N3 from neutral Be C2H+N1H+St N1C2+C5N7-C4N9 Im Bre+St N1C2+Be N1H+C2H

from neutral St N7C8-C8N9-C4C5+ C2N3+Be C8H+C2H

Py ring torsion+Be C2H

St C6O+Be N1H St C4C5-C2N3+Be C2H

assignment

-15% St NC 14% St NC 34% Be HCN -11% St NC 37% St NC -17% Be OCN 25% Be HCN -14% Be CNC 18% Be CNC

-12% St NC -22% St NC 23% St NC

-22% St NC 29% Be HCN

12% St CC -21% St NC -27% Be HCN

33% St NC -13% Be NCN

12% St NC 11% Be HCN 25% Be CCN 11% Be NCC

26% St NC -25% St NC 29% Be HCN

-35% St NC -13% St CC 18% St NC

82% St OC -21% St NC 36% St CC

PED %

TABLE 7: Experimental Resonance Raman Shifts (λexc ) 260 nm) of Protonated Hypoxanthine at pH ) 1.5 and IMP at pH ) 1.0 in Water and D2O and Computed (B3LYP/ 6-31G(d,p)) Vibrational Wavenumbers of Protonated Hypoxanthine (H1, H7, and H9, Figure 2(e) and Figure 2(h)) with Mode Assignmentsa

6-Oxopurines in Solution J. Phys. Chem. B, Vol. 113, No. 45, 2009 15111

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TABLE 8: Experimental Resonance Raman Shifts (λexc ) 260 nm) at pH ) 6.0 in Water and D2O and Computed (B3LYP/ 6-31G(d,p)) Vibrational Wavenumbers of Neutral Xanthine (H7 Tautomer, Figure 3(a)) up to 600 cm-1 with Mode Assignmentsa UVRR DFTb in H2O N1H, N3H, N7H 1709

1707

1693 1604

1680 1620 1590

assignment

St C2O+C6O Be N3H+N1H St C6O+C20+Be N1H St N3C4-C4C5+Be N7H+C8H St N3C4-C5C6-N7C8+ Be N3H+N7H+C8H St C8N9-N7C5+ Be C8H+N7H

1477

1470

1454

1450

Be N3H+N7H+C8H+St C5N7-C4N3

1416

1413

Be N7H+N3H+C8H St N7C8-C5C6-N1C2

1383

Be N1H+N7H+St N7C8-N9C4

1331

1327

1283

1285

Be N1H+C8H+St N1C2C2N3-C4C5-N9C8 Be N3H+St C5N9-C4N7

1263

1269

1212

1200

1132

1124

1036

1099

951

980 952 840

644 a

Be C8H+St N1C2-N3C4C5C6+C8N9 Be C8H+N7H+C4C5N9 Be N1H+N7H+St C6N1+N7C8Be N7H+C8H+St N7C8 Be N1H+N3H+St N1C2C2N3 St N7C8N9

831

Be N3H+C8H+St N1C2N3C4-C8N9-C8N7 (OOP) Be C8H

743

(OOP) purine ring

733 709

(OOP) Py ring (OOP) Be N1H+C6O+ N7H+C8H+St C4N9

621

purine ring Bre

PED %

UVRR DFTb in D2O N1D, N3D, N7D

assignment

PED %

75% St OC

1688

1698

St C2O+C6O

67% St OC 11% St OC

73% St OC 55% St CC -13% St NC

1651 1595

1669 1613

St C6O+C2O St N3C4-C4C5

-12% St OC 64% St OC -61% St CC 20% St NC

13% St NC 40% Be CCN -23% St NC 27% St NC 22% Be HCN

1551

1568

37% Be CCN

1448

1463

St N3C4-C5C6-N7C8+ Be C8H+N7H St C8N9-N7C5+ Be C8H

10% St CC -15% St NC -11% Be NCN 23% Be HNC 10% St NC 11% St NC -10% St NC 22% Be HNC 66% Be HNC

1422

1406

Be N3H+C8H+St C5N7-C4N9

1406

1356

1226

1228

28% St NC 12% St NC -10% St NC

1323

1325

27% St NC 43% Be HNC

1128

1137

-13% St NC -18% St NC -12% Be HCN -16% Be CNC -13% Be HNC 34% Be HCN -10% Be CNC -15% St NC 35% St NC

1275

1267

1150

1170

Be C8H+N7H+St N7C8C5C6-N1C2 Be N1H+C8H+St N1C2+ N7C8 Be C8H+St N1C2-C2N3C4C5-N9C8 Be N3H+St N1C6-C2N3+ C5N9-C4N7 Be C8H+St N1C2-N3C4C5C6+C8N9 Be C8H+C4C5N7

1048

1010

997

870

971

842

-32% St NC 29% Be HNC 12% Be HCN 25% St NC 22% St NC 10% Be HNC 11% Be HNC 58% Be NCN

Be N1H+N3H+purine ring torsion Be N7H+C8H+ St C8N9+N1C2-C2N3 Be N1H+N3H+N7H+ St N1C2-C2N3

950

St N7C8N9+Be N7D

45% Be CNC 13% Be NCN

770

Be N3H+N1H+N7H+ St N3C4-C8N9-C8N7

87% Tor HCNC -12% Tor CNCN -24% Tor CCNC 37% out ONCCC 22% out ONN -72% out ONNC 19% Tor HNCN -10% Tor CCNC -33% out ONCC -14% out NCNC

831

(OOP) Be C8H

743

(OOP) purine ring

732 695

(OOP) Py ring (OOP) Be C6O+C8H+St C4N9

617

purine ring Bre

640

27% St NC -20% St NC -23% Be HCN 10% Be CCN -10% St CC 31% St NC -15% St NC 12% Be NCN 40% St NC -11% Be DNC -12% St NC 19% St NC 10% Be HCN 22% Be DNC 27% St NC 13% St NC -10% Be NCN 11% Be HCN 10% Be CCN 26% St NC -11% St NC -14% St NC 21% Be DNC -10% St NC 25% St NC 10% Be HCN 14% Be CNC 33% Be HCN -16% Be CNC -25% Be DNC 26% Be DNC -12% Be OCN -11% Be NCN 41% Be DNC 14% Be CNC -12% St NC -10% St NC 21% Be DNC -24% Be DNC 17% St NC 43% Be NCN 10% Be DNC 10% St NC -23% Be DNC 23% Be CNC 86% Tor HCNC -11% Tor CNCN 24% Tor CCNC -37% out ONCC -24% out ONNC -68% out ONNC

Abbreviations: St: stretch; Be: Bending; Bre: breathe; Py: pyrimidine; (OOP): out of plane; Tor: torsion. b Scaling factor used: 0.99.

as compared to that of the free base. Hence, the computed spectrum of the xanthine N9H tautomer serves as a good model for XMP. Deprotonated Xanthine (X-) and Xanthosine Monophosphate (XMP-). The UVRR spectra of xanthine (Figure 5(c), pH 10) and XMP (Figure 6(c), pH 11), respectively, are similar indicating that deprotonation occurs at the same position in both molecules. To identify the site of deprotonation, vibrational spectraofallthreepossiblestructureswerecomputedsdeprotonation at either N3, N7, or N1. The observed spectra are found to be in agreement with the computed spectrum of deprotonated

xanthine formed via loss of the proton at N3 (Table 10 and Figure 1(m)). Thus, the solution structure of xanthine at pH 10 is indeed the 2,6-diketone form with the deprotonation at N3. Shugar and co-workers21 have suggested that while both xanthosine and xanthine anions are formed by dissociation of the proton at the N3 position the xanthine anion is expected to exist in a tautomeric equilibrium between X- with the hydrogen on the imidazole on N7 and X- with the hydrogen on N9. The experimental Raman spectra, however, do not show the presence of two tautomers, and all observed bands can be accounted for by a single X- tautomer with the hydrogen atom at N9 in


Figure 6. Resonance Raman spectra of XMP in the region 1100-1750 cm-1: (a) neutral, in H2O, pH 2.0; (b) neutral, in D2O, pH 2.0; (c) deprotonated in H2O, pH 11.0; and (d) deprotonated in D2O, pH 11.0; obtained with 260 nm excitation.

xanthine. The spectrum of deprotonated XMP, XMP-, correlates well with the X- spectrum indicating that the same protonation state of the nucleobase is present in both xanthine at pH 10 and xanthosine mononucleotide at pH 11 (Table 10). Dianionic Xanthine. Further increase in the pH leads to loss of another proton from xanthine. We computed spectra of the two possible anions with loss of the N1 or N7 proton in addition to the N3 proton dissociated at the first deprotonation. The structure of xanthine at pH 13 is found to be the 2,6-diketone form with dissociation of the protons at N3 and N7, while the hydrogen at N1 is still present Figure 1(n). The comparison of the UVRR spectrum with the computed spectrum is given in Table 11. Carbonyl Stretches. Xanthine and XMP are expected to contain two carbonyl stretching bands corresponding to the C2dO and C6dO groups. Indeed, the modes appear at 1709 and 1693 cm-1. Correspondingly, two modes are predicted from calculations with the higher frequency mode corresponding to the band at 1709 cm-1 containing contribution from stretching of both carbonyls and coupled to bending of both N1-H and N3-H bonds. The predicted coupling of N-H bends to this mode is borne out by the downshift observed in the experimental spectrum of xanthine in D2O by 21 cm-1(Table 8). This is a low-intensity, broad band, typical of carbonyl modes in Raman spectra. In XMP, the mode appears at 1712 cm-1 and downshifts to 1696 cm-1 in D2O indicating that the normal mode composition is similar to that in the free nucleobase. Upon deprotonation of xanthine, the CdO bonds are expected to weaken, and correspondingly there is a downshift to 1557 cm-1 in X-. This band is absent in the spectrum of XMP- in H2O but observed at 1545 cm-1 in the spectrum of XMP- in D2O. The contribution from the N-H bend is retained in the modes (Figure S1D, Supporting Information) as evidenced by a further decrease in the wavenumber in X- to 1549 cm-1. The weakening in this

J. Phys. Chem. B, Vol. 113, No. 45, 2009 15113 mode can be explained by the effects of the negative charge on the molecule. This band is not observed in X2- in H2O although in D2O the band is observed at 1632 cm-1 (Figure 5(f), Table 11). The upshift in the CdO stretching mode in X2- as compared to that in X- is an indication of the coupling of the CdO mode to the N9-H bend in X-. Once the N9-H is dissociated, the CdO stretching band moves up to a typical value for this mode. The mode at 1693 cm-1 in xanthine is the second CdO stretching mode with a larger contribution from the C6dO group. This mode is predicted to be coupled to N1-H, and the large 42 cm-1 downshift in this band on isotope labeling confirms this prediction (Table 8). In XMP, the band appears at 1696 cm-1 in H2O and 1659 cm-1 in D2O. Deprotonation causes weakening of the double bond lowering the computed Raman shift to 1674 cm-1 in xanthine and 1652 cm-1 in XMP-. Further raising the pH causes dissociation of the H9 proton of xanthine, and the CdO stretching mode is now observed at 1693 cm-1 in D2O due to uncoupling of the N9-H bend. The coupling of CdO stretching to the N9-H bend we infer is corroborated by the shifts observed in XMP-. In XMP-, H9 is replaced by the ribose moiety, and the CdO stretching mode undergoes only a minor shift of 3 cm-1 upon H/D exchange (1652 cm-1 in H2O to 1649 cm-1 in D2O). Pyrimidine Ring Stretch. The high-intensity band at 1604 cm-1 is assigned to the pyrimidine ring N3C4-C4C5 stretching coupled to N7-H, C8-H, and N3-H bending. In XMP, this band appears at 1612 cm-1 in H2O (1607 cm-1 in D2O) showing a much weaker contribution from N-H bends than in the carbonyl modes. In the deprotonated forms, this band appears at 1592 cm-1 in X- and 1573 cm-1 in XMP-. Upon second deprotonation of xanthine, this band appears at 1519 cm-1 and is weakened in intensity as well as frequency. Triene Stretch. Observed at 1579 cm-1 in XMP, the N3C4-C5C6-N7C8 triene stretch is coupled to the N3-H and N9-H bends and downshifts to appear at 1555 cm-1 in D2O. A band corresponding to this mode in xanthine is predicted by DFT at 1590 cm-1 (Table 8). Although this band is not observed in H2O, it gains intensity in D2O to appear at 1551 cm-1 at pD 6. In the deprotonated forms, the mode is weak and appears at 1467 cm-1 (X-) and 1469 cm-1 (XMP-) for the two molecules which may be explained by the modified electron density on the rings in this state. In the xanthine double deprotonated state, X2-, this mode is further weakened and appears at 1420 cm-1. Isotopic exchange from hydrogen to deuterium leads to a gain of intensity indicating decoupling that influences the symmetry of the mode. In Plane Imidazole Ring Distortion. The band at 1477 cm-1 is assigned to a vibrational mode comprised of the imidazole ring, N7-H, and C8-H from comparison with the DFT calculations. The contribution of the N7-H bend leads to a significant downshift in this band on deuterium labeling. The corresponding mode is observed at 1555 cm-1 in XMP. In the deprotonated forms, this band appears at similar positions in both molecules 1528 and 1530 cm-1, respectively, for X- and XMP-. In the twice ionized (i.e., deprotonated) xanthine, X2-, the mode appears at 1498 cm-1. No downshift in this mode is observed on deuterium labeling of the deprotonated states suggesting that N7-H is no longer coupled to the mode. N-C Stretch and Imidazole Ring Hydrogen Bend. A lowintensity band at 1454 cm-1 in xanthine shows a large downshift to appear at 1422 cm-1 in D2O and is assigned to N3-H, N7-H, C8-H and bending, coupled to C5N7-C4N3 stretching. In XMP, this mode appears at 1459 cm-1 and shows similar

15114


Gogia et al.

TABLE 9: Experimental Resonance Raman Shifts (λexc ) 260 nm) of Neutral XMP at pH ) 6.0 in Water and D2O and Computed (B3LYP/6-31G(d,p)) Vibrational Wavenumbers of Neutral Xanthine (H9 Tautomer, Figure 3(d)) with Mode Assignmentsa UVRR DFTb in H2O N1H, N3H, N9H

assignment

1712

1709

St C2O+C6O+Be N3H+N1H St C6O+C20+Be N1H St N3C4-C4C5+Be N9H

1696 1612

1700 1634

1579

1571

1555 1488

1524

1459

1418

1421

1394

1387

1371

1343

1332

Be C8H+N3H+St N7C8

1320

1301

St N1C2-N3C4-C5N7+Be C8H

1220

1260

Be N3H+N9H+C8H+St C4N7-C5N9

1206

1183

1161

1102

Be C8H+N9H+St N1C6-C5N7-C4N9-N3C4 St N1C6-C6C5-C4N9+Be N1H+C8H

St C2N3-C4C5-N7C8+Be N3H+N9H+C8H not assigned St N7C8+Be C8H+N9H+N3H Be N9H+N3H+N1H+St C8N9-N7C5 Be N1H+N9H+St C5N7-C4N9-N1C6 Be N1H+N9H+St C8N9-C4C5

PED %

UVRR DFTb in D2O N1D, N3D, N9H 1696

1705

St C2O+C6O

32%St OC 45% St OC

-17% St OC 59% St OC -21% St CC -13% St NC 29% St NC -10% Be HNC 15% Be NCC -19% St CC 11% St NC 20% Be NCC

1659 1607

1686 1630

St C6O+C2O St N3C4-C4C5+Be N9H

1555

1556

66% St NC

1518 1480

1518


1433

1377

St C2N3-C4C5-N7C8+ Be C8H not assigned St N7C8-C4C5-N3C4+Be C8H+N9H Be N9H+St C8N9-C4C5

46% St OC -35% St OC -22% St CC -12% St NC 27% St NC -10% Be HNC 16% Be NCC -15% St NC -17% St CC 10% St NC 18% Be NCC

11% St NC 13% St NC 27% Be HNC 10% Be HNC 39% Be HNC -19% Be HNC

1422

1388

1380

11% St NC 12% Be HNC 42% Be HCN -15% St NC 31% St NC 10% Be CNC 11% St NC 32% Be HNC -11% Be HNC 10% Be NCC 12% St NC -17% St NC -25% Be HCN -10% St NC 43% St NC

1227

21% St NC 14% St NC -10% St NC -11% St NC 25% St NC 24% Be DNC

1277 1335

1319

Be C8H+St C5N7+C2N3

1311

1295

St N1C2-N3C4-C5N7+Be C8H from sugar Be N3H+N9H+St N1C6

13% St NC -18% St NC 42% Be HCN 35% St NC 11% Be CNC

1112 1203

1170

1157

999

Be C8H+N9H+St C5N7-C4N9-N3C4 Be N1H+N3H+Py ring

1056

St C8N9+Be N8H+C8H

St C8N9+Be N8H+C8H

978

St N1C2N3

17% str NC 35% str NC 10% be HNC

1069

853

N1C2N3

1014

938

St N7C8N9

1029

937

St N7C8N9

877

835

St N1C2-N3C4-C4N9C5N7+Be C8H+N9H (OOP) Be C8H

-18% St CC 41% Be CNC -17% Be NCC -12% Be NCC 18% St NC -15% Be NCC 15% Be NCC 15% Be NCN 78% Tor HCNCN 10% Tor NCC 18% Tor CNCC -57% out ONCC 88% out ONN

850

780

801

788

Be N3H+N1H+C8H+St N1C2-N3C4-C4N9-C5N7 (OOP) Be C8H

756 732 692 685

668

a

(OOP) Be C8H+C6O+St C5C6 (OOP) St C2N1+Be N1H+C2H (OOP) Be N1H+C8H+St C4C5

680

Be N1H+C2H+N3H+Py ring torsion

650

(OOP) Be C8H+N9H+St N7C8-C8N9

617

purine ring Bre

42% Tor HNCN 11% Tor NCCN -11% out ONCCC 16% out NNC -25% Be OCN -24% Be OCN 16% Be NCC -11% Be NCN 11% Tor HNCN -13% Tor HCNCC 59% Tor CNC

59% St NC

St C5N7-C4N9-N1C6+Be N9H+C8H Be N1H+N9H+C8H

1068

788

PED %

56% St OC 19% St OC

1262

1057

assignment

755 483

677

661

(OOP) Be C8H+C6O+St C5C6 (OOP) St C2N1+Be C2H

665

(OOP) Be N1H+C6O+St C4C5

634

Be N1H+C2H+N3H+Py ring torsion

647

(OOP) Be C8H+N9H+St N7C8-C8N9

613

purine ring Bre

15% St NC -11% St NC -15% St NC 25% Be DNC -18% St NC 13% St NC 23% Be HCN 10% Be CNC -22% Be DNC 24% Be DNC -14% Be OCN -10% Be NCC 25% Be CNC 32% Be HNC 22% Be NCC 11% St NC 17% St NC 23% Be DNC 10% Be DNC 10% Be NCC 16% St CC -41% Be CNC 13% Be NCC 15% Be NCC -10% St NC -11% St NC 20% Be DNC 78% Tor HCNCN 11% Tor NCC 18% Tor CNCC -54% out ONCC 90% Tor DNC 28% Tor CNCC -16% Tor NCCN 34% out ONCC -15% out NNCC -12% Be CNC 20% Be OCN 16% Be OCN -13% Be NCC 13% Tor HNCN -15% Tor HCNC 41% Tor CNCCN 14% Tor NCC -17% St NC -14% St NC -13% St NC -11% Be CNC


isotope-induced shift to xanthine (1433 cm-1) indicating a similar normal mode composition. The high Raman intensity of this mode makes it an ideal candidate as an imidazole ring marker. In the deprotonated states, X- and XMP-, a change in the composition of this mode has been calculated such that it comprises N9-H and N1-H bending coupled to C8N9-C4C6 stretching with bands at 1401 (X-) and 1391 cm-1 (XMP-). On the basis of DFT prediction, the bands at 1212 and 1217 cm-1 in the D2O spectra of anionic xanthine and XMP, respectively, have been assigned to this mode. This large perturbation in D2O suggests that this mode is largely comprised of the N-H bending in these states. The mode is absent in the

double deprotonated state X2-. We do observe a medium intensity band in both X- (1425 cm-1 H2O, 1426 cm-1 D2O) and XMP- (1391 cm-1 in H2O, 1389 cm-1 in D2O) which cannot be assigned to any of the predicted modes. Calculations predict a large downshift for the mode at 1388-1192 cm-1, hence the observed band which is not sensitive to isotope labeling cannot be assigned to this mode. Another mode attributed to N-H bending is observed at 1416 cm-1 in xanthine. Contributions from N7-H and N3-H bend as well as N7C8 stretching and pyrimidine ring vibrations make it a complex mode that reflects the combined effect of electron distribution of both rings. Computational calculations overes-

(OOP)Be C8H+N9H+St N7C8

(OOP)Be N1H Purine ring Bre

1299

1244

930 830

772 758 722 695

689

655

642 620

1322

1298

1242 1196

1174

1079

1031

1058

873 829

805 782 719 683

1332

1301

1279

1188

1144

1035

963

945 845

a

981

1054

1122

1148

1331

Be N1H+N9H+St N1C2-C2N3

St N9-C1′ Be C8H+N9H+N1H+St C2N3-N1C6-C5C4 Be C8H+N9H+St C2N3-C4N9-C5N7 St C6N1-C2N3-C4N9+C5N7+Be N1H+C8H+N9H St C8N9+Be N9H+C8H

Be C8H+N1H+St C5N7

Be N1H+C8H+N9H+St N1C2

Be C8H+St C6C5-C4N9-C8N7

St C4C5-C5C6-C2N3+N7C8+Be N9H+C8H not assigned Be N9H+N1H+St C8N9+C4C5

1549

62% St OC -10% St OC 10% Be HNC 34% St NC -12% Be CNC 52% St NC 11% Be CNC -14% Be HCN 13% St NC 20% Be NCC -10% Be CNC -18% Be CNC

20% Tor HNCN -13% Tor HCNC 15% Tor CNCN -42% Tor CNCN N 79% Tor HNC 10% St NC 11% St NC 23% St NC 18% Be CNC

15% St NC 30% St NC 16% Be HNC -11% Be CNC 60% Be NCN 28% Be CNC 10% Be NCC 14% Be NCN 40% out ONCCC 26% out NNC -91% out ONNC 47% Tor HCNC -32% out ONCC -14% Tor HNCN 32% Tor HCNC -10% Tor CNCN 16% out ONCC -17% out NNCC -12% St NC -26% Be OCN -23% Be OCN -10% Be NCN

51% St NC 34% Be HNC

642

941

963

838

1107

1151

1266

1300

-14% St NC -11% St NC 32% St NC -18% Be HCN -12% St NC -11% St NC 12% St NC -15% Be HCN -18% Be HCN -12% Be CNC 20% Be CNC 19% St NC 38% St NC

1316

1372

51% Be HNC


1426 1212

1463

1574 1525

1652


XMP

670

802 787 715 692

868 821

1037

935

1056

1136

1246 1193

1292

1316

1330

1389 1217

1468

1575 1528

1545

1649

in D2O

Xan

UVRR

59% St OC -12% Be CNC

PED %

476 610

642

644

771 758 717 689

930 825

953

815

1087

1138

1234

1301

1308

1339

1180

1473

1599 1532

1609

1640

N1D, N9D

DFTb assignment

(OOP)Be N1H purine ring Bre

(OOP)Be C8H+N9H+St N7C8

Be N1H+Py ring

St C6N1-C2N3-C4N9+C5N7+Be N1H+C8H St C8N9+N1C2+C2N3C4+Be N9H+N1H+C8H Be N1H+N9H+C8H+St N1C2+C2N3C4 St N1C8N9 St N1C2-C2N3-C4N9-C5N7+Be N1H+N9H (OOP)Be C8H+C6O+St C4C5 (OOP) St C2N3+Be C2O (OOP)Be C8H+C6O Be C8H+Py ring torsion

St N9-C1′ Be C8H+N9H+N1H+St C2N3-N1C6-C5C4 Be C8H+St C2N3-C4N9-C5N7

Be C8H+St C6C5-C4N9-C8N7+C2N3 Be C8H+N9H+St C2N3-C4C5-N9C8 Be C8H+St C5N7

St C4C5-C5C6-C2N3+N7C8+Be C8H not assigned Be N1H+St N1C2+N3C4

St N3C4-C5C6+C4N9+Be N9H Be C8H+St N7C8+C5C6-N3C4

St C6O+C2O+Be N1H

St C6O+C2O+C5C6+C2N3


669

Be N1H+Py ring

1345

1334

1375

647

St N7C8N9+Be N1H St N1C2-C2N3-C4N9-C5N7+Be C8H (OOP)Be C8H+C6O+St C4C5 (OOP) St C2N3+Be C2O (OOP) Be C8H+C6O+C2H+N9H Be N1H+C8H+Py ring torsion

1374

1391

1425 1401

1476

1469

1467

St N3C4-C5C6+C4N9+Be N9H Be C8H+St N7C8+C5C6-N3C4

1613 1534

1573 1530

St C2O+C6O+Be N1H

St C6O+C2O+Be N1H+St C5C6

1592 1528

1641

N1H, N9H

assignment

1623

1652

XMP

DFTb

1557

1674

in H 2O

Xan

UVRR

N 91% Tor DNC 10% St NC 10% St NC 23% St NC 21% Be CNC

-13% St NC -15% Be DNC 27% Be DNC 13% Be OCN 17% St NC 46% Be NCN 10% St NC 12% St NC 13% Be CNC 15% Be DNC 15% Be DNC 39% out ONCCC 24% out NNC -91% out ONNC 59% Tor HCNC -27% out ONCC 28% Tor HCNC -12% Tor CNCN 27% out ONCC -21% out NNCC -12% St NC 19% Be DNC -21% Be OCN -20% Be OCN -10% Be NCN 19% Tor CNCN -53% Tor CNCN

-24% Be HCN -12% Be CNC 23% Be CNC 18% St NC 13% St NC -16% Be OCN -11% Be CNC 35% Be DNC

13% St NC -10% Be NCN

-12% St NC 18% St NC 24% St NC -11% Be DNC -15% St NC 19% St NC -24% Be HCN -12% Be CNC 13% Be NCN

-25% St NC 30% St NC -20% Be DNC 28% St NC 20% Be HCN

11% St NC 18% Be NCC -11% Be CNC -21% Be CNC

38% St NC -13% Be CNC 55% St NC -16% Be HCN

18% St OC 50% St OC -11% Be CNC 56% St OC -22% St OC

PED %

TABLE 10: Experimental Resonance Raman Shifts (λexc ) 260 nm) of Xanthine at pH ) 10.0 and XMP pH ) 11.0 in Water and D2O and Computed (B3LYP/6-31G(d,p)) Vibrational Wavenumbers of Deprotonated Xanthine (H9 Tautomer, Figure 3(b) and Figure 3(e)) with Mode Assignmentsa

6-Oxopurines in Solution J. Phys. Chem. B, Vol. 113, No. 45, 2009 15115

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Gogia et al.

TABLE 11: Experimental Resonance Raman Shifts (λexc ) 260 nm) at pH ) 13.0 in Water and D2O and Computed (B3LYP/ 6-31G(d,p)) Vibrational Wavenumbers of Double Deprotonated Xanthine (Figure 3(c)) with Mode Assignmentsa UVRR DFTb in H2O N1H

assignment

PED %

UVRR DFTb in D2O N1D

assignment

1588

St C6O+C2O

45% St OC 11% St OC -13% St CC 12% Be NCC

1693

1588

St C6O+C2O

1561

St C2O+C6O+Be N1H

1632

1542

St C2O+C6O

1519

1539

St C4C5-N7C8-C8N9C4N3-C5C6+Be C8H

1521

1539

St C4C5-N7C8-C8N9C4N3-C5C6+Be C8H

1498

1487

1499

1487

1420

1427

1419

1426

1349

1341

St C4C5-N7C8-C8N9C4N3+Be C8H St N7C8-C5C6-C4C5C2N3+Be C8H+C6O St C4N9-C5N7-C5C6-N9C8

-10% St OC 45% St OC -15% St NC 15% Be HNC 15% St NC 13% St NC -12% St NC 12% St NC -14% Be HCN 20% St NC -23% St NC 21% Be CNC -14% St OC -10% St CC 15% Be HCN 19% Be CNC 39% St NC 17% St NC

1352

1341

1312

Be N1H+C8H+St C5N7

St C4C5-N7C8-C8N9C4N3+Be C8H St N7C8-C5C6-C4C5C2N3+Be C8H+C6O St C4N9-C5N7-C5C6N9C8 Be N1H+St C5N7

1286

St N1C2-C2N3-C4C5+ C8N9+Be N1H+C8H Be C8H+N1H+St N7C8N7C5

1275

1280 1212

1214 1167

1134

1133

1042

990

St C8N9+Be C8H Be C8H+St C5N7-C6N1C2N3 St C6N1+C4N3+Im Bre+Be C8H Be N1H+St N1C2N3

961

955

St N7C8N9+Be N1H

839

St N1C2-N3C4-N9C8N7

819 780

(OOP)Be C8H (OOP) St C4C5C6+Be N1H

755 727 706

(OOP) St C2N3 (OOP) Be C6O+C8H+Be C4N9 Be N1H+Py ring torsion

674

(OOP) Be C8H+St N7C8

624

purine ring Bre

652

-17% St OC 13% St NC 34% Be HNC 24% St NC 17% Be HNC -11% Be NCN 32% St NC -11% St NC -12% Be HNC 25% Be HCN 54% St NC 11% St NC -12% St NC 26% St NC -16% Be HCN -13% St NC -14% St NC 25% St NC 11% Be CNC -10% Be HNC -17% Be NCN 14% Be NCN -14% Be NCC 12% St NC -10% Be CNC 38% Be NCN -13% Be CNC -15% Be CNC 17% Be CNC -10% Be OCN 18% Be NCC C 88% Tor HCN -12% Tor CNCC -26% Tor CNCN -20% out ONCCC 20% out NCN

1297 1279

1282 1181

St N1C2-C2N3-C4C5+ C8N9+Be C8H Be C8H+N1H+St C6N1C2N3-C5N7

PED % 41% St OC 16% St OC -12% St NC -12% St CC 11% Be NCC -19% St OC 43% St OC -14% St NC 15% St NC 14% St NC -12% St NC 11% St NC -14% Be HCN 20% St NC -23% St NC 23% Be CNC -13% St OC -11% St CC 16% Be HCN 16% Be CNC 39% St NC 18% St NC 12% St OC 20% St NC 21% St NC -13% St NC 30% St NC 10% St NC 31% Be HCN 10% St NC 40% St NC 10% Be DNC

874

St C8N9+Be C8H Be C8H+N1H+St C5N7-C4N3 St C6N1+C4N3+Im Bre+ Be N1H Be N1H+St N1C2N3

48% St NC -12% Be NCN -12% St NC -13% Be HCN 11% Be CNC 12% St NC 10% Be DNC 20% Be OCN 13% Be CCN 32% Be DNC 17% Be OCN -12% Be NCN

958

St N7C8N9

10% St NC 53% Be NCN -11% Be CNC

832

St N1C2-N3C4-N9C8N7

-14% Be CNC 19% Be CNC 22% Be NCC

819 779

Be C8H (OOP) (OOP) St C4C5C6

-86% out ONNC -27% Tor CNCNC 48% out ONC 10% St CC 12% St NC 17% Be OCN 24% Be OCN

754 727 653

(OOP) St C2N3 (OOP)Be C6O+C8H+ Be C4N9 Be N1H+Py ring torsion

11% Tor HCNC 64% Tor CNCN -18% out ONCC 15% St NC 15% St CC 18% Be CNC

674

(OOP) Be C8H+St N7C8

623

purine ring Bre

C 88% Tor HCN -11% Tor CNCC -25% Tor CNCN -10% Tor CNCN -23% out ONCCC 23% out NCN -90% out ONNC -28% Tor CNCNC 47% out ONC -11% St NC 20% Be DNC -11% Be OCN -14% Be OCN 11% Tor HCNC 64% Tor CNCN -18% out ONCC 15% St NC 18% St CC 15% Be CNC

1225 1154

1216 1155

1108

1106

838 962

643

a

Abbreviations: St: stretch; Be: Bending; Im: imidazole; Bre: breathe; Py: pyrimidine; (OOP): out of plane; Tor: torsion. b No scaling factor used.

timate the N-H coupling in this mode leading to a much larger predicted downshift (57 cm-1) than experimentally observed (10 cm-1; Table 8). The 1421 cm-1 band in the neutral XMP spectrum corresponds to this mode (Table 9). The bands at 1375, 1334, and 1349 cm-1 in deprotonated xanthine, deprotonated XMP, and double deprotonated xanthine, respectively, correspond to this mode and show a downshift as in all ring modes upon deprotonation. Purine Ring Vibrations. Purine ring vibration leads to a mode at 1331 cm-1 which couples to the C8-H bend and the pyrimidine ring with N1-H bend. Deuteration alters the mode description and band position by a small amount to 1323 cm-1. In XMP, this mode appears at 1387 cm-1 at a surprisingly low intensity. From DFT, a change in the mode description is predicted on deuterium labeling accompanied by a large downshift of 144 cm-1. The two experimental bands at 1380 and 1277 cm-1 are potential candidates for this mode. However, from comparison of the intensities of the bands, we assign the band at 1380 cm-1 in D2O to this mode. The resulting downshift of 7 cm-1 in this mode is in contrast with the calculations suggesting that no change in the mode composition takes place.

However, there is no corresponding computed mode for the band at 1277 cm-1. In the deprotonated states, the bands appear at 1332 (X-) and 1322 cm-1 (XMP-). Although a band corresponding to this mode has been predicted by DFT in dianionic xanthine, none was observed in the recorded UVRR spectrum. It is possible that this band is below signal-to-noise in the current spectrum since bending motions have low intensity in Raman spectra. C-H Bending Mode. The band at 1263 cm-1 in xanthine is a marker of C8-H bending and pyrimidine ring stretching with a weak coupling to the N7-H bend. In D2O the band undergoes an upshift to 1275 cm-1, probably due to uncoupling of the N7-H bend. In neutral XMP, a corresponding, intense band is observed at 1320 cm-1. Deprotonation leads to downshifts to 1279 and 1196 cm-1 in X- and XMP-, respectively. Again, the mode is not observable in X2-. The bands at 1211 in X and 1220 cm-1 in XMP are lowintensity modes but show a high sensitivity to deuteration due to contributions from the N7-H bend in addition to the C8H bend.

6-Oxopurines in Solution Conclusions Oxypurines can exist in many possible tautomers in solution. Knowing the precise tautomers of these molecules is crucial to understanding the mechanisms of the many enzymes of which these are substrates. Several computational and spectroscopic studies have been carried out in the past on the neutral states of purines. However, unequivocal determination of the solution tautomers of their deprotonated and protonated forms has remained unresolved. We have used a powerful combination of resonance Raman spectroscopy and quantum chemical calculations to: (1) identify the solution state tautomers of 6-oxopurine bases and nucleotides and their structures; (2) make detailed normal mode assignments from computed and experimental isotopic shifts of the nucleobases and nucleotides; and (3) identify markers of protonation and tautomeric states. The hypoxanthine anion is formed via deprotonation of the H7/H9 protons leading to a single tautomeric species of the anion in solution. Anions of IMP and GMP are formed via loss of the N1 protons. At low pH, hypoxanthine is protonated at N1, N7, and N9. Xanthine on the other hand is a single N7 tautomeric species in solution at physiological pH. The monoanionic form of xanthine and xanthosine monophosphate are formed via loss of the N3 proton. Interestingly, the pKa of the N3 proton in XMP is lower than that of the free base, and XMP is an anion at physiological pH.21 Raman shifts show systematic variation with change in the protonation state. Deprotonation causes a downshift in all the modes corresponding to carbonyl stretch and ring vibrations in 6-oxopurines. The three purines studied here remain in the ketone form at higher pH although the C6dO carbonyl bond is weakened. In xanthine and XMP, however, the additional carbonyl group at C2 is weakened considerably in response to the deprotonation at N3 and consequent delocalization of the electrons in the ring, while the C6dO is relatively less affected. The pyrimidine and imidazole ring modes couple to the N-H bending vibrations and are expected to be sensitive markers of hydrogen bonding contacts of these nucleobases. These analyses provide a set of diagnostic markers for identification of the ionization and protonation states of 6-oxopurines. The experimental trends on protonation and deprotonation are reliably reproduced by calculations. Although the modes which decouple from N-H bending vibrations upon deuteration are not predicted well by DFT, effects of isotope labeling are reliable for most ring modes. Thus the computed normal mode composition is corroborated by experimental isotope-induced shifts. Acknowledgment. This research was supported by the Department of Biotechnology, Department of Science and Technology, India. M.P. is a recipient of the Innovative Young Biotechnologist Award of the Department of Biotechnology, India. Supporting Information Available: Computed (DFTB3LYP/6-31G(d,p)) bond distances and Mulliken charges of the nucleobases guanine, hypoxanthine, and xanthine are given in Tables S1-S6. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Wen, Z. Q; George, J; Thomas, J Biopolymers 1998, 45, 247–256. (2) Sokolov, L.; Wojtuszewski, K.; Tsukroff, E.; Mukerji, I. J. Biomol. Struct. Dyn. 2000, 2, 327-334. (3) O’Connor, T.; Johnson, C.; Scovell, W. M. Biochim. Biophys. Acta 1976, 447, 495.

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