Preferential Protonation and Methylation Site of ... - ACS Publications

J. Phys. Chem. B 2008, 112, 3259-3267

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Preferential Protonation and Methylation Site of Thiopyrimidine Derivatives in Solution: NMR Data Artem V. Kozlov,† Vyacheslav E. Semenov,† Anatoliy S. Mikhailov,† Albert V. Aganov,‡ Michael B. Smith,§ Vladimir S. Reznik,† and Shamil K. Latypov*,† Institute of Organic and Physical Chemistry, ArbuzoV Str. 8, Kazan, 420088, Russian Federation, Kazan State UniVersity, Kazan, 420008, Russian Federation, and UniVersity of Connecticut, 55 North EagleVille Road, 4-60, Storrs, Connecticut 06269-3060 ReceiVed: NoVember 16, 2007; In Final Form: January 4, 2008

Protonation (alkylation) sites of several thiopyrimidine derivatives were directly determined by 1H-15N (1H13 C) heteronuclear single quantum coherence/heteronuclear multiple bond correlation methods, and it was found that in all compounds, protonation (methylation) occurred at the N1 nitrogen. GIAO DFT chemical shifts were in full agreement with the determined tautomeric structures. According to ab initio calculations, the stability of the different protonated forms and methylated derivatives was favored due to thermodynamic control and not kinetic control.

Introduction An important method used in the rational design of new active compounds is to combine molecules or fragments possessing desired properties into macrocycles by different spacers. This approach assumes the preorganization of active moieties in a rational way, with the possibility of incorporating new characteristics for derivatives. Further, linked systems are of considerable intrinsic interest since a number of systems have now been demonstrated to give rise to new derivatives whose properties may be somewhat more than the sum of the parts.1 To this end, the incorporation of nucleic acid bases into macrocycles with different spacers is very promising since most are widely implicated for a variety of biological activities.2 In addition, the introduction of different electron-donating or -withdrawing substituents into the nucleic acid may change the acid-base character of the nucleobases and their tautomeric composition and can modify the reactivity and complexion ability of these compounds to target molecules.3,4 These ideas prompted us to synthesize thio-analogues of pyrimidine and their macrocyclic derivatives. These rare 2(4)thiouracil tautomers may have important regulating functions in carcinogen, neoplastigen, tumorigen, or teratogen processes and may be responsible for spontaneous point mutations.5 Therefore, the rational design of such compounds is dependent on understanding their conformational and tautomeric properties. The character of interactions of nucleic acids with different target compounds of biological interest is important, and there are a number of publications dealing with this subject.6-9 Even the A-T or G-C base pair stabilities in different models of DNA/RNA structures (Watson-Crick, Hoogsteen, and JanusWedge) have been reevaluated recently.10-13 The structure and stability of these complexes are related to the tautomeric structure of the nucleic acid, the dependence of tautomeric and supramolecular structures of substituents on the heterocycle, the * To whom correspondence should be addressed. Tel.: +7 843 2727484; fax: +7 843 2732253; e-mail: [email protected]. † Institute of Organic and Physical Chemistry. ‡ Kazan State University. § University of Connecticut.

influence of solution properties on structure, etc. Moreover, the influence of protonated (charged) forms (even in trace quantities) can be important to the overall structure and properties of such systems.14-16 Therefore, proton transfer and tautomerism are subjects of long-standing interest.17-19 In principle, protonation can be considered as a gentle method to prepare target molecules with the requisite noncovalent or covalent bonds. In general, proton-transfer reactions are important in chemical processes and in biomolecular processes of living organisms.20,21 The latter include most enzymatically catalyzed reactions (e.g., ATP hydrolysis/synthesis by F1F0-ATP synthase). Furthermore, the protonation state of chemical groups (e.g., nucleic acids) is fundamentally related to their biomolecular function. Therefore, there has been interest in this phenomenon, and the site of protonation and stability of different forms are also subjects of long-standing interest.22-24 In most cases, our attention was focused on heterocyclic and exocyclic nitrogen atoms and to a lesser extent on oxygen as the most probable sites of proton binding. However, it was recently demonstrated that carbon also can serve as an effective proton acceptor.25,26 The need to monitor protonation of pyrimidine/purine acids or DNA/RNA prompted the development of experimental approaches, including the determination of the site of protonation or alkylation,27,28 where UV and IR spectroscopic methods have been used widely. We also explored the use of 1H NMR chemical shift (CS) data to determine the site of protonation by a change of CSs during titration by an acid. However, these methods are not very sensitive to the changing electronic structure during proton binding, and sometimes, the relation between changing the frequency (UV, IR, and 1H NMR) and the protonation site is not straightforward.29,30 Theoretically, 13C NMR is a more sensitive probe to detect these structural changes, and 15N NMR would be the safest method, but 1-D 15N spectra at natural abundance cannot be acquired for the majority of the cases.31,32 Inverse detected long-range 1H-13C and, in particular, 1H15N heteronuclear CS correlation techniques have revolutionized the use of NMR as a structural probe at natural abundance.33

10.1021/jp710952r CCC: $40.75 © 2008 American Chemical Society Published on Web 02/20/2008

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Figure 1. Structures of investigated thiopyrimidine derivatives.

Kozlov et al. conformational and supramolecular structure of its derivatives.34 Therefore, we paid special attention to the structure of protonated and methylated forms in solution. Here, we present our results of an investigation into the preferential site of protonation (methylation) of thiopyrimidine derivatives by NMR methods. In addition, the scope and limitation of computational methods for the evaluation of NMR CSs and their usage in tautomeric structure identification are considered. Finally, the ability of different models to predict the protonation (alkylation) reactions sites is compared. Materials and Methods

Figure 2. Conformational equilibrium of 1.

Indeed, NMR methods provide a direct way for monitoring protonation (alkylation) sites due to spin-spin connectivities. Preliminary data suggest that the contribution of the protonated form of thiopyrimidine plays an important role in the

Synthesis. 2-Methylthio-4-decylamino-6-methylpyrimidine (1) was synthesized by amination of 1,2,3,4-tetrahydro-2,4dithio-6-methylpyrimidine, followed by reaction with dimethyl sulfate. The purity of the compounds was validated by NMR, mass spectrometry, and elemental analysis. 2-Methylthio-4amino-6-methylpyrimidine (2),35 2-benzylthio-4-amino-6-methylpyrimidine (3),35 2-methylthio-4-N,N-dimethylamino-6methylpyrimidine (4),36 and N,N-bis(2-methylthio-6-methylpyrimidine-4-yl)hexylenediamine (5)37 were prepared according to procedures reported previously. Trifluoroacetic acid was

Figure 3. 1-D 1H NMR spectra in CDCl3 of 1 (T ) 303 and 233 K), 1a (T ) 303 and 233 K), and 1b (T ) 303 K). Tosylate signals in spectrum of 1b are indicated by asterisks.

Thiopyrimidine Derivatives in Solution

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TABLE 1: Principal 1H, 13C, and 15N Experimental CSs for 1-5 in CDCl3a nucleus

1 (233 K)

1a (233 K)

1b (303 K)

2 (303 K)

N1 N3 N7 C2 C4 C5 C6 CH3(C6) CH3(S)/CH2(S) CH3(+N1) H(C5) H(N7)/CH3(8) H(N7)/CH3′(8) CH3(C6) CH3(S)/CH2(S) H(+N1)/CH3(+N1)

241.50 223.30 84.00 170.63 161.67 99.64 163.76 23.65 14.09

154.50 218.20 112.20 165.87 160.81 100.67 152.48 18.23 13.48

249.98 228.24 68.20 171.37 162.73 99.22 166.05 23.75 13.84

166.33 nab na 167.06 163.11 99.92 157.50 18.94 13.46

248.76 227.27 68.76 170.45 162.81 99.58 166.02 23.71 35.04

5.83 4.77

6.28 8.89

146.67 223.32 116.14 167.06 158.91 104.37 153.63 20.12 15.47 35.81 7.06 9.76

2.22 2.49

2.27 2.53 14.15

2.41 2.59 3.64

5.97 4.79 4.79 2.28 2.50

6.27c 6.98c na 2.44c 2.58c 14.23c

5.97 4.83 4.83 2.28 4.37

nucleus

4 (233 K)

N1 N3 N7 C2 C4 C5 C6 CH3(C6) CH3(S)/CH2(S) CH3(+N1) H(C5) H(N7)/CH3(8) H(N7)/CH3′(8) CH3(C6) CH3(S)/CH2(S) H(+N1)/CH3(+N1)

238.00 226.12 67.39 169.81 161.57 96.54 164.46 24.06 14.12 5.99 3.02 3.24 2.32 2.53

4a (233 K)

4b (303 K)

155.98 224.00 93.74 165.82d 160.45d 97.20d 155.98d 18.85d 13.49d

149.44 na 94.65 166.06 158.26 101.35 157.10 21.18 15.85 36.45 6.80 3.24 3.28 2.58 2.58 3.67

6.22 3.26 3.42 2.44 2.60 14.18

2a (303 K)

5 (233 K) 243.74 225.91 84.20 170.40 161.72 99.79 163.38 23.45 14.06

3 (303 K)

5a (303 K) 155.46 na na 166.55 na 101.25 152.95 18.12 na

5.93 5.41

6.39 9.01

2.24 2.52

2.30 2.55 14.08

3a (303 K) 170.85 na 94.40 165.61 162.87 100.41 157.73 19.19 35.11 6.26 na na 2.27 4.35 na 5b (303 K) 147.64 223.36 113.94 167.47 159.08 104.05 154.09 20.21 15.70 36.02 6.83 9.53 2.35 2.58 3.61

a In the case if two conformers are presented in the spectra, CSs are given for the Z form, and all CSs can be found in the Supporting Information. Because the low solubility of protonated forms S/N in NMR spectra are essentially worse in these cases, no signals for some nuclei were obtained. c T ) 233 K. d T ) 303 K. b

purchased from Lancaster and distilled before use. The protonation of the substituted pyrimidines proceeded by the addition of a stoichiometric amount or excess of CF3COOH in CDCl3 solutions of 1-5. Methylated derivatives of 1, 4, and 5 in the form of p-toluenesulphonates (tosylates) were synthesized as follows. The substituted pyrimidines 1, 4, and 5 (2 mmol) were stirred with the methyl ester of p-toluenesulphonic acid (8 mL) at 100 °C for 5 h, followed by the addition of ether and then filtration. The tosylated products were washed with ether and dried. The purity of the tosylates was confirmed by NMR and elemental analysis. Unfortunately, 2b and 3b were not isolated due to the dissociation of the thiopyrimidine derivatives (2 and 3) under methylation. NMR Spectroscopy. All NMR experiments were performed using a Bruker Avance-600 spectrometer (14.1 T) equipped with a 5 mm diameter gradient inverse broad band probehead. Frequencies were 600.13 MHz in 1H, 150.90 MHz in 13C, and 60.81 MHz in 15N experiments. Solutions for probes were made at a concentration of 100 mM by being dissolved in 0.6 mL of CDCl3 (99.5% D, Deiton) and were placed in standard NMR tubes (Norell) and then sealed. CSs were reported on the δ (ppm) scale and were relative to the 1H and 13C signals of tetramethylsilane (TMS) (0.00 ppm). 15N CSs were referenced to the 15N signal of CH3CN (235.50 ppm). Experiments were carried out using a Bruker variable temperature unit BVT3000 (with BTO2000, accuracy: (0.01 °C) at 303 K, and if necessary, conformational or proton exchange at 233 K was slowed using a liquid nitrogen evaporator.

Calculations. The ab initio quantum chemical calculations were performed using Gaussian 9838 on an IBM PC compatible computer. Full geometry optimizations to a minimum energy were executed at the ab initio HF/6-31G level for all models. CSs were determined by the GIAO method within the DFT framework using a hybrid exchange-correlation functional, B3LYP, at the 6-31G(d) level unless otherwise noted. All data were referred to TMS (0.00 ppm) for 1H and 13C and CH3CN (235.50 ppm) for 15N CSs that were calculated in the same conditions. NPA charges were calculated at the HF/6-31G level. To obtain proton affinities (PA) and Gibbs free energy data, calculations at the RB3LYP/6-31G(d) level were employed. Gibbs free energy values were calculated at standard conditions (T ) 298.15 K and P ) 1.00 atm). Results and Discussion The structures of thiopyrimidine derivatives are illustrated in Figure 1. Structure Elucidation and Protonation/Methylation Site. Assignment of the 1H and 13C spectra of 1 and its protonated and methylated forms (1a and 1b, Figure 3) will be considered in detail. For other compounds, the analysis was carried out in a similar way but is otherwise not mentioned. The 1H spectrum of 1 in CDCl3 consists of signals assigned to the protons of the methyl and methylene groups, the broadened lines of NH, NCH2, and an aromatic proton (H5). The essential broadening of these signals is due to rotation around the C4-N7 bond, which is intermediate on the NMR chemical shift time scale (1 (T )

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Figure 4. Principal HMBC correlations: (a) 1H-13C for 1a; (b) 1H15 N for 1a; and (c) 1H-13C and 1H-15N for 1b.

303 K), Figure 3).34 Decreasing the temperature leads to coalescence of the lines, and finally, at ca. T ) 233 K, the 1H NMR spectra includes a set of sharp signals that can be ascribed to a two-component equilibrium of the Z and E forms (Figure 2) at a slow exchange on the NMR chemical shift time scale (1 (T ) 233 K), Figure 3). Complete assignments of the 1H, 13C, and 15N NMR spectra of these derivatives are straightforward based on a variety of correlations (2-D COSY, heteronuclear single quantum coherence (HSQC) (1H-13C and 1H-15N), and heteronuclear multiple bond correlation (HMBC) (1H-13C and 1H-15N)) starting from a well-documented reference group, for example, H5. Related 1- and 2-D NMR spectra can be obtained in the Supporting Information. Principal CSs are given in Table 1 (all CSs can be found in the Supporting Information). Protonation leads to slowing the rotation around the C4-N7 bond with respect to rate in neutral molecules (unprotonated), so even at room temperature, two set of signals are observed separately in the 1H NMR spectrum due to the Z and E forms (1a (T ) 303 K), Figure 3). Strong H5/NH NOE and no NOE between H5 and CH2(8) unambiguously demonstrate that the dominant form is Z (details in the Supporting Information). At a lower temperature (T ) 233 K), a new proton signal appears at ca. 14 ppm, presumably due to the N+H proton (1a (T ) 233 K), Figure 3). Methylation similarly slows rotation around the C4-N7 bond, we observe sharp lines in the 1H NMR spectrum at 303 K, and the signal of NH moves to a lower field (ca. 10 ppm) (1b (T ) 303 K), Figure 3). However, in this case, the equilibrium is completely biased toward the Z form. Its conformational structure is proven by indicative NOE interactions (Supporting Information). The protonation and methylation phenomena also have some impact on the 13C NMR spectra. A combination of 2-D HSQC/HMBC (1H-13C) and HSQC/ HMBC (1H-15N) was used to determine directly the preferential

Kozlov et al. protonation and methylation site of 1. To have all signals at a slow exchange regime on the NMR chemical shift time scale, all 2-D experiments for 1 and 1a were carried out at a low temperature (T ) 233 K). The key 1H-13C correlations (Figure 4a) led to the univocal establishment of the protonation site using connectivities of the N+H proton (14.15 ppm) to a variety of carbons (C5, C2, C6, and Me(C6)), which have well tabulated CSs. Moreover there are a number of HSQC and HMBC (1H-13C) correlations from these carbons to other protons that allows determination of the overall structure of the molecule. Finally, independent proof of the structure of 1a was obtained by a combination of 2-D 1H-15N HSQC and HMBC experiments (Figure 5). First of all, starting with data for several protons with well-tabulated values (independently established) (N-CH2-CH2-, H5, Me(C6), and amide NH from NOESY/ COSY), CSs of three nitrogen atoms were established based on 1H-15N HMBC (long-range) correlations (key correlations also are shown in Figure 4b). The 1H-15N HSQC spectra revealed those protons that are attached directly to nitrogen (N1 and N7). Thus, it was shown that in 1, protonation occurred at the N1 position. We also measured 1H, 13C, and 15N CSs for the protonated form. In the case of methylated derivatives of nucleic acids, the key correlations are the N+Me to vicinal C2/C6 carbons in the HMBC spectra (Figure 4c). The Me/N cross-peak in the 1H15N HMBC spectrum leads to the unequivocal assignment of N1. In addition, the set of 1H-15N HMBC correlations from exocyclic aliphatic group protons to the exocyclic NH nitrogen indicates an amine type of nitrogen and that the methylated form exists in the tautomeric form shown in Figure 4. In the same way, we established the site of protonation for 2-5 and the site of methylation for 4 and 5. Chemical Shift Analysis. NMR CSs are summarized in Table 1. As one can see, there are major effects due to protonation/ methylation on 15N CSs, while to a lesser extent on 13C and 1H CSs. In general, the observed shifts are in qualitative agreement with data on the alkylation of some pyrimidine and purine bases.39,40 Namely, the most significant changes have been observed when nitrogen was directly protonated (high-field shifts by ca. -85 ppm for protonation and by ca. -95 ppm for methylation). The CSs of exocyclic nitrogen (N7) connected to carbon in the para position to the protonation atom in all compounds also suffered significant changes, but in the opposite direction (low-field shifts by ca. 30 ppm).

Figure 5. Superposition of 2-D 1H-15N HSQC (square cross-peaks) and 1H-15N HMBC (round cross-peaks) spectra of 1a at T ) 233 K.

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TABLE 2: R2 Values for 1a/b-5a/ba,b

a In parentheses are data for HF/6-31G//HF/6-31G GIAO CSs. b Full set of correlation analysis results for all compounds can be found in the Supporting Information. †Because of low solubility, no full set of experimental data was available to calculate the value.

Protonation of 1-5 led to considerable upfield shifts of 13C CSs, particularly in the position β to the protonation atom. These upfield protonation shifts may be mainly attributed to the electric field of the charged group and its gradient at the site of the observed carbon, while in the γ positions, the contributions of inductive and steric effects will be more relevant. In nitrogen heteroaromatics, upfield protonation shifts are found for ortho carbons, while those in meta positions are deshielded when protonated.41,42 Comparisons of Calculated Chemical Shifts with Experimental Values. The question as to whether the GIAO (nonempirical) predictions can account for these changes in electronic structure is of interest.43-47 Therefore, GIAO calculations of CSs on optimized (for gas phase) structures were carried out (data of calculation can be found in the Supporting Information) and compared with experimental results. To begin with, we ran the calculation of CSs on the B3LYP/6-31G(d) level and geometry optimization on the HF/6-31G (B3LYP/6-31G(d)// HF/6-31G) level because for some nitrogen containing heteroaromatics, it resulted in good quality CSs for a reasonably low cost of calculations.48 In addition, the combination of basis

sets was also probed for comparison reasons. The results of the linear regression analysis comparing experimental shifts with GIAO CSs are summarized in Tables 2and 3. 1H NMR. As one can see from Table 2, only for the C5 protonated form is the R2 value and, respectively, other statistical parameters (see Supporting Information) notably worse than the ones for forms protonated at other positions, where these values are quite high and of similar value (0.988, 0.985, and 0.994). Therefore, if proton CSs were used, the N7/N3/N1 position cannot be distinguished using only these data. Moreover, for various tautomeric forms, correlation coefficients (Table 3) also differ insignificantly and therefore cannot be used for an assignment with confidence. 13C NMR. Inspection of Table 2 shows that analysis of 13C CSs changes is clearly safer than the similar analysis of proton CSs. Data obtained from statistical analysis shows a small but valuable preference for the N1 protonated form (e.g., R2 0.992 vs 0.978). At the same time, the analysis of the methylation position could not be performed using 13C CSs data only because the experimental CSs show a correlation for both N1 and N3 methylated forms (R2 ) 0.991 and 0.990).

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Kozlov et al.

TABLE 3: R2 Values for 1, 1a, and 1b (Protonation/Methylation at Position N1) for Different Tautomeric Formsa,b

a

In parentheses are data for HF/6-31G//HF/6-31G GIAO CSs. b Full set of correlation analysis results can be found in the Supporting Information.

The use of 13C NMR for tautomeric form assignments of neutral (unprotonated) compounds fails to differentiate the N7 and N3 tautomers (Table 3), but for both protonated and methylated forms, 13C can be applied with some precaution since the differences in the R2 values are not large. To ensure that these are not due to limitations of the employed method, we performed calculations of CSs for more light combinations (HF/6-31G//HF/6-31G, data for 1 in Tables 2 and 3). In general, all tendencies were very similar to those commented on previously. Therefore, the fact that 13C and 1H CSs depend less on the tautomeric form in these compounds is likely due to intrinsic reasons. 15N NMR. A comparison of experimental versus calculated 15N CSs clearly demonstrates that we can unequivocally establish the protonation or methylation site and tautomeric forms. The calculated 15N CSs for correct protonated or methylated structures correlated quite well with experimental CSs (R2 ) 0.97-0.99), while the calculated CSs for structures that proved to be incorrect showed a poor correlation with experimental data (R2 ) 0.6 or less). Thus, 15N CSs definitely can be used for spectra-structural correlations in the case of such tautomeric variety. It is clear that the 15N CSs are the most sensitive to tautomeric differences and therefore are the best choice for such structural tasks. Preferred Protonation Sites: Theoretical Considerations. Actually, the protonation/methylation takes place at the heterocyclic N1 nitrogen in all cases, although, hypothetically, it can occur at several basic atoms in the title molecules. Therefore, it is of interest to see if any theory can take into account such selectivity. In general terms, both the electronic disposition of the nitrogen/carbon atoms and the stability of the resulting ion can constitute a driving force. Therefore, to check the importance of these factors, ab initio calculations of natural population analysis (NPA) charges49 and the proton affinity (PA) were performed on derivatives 1-5.

TABLE 4: NPA Charges of Most Electronically Negative Atoms of 1-5a nucleus N1 N3 N7 C5 a

1

2

3

4

5

-0.64 -0.64 -0.69 -0.42

-0.63 -0.63 -0.88 -0.43

-0.61 -0.65 -0.88 -0.44

-0.64 -0.64 -0.51 -0.43

-0.62 -0.65 -0.69 -0.43

HF/6-31G//HF/6-31G.

Charge Distribution. The differences in NPA charges between gas- and solvated-phase structures are negligible.50 Therefore, NPA charges (HF/6-31G//HF/6-31G) were calculated only in vacuum (Table 4.). The heterocyclic (N1 and N3) and, to a greater extent, exocyclic (N7) nitrogen atoms are negative. Moreover, the charge on C(5) is also negative in all bases. Inspection of Table 4 demonstrates that only on N7 is there a noticeable charge variation in the 1-4 series, presumably due to electronic effects of the R1 and R2 substitutions. Indeed, for the substitution of R2 by a proton (2 and 3, H is less electronegative than Me), the NPA charges on N7 increase with respect to the value in 1, contrasting with R1 ) R2 ) Me (4), where the NPA charge on N7 decreases. In general, in all compounds, except 4, the NPA values indicate more negative N7 nitrogen atoms than N3 and N1, which suggest that N7 would be more favorable for protonation if kinetic control took place. Thus, by arguing only NPA charge considerations, the predicted trend of protonation/methylation disagrees with experimental results. Therefore, because N1 protonation/methylation is favored in all cases, we attempted to find signs of thermodynamic stability of these protonated/methylated forms. PA. To obtain an indication of the relative stability of the different protonated forms and methylated derivatives, total energies were calculated and compared. In a gas-phase environment that is more frequently used, the selected quantity is associated with PA, defined as the negative of the enthalpy change at standard conditions (i.e., temperature and pressure).51

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TABLE 5: PA,a Relative Proton Affinities (-∆PA),a and Relative Gibbs Free Energies (∆G)a for Different Protonated and Methylated Forms of 1-5

a In kJ/mol, RB3LYP/6-31G(d)//HF/6-31G, values belong to the most stable studied conformers. This theory level gives a good correlation with experimental data for water: PA ) 697.6 kJ/mol vs PA (expt) ) 697.0 kJ/mol.54

TABLE 6: Relative Gibbs Free Energiesa of 1, 1a, and 1b (Protonation/Methylation at Position N1) for Different Tautomeric Forms

a

In kJ/mol, B3RLYP/6-31G(d)//HF6-31G, all data belong to Z conformers.

At the same time, the Gibbs free energy difference (∆G) can be directly compared with the protonation distribution measured by NMR. Because of the low dielectric constant of CHCl3 (4.8 at 293 K), it seems reasonable to carry on the calculations in a vacuum. Therefore, both quantities were calculated at the RB3LYP/6-31G(d) level52,53 (Table 5).

In general, changes in both quantities were in parallel for all compounds. For all available variations of substitution (at exocyclic nitrogen and at sulfur), protonation/methylation showed a notable preference for the N1 position (Table 5). Aliphatic substitution on N7 led to some additional stabilization of the N1 protonation/methylation site. The substitution of Me

3266 J. Phys. Chem. B, Vol. 112, No. 10, 2008 (C2) at S by Bz (3) essentially destabilized the N3 protonation site. In all cases, in contrast to NPA charge predictions, the exocyclic N7 position is clearly the unfavorable site of protonation/methylation. Finally, to estimate the relative stability of other tautomers, their energies were calculated relative to 1 (Table 6). The tautomer I is much more stable than other ones in the neutral molecule and in the protonated/methylated form, in full agreement with experimental results. Thus, gas-phase data were in reasonable agreement with solution results for pyrimidines,50 and one can conclude that only one tautomer can be expected to predominate experimentally in solution. Analysis based on thermodynamic parameters describes adequately the observed preferences on protonation sites in our case. Moreover, taking into account examples on other pyrimidine protonations,50 this approach has a reasonable value for prediction. Our calculations confirm some examples taken from the literature.55 Conclusion The protonation and methylation of a series of substituted thiopyrimidine derivatives was investigated. The site of the protonation/methylation was directly established by HMBC 1H13C and 1H-15N experiments. In all cases, protonation occurred at the N1 position. The scope and limitation of the theoretical CSs (GIAO DFT) for determining the site of protonation/ methylation was analyzed. 15N CSs showed good spectrastructure correlations, while 13C CSs have to be used with care and 1H CSs cannot be used in all cases. Protonation in the title molecules was also studied theoretically by using B3LYP/6-31G(d) quantum chemical methods. The results of quantum chemical calculations allowed us to conclude that the charge (NPA) distribution data do not allow correct predictions of the site of protonation, while calculated thermodynamic parameters are in good agreement with experimental observations. Acknowledgment. Financial support from the RFBR (0503-32558-a) is acknowledged, and S.L. acknowledges the Russian Science Support Foundation for a doctoral grant. This investigation was carried out in the NMR Department of the Federal Collective Spectral Analysis Center for Physical and Chemical Investigations of Structure, Properties and Composition of Matter and Materials (CKP SAC) and the Federal CKP for Physical and Chemical Investigations of Matter and Materials (FCKP PCI) (State Contracts of the Russian Federation Ministry of Education and Science 02.451.11.7036 and 02.451.11.7019). Supporting Information Available: 1- and 2-D NMR spectra, experimental and calculated CSs, and their correlation analysis. This information is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Gloe, K. Macrocyclic Chemistry. Current Trends and Future PerspectiVes; Springer: Berlin, 2005. (2) Murakami, Y.; Kikuchi, J.-I.; Hisaeda, Y.; Hayashida, O. Chem. ReV. 1996, 96, 721. (3) Nugent, R. A.; Schlachter, S. T.; Murphy, M. J.; Cleek, G. J.; Poel, T. J.; Wishka, D. G.; Graber, D. R.; Yagi, Y.; Keiser, B. J.; Olmsted, R. A.; Kopta, L. A.; Swaney, S. M.; Poppe, S. M.; Morris, J.; Tarpley, W. G.; Thomas, R. C. J. Med. Chem. 1998, 41, 3793. (4) Kool, E. T. Chem. ReV. 1997, 97, 1473. (5) Leit, A.; Adamowicz, L. J. Am. Chem. Soc. 1990, 112, 1504. (6) Muller-Dethlefs, K.; Hobza, P. Chem. ReV. 2000, 100, 143 and references cited therein.

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