13C Chemical Shift Tensors in Hypoxanthine and ... - ACS Publications

ACS2GO © 2018. ← → → ←. loading. To add this web app to the home screen open the browser option menu and tap on Add to homescreen...
0 downloads 0 Views 218KB Size
Download PDF
J. Phys. Chem. A 2010, 114, 1985–1995

1985

13

C Chemical Shift Tensors in Hypoxanthine and 6-Mercaptopurine: Effects of Substitution, Tautomerism, and Intermolecular Interactions Katerˇina Malinˇa´kova´,† Lucie Novosadova´,† Manu Lahtinen,‡ Erkki Kolehmainen,‡ Jirˇ´ı Brus,§ and Radek Marek*,† National Center for Biomolecular Research, Masaryk UniVersity, Kamenice 5/A4, CZ-62500 Brno, Czech Republic, Department of Chemistry, UniVersity of JyVa¨skyla¨, P. O. Box 35, FIN-40014, Finland, and Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, HeyroVske´ho na´m. 2, CZ-16206 Prague, Czech Republic ReceiVed: October 21, 2009; ReVised Manuscript ReceiVed: December 10, 2009

Principal values of the 13C chemical shift tensor (CST) are measured for two biologically interesting and structurally related compounds, hypoxanthine and 6-mercaptopurine, and differences in the values are discussed with an attempt to reveal chemical shifts sensitive to substitution and prototropic tautomerism in the purine ring. Furthermore, methods of density-functional theory (DFT) are used to calculate principal values of the 13 C chemical shift tensor and orientations of the principal components. Values calculated for isolated molecules are compared to those for several supramolecular clusters and then to experimental data to investigate the degree of modulation of the 13C CSTs by molecular packing. Focusing on the protonated carbons, C2 and C8, which are crucial for relaxation measurements, we show that neglecting intermolecular interactions can lead to errors as large as 30 ppm in the δ22 principal component. This has significant implications for the studies of molecular dynamics, employing spin relaxation, in large fragments of nucleic acids at high magnetic fields. Introduction Noncanonical purine base hypoxanthine (1a) and its thio analogue 6-mercaptopurine (2b), see Chart 1, have been extensively studied for their numerous biological implications. Hypoxanthine is an intermediate of purine metabolism in living systems. It most commonly arises from the oxidative deamination of adenine. Subsequently, it is a substrate of the metalloenzyme of xanthine oxidase in uric acid production. At the same time, it can play the role of precursor of the canonical purine bases in the salvage pathway for purine nucleotide synthesis. Additionally, it can be formed when a cell is under oxidative stress; nitrogen loss in adenine occurs in this case either spontaneously by hydrolysis or at a much higher rate due to reaction with free radicals.1,2 Such a conversion leads to point mutations, which are associated with carcinogenesis and cell death. This inevitable damage is counteracted by glycosylase enzymes, which cleave damaged bases from DNA. As a constituent of nucleic acid, it is occasionally found in the anticodon of tRNA where it is present in the form of its nucleotide inosine.3 Thiopurines, such as 6-mercaptopurine, are cytotoxic and immunosuppressant compounds, which are used to treat childhood acute lymphoblastic leukemia, inflammatory bowel disease, and transplant rejection.4-6 They act as prodrugs, which are metabolized to give thioguanine nucleotides that exert their therapeutic effects by incorporation into DNA or inhibiting purine synthesis. Recently, it was discovered that thioguanine in the context of a thioG-C base pair is responsible for only modest, localized changes in DNA structure. Nevertheless, * Corresponding author. Phone: +420-549495748. Fax: +420-549492556. E-mail: [email protected]. † Masaryk University. ‡ University of Jyva¨skyla¨. § Academy of Sciences of the Czech Republic.

CHART 1: Structures and Numbering Scheme for Hypoxanthine (1a; 1b, the Tautomer Used in Calculations Only) and 6-Mercaptopurine (2b, Crystallizes as Monohydrate 2b · H2O)

dynamics of this base pair is significantly increased and stability decreased when compared to the canonical G-C pair,7 which inevitably leads to modifications in specific DNA-processing enzyme activities and DNA-protein interactions. Apart from this biological importance, direct reaction between 6-mercaptopurine and CdII and deprotonation of the resulting polymer has proven to be useful route for isolation of one-dimensional systems on surfaces, which are currently investigated for their potential use as nanomaterials.8 Hypoxanthine and 6-mercaptopurine are nice examples of very similar structures with different biological effects. Explanation for their different activity can be found by closer inspection of interatomic interactions at molecular and supramolecular levels. Substitution of the exocyclic oxygen in hypoxanthine by sulfur results in different protonation patterns and hydrogenbonding interactions. This is documented in studies on the

10.1021/jp9100619  2010 American Chemical Society Published on Web 01/07/2010

1986

J. Phys. Chem. A, Vol. 114, No. 4, 2010

prototropic tautomerism of purine derivatives.9,10 The N7-H/ N9-H tautomeric ratios were determined in DMSO-d6, DMFd7, and methanol-d4 solutions with similar results for all of the solvents. While for hypoxanthine the two tautomers are almost equally populated at 303 K, with only a slight preference of the N7-H form, for 6-mercaptopurine the N7-H tautomer is much more favored (N7-H/N9-H ≈ 4/1).9,10 In contrast to solution, the N9-H tautomer was found to be dominant in the solid state. Hypoxanthine and anhydrous 6-mercaptopurine11 exist as N9-H tautomers in crystal, and hypoxanthine exists as either triclinic12 or monoclinic13 polymorph. Nevertheless, 6-mercaptopurine tends to crystallize as monohydrate with proton attached to nitrogen N7,11,14,15 which also indicates that its N7-H tautomer is more favored than the N7-H form of hypoxanthine. One of the most successful techniques for providing structural information including intermolecular interactions and dynamic behavior is NMR spectroscopy. Circulation of current within electronic clouds of molecules gives rise to a local magnetic field at nuclei of interest, which is dependent on the molecular orientation relative to the applied magnetic field. The orientation dependence of this local field can be represented by a second rank chemical shift tensor (CST). The 13C and 15N CST principal components are sensitive probes of the electronic environment around the nucleus of interest. Their magnitudes and orientations within the molecular framework can be directly correlated with the local molecular structure. However, when the NMR experiments are performed on powdered solids, determination of the CST orientation is unavailable. For these samples, NMR chemical shift calculations16 can be used to obtain the CST orientation in the molecular framework. Density-functional theory (DFT) with B3LYP is very frequently used for these calculations, offering good accuracy together with acceptable computational costs.17 Among other applications, knowledge of CSTs is required to account for CSA relaxation mechanisms. This is important for a range of biomolecular NMR applications including the interpretation of spin relaxation data in terms of angular constraints18,19 and dynamical parameters.20 Of particular interest are the CSTs of the nucleobase carbons C2 and C8 in purine, and C5 and C6 in pyrimidine, which are used for probing the fast dynamics of nucleic acids by spin relaxation measurements.21,22 For these studies, experimental values for the principal components of the nucleobase 13C CSTs, reported previously for mononucleotide powders,23 are widely used. In this study, we examined whether it is possible to use mononucleotide CST data for larger molecules and supramolecular clusters, where hydrogen bonding and stacking interactions play a role. Although the sensitivity of 15N CSTs to intermolecular interactions is well known,24 for 13C these effects are often neglected. By an example of two purine-based compounds, we want to show how the 13C CSTs of the crucial carbons for relaxation measurements, C2 and C8, change due to local structural changes like substitution on the purine ring, tautomerism, hydrogen bonding, and stacking interactions. Combination of solid-state NMR experiments and quantum chemical calculations is used for this purpose. In advance, we analyze these effects also for the quaternary carbons of the purine ring, to supplement the literature with CST data for hypoxanthine and 6-mercaptopurine, which are not less important than the canonical nucleobases, but less studied. Furthermore, we examined the differences in electronic properties of these two structurally very similar but biologically different molecules.

Malinˇa´kova´ et al. Experimental Section Samples. Hypoxanthine and 6-mercaptopurine monohydrate were purchased from Sigma-Aldrich and used without further purification. X-ray Powder Diffraction. The X-ray powder diffraction data were measured with PANalytical X’Pert PRO diffractometer in Bragg-Brentano geometry using the step-scan technique and Johansson monochromator to produce pure Cu KR1 radiation (1.5406 Å; 45 kV, 30 mA). The data were collected by an X’Celerator detector using continuous scanning mode in 2θ range of 4-60° with a step size of 0.017° and counting times of 140 s per step. Programmable divergence slit (PDS) was used in automatic mode to set the irradiated length on sample to 10 mm together with a fixed 10 mm incident beam mask. Soller slits of 0.02° rad were used on incident and diffracted beam sides together with anti-scatter slits of 4° and 13 mm, respectively. The diffraction data were converted from automatic slit mode (ADS) to the fixed slit mode (FDS) data in PANalytical HighScore Plus v. 2.2d software package before further analyses. Lightly hand-ground powder sample was prepared on a siliconmade zero-background holder using petrolatum jelly as an adhesive. The sample was spun during the measurement to reduce orientation effects of the crystallites. The ICDD PDF-2 powder diffraction database25 implemented in Highscore Plus was used for the search-match phase identification analyses. The simulated X-ray diffraction (XRD) patterns used for comparison were generated by the program Mercury26 using the CIFs taken from the Cambridge Structural Database.26 To further evaluate structural consistency between the bulk powder and single crystals, the Le Bail method27 (whole profile unit cell refinement involving overlapping reflections, extraction of |F|hkl’s for structure solution, and space group determination) was made using the unit cell settings of the single crystal structure as the basis of the refinement (Supporting Information). Solid-State NMR Spectroscopy. Solid-state NMR experiments were performed at room temperature on a Bruker AVANCE-400 spectrometer operating at frequencies 400.13 MHz (1H) and 100.61 MHz (13C) and on a Bruker AVANCE500 spectrometer operating at frequencies 500.13 MHz (1H) and 125.77 MHz (13C). A Bruker 4 mm CP/MAS probe was used for all of the measurements. The 13C chemical shifts were referenced to crystalline R-glycine as a secondary reference (δst ) 176.03 ppm for carbonyl carbon). 13C CP/MAS spectra were recorded with a 3 ms contact time and a recycle delay of 240 s for hypoxanthine sample and 30 s for 6-mercaptopurine monohydrate. The ramped amplitude (RAMP) shape pulse was used during the cross-polarization and two-pulse phase-modulated (TPPM) decoupling during the acquisition. Spectral assignments were done with the use of NQS28 and CPPI29 pulse sequences and from the knowledge of solution-state chemical shifts.11 Program DMFIT30 was used to obtain principal components of the 13C CSTs from the MAS sideband patterns. Calculations. Geometries of isolated molecules built for the study of tautomerism and substitution effects were pre-optimized by the MM+ and AM1 methods. For the theoretical evaluation of intermolecular effects, geometries determined by singlecrystal X-ray diffraction analysis (obtained from the Cambridge Structural Database) were used. Proton positions in the case of X-ray structures and positions of all atoms in the case of isolated molecules were optimized within the Gaussian 03 code.31 Subsequently, nuclear shieldings were calculated. All of the calculations were performed within the density-functional theory (DFT).32 The hybrid exchange-correlation B3LYP33 functional was used in the 6-31G* basis set for geometry optimizations

13C

Chemical Shift Tensors in Hypoxanthine and 6-Mercaptopurine

and 6-311G** basis set for calculations of the nuclear shieldings. Components of the shielding tensors were computed by the gauge-including atomic orbital (GIAO) method.34 Subsequently, chemical shifts were calculated using the following equation: δi ) σst - σi + δst. The R-glycine cluster35 was built (see Figure S1 and Table S19 in the Supporting Information), and its shielding was calculated (σC13 of the CdO group: σst ) 0.5 ppm) by the same methods. The experimental chemical shift δst ) 176.03 ppm was employed for carbonyl carbon in R-glycine. Finally, root-mean-square deviations (rmsd) between theoretical and experimental data were calculated. Results and Discussion The basic framework topologies for hypoxanthine12 and 6-mercaptopurine monohydrate11 were obtained from the Cambridge Structural Database (CSD). Identity of the powder samples used for CP/MAS experiments with the geometries deposited in CSD was confirmed from X-ray powder diffraction data (see Experimental Section and Supporting Information). Slow Spinning 13C Spectra. Principal components of the 13C CSTs in the hypoxanthine and 6-mercaptopurine monohydrate samples were determined by using the one-dimensional CP/ MAS experiment. More sophisticated two-dimensional methods like 2D PASS turned out to be inefficient due to extremely long T1 proton relaxation times of the samples (more than 240 s in the case of hypoxanthine). A small number of the carbon atoms in the samples and their limited overlap in the 1D spectrum (only C2 and C8 carbon signals in the 6-mercaptopurine monohydrate sample overlap so much that the analysis of the sideband pattern is precluded) also makes the slow spinning 1D CP/MAS experiment the method of choice. Assignment of isotropic chemical shifts was done using the NQS and CPPI editing techniques and by comparison with calculated data. Obtained values are in overall agreement with chemical shifts determined in DMSO and DMF solutions,9-11 and also with solid-state isotropic chemical shifts for 6-mercaptopurine monohydrate reported previously.11 For the analysis of the MAS sideband pattern, the DMFIT program30 was used. Throughout this program, the Haeberlen-Mehring36,37 convention for description of the chemical shift tensor is used. Following this convention, three parameters of the chemical shift tensor can be determined from MAS sideband pattern: δiso, the isotropic value, is the average value of the principal components and corresponds to the center of gravity of the line shape; CSA ) δzz - δiso, the anisotropy, describes the largest separation from the center of gravity; and η ) (δyy - δxx)/(δzz - δiso), the asymmetry, indicates by how much the line shape deviates from that of an axially symmetric tensor. δiso can be determined from the centerband position and the two other parameters from an analysis of sideband intensities.38 From the definition of these three parameters, the principal components of the chemical shift tensor follow: δzz ) CSA + δiso, δyy ) δiso - CSA(1 - η)/2, δxx ) δiso - CSA(1 + η)/2. The principal components obtained from the DMFIT program were subsequently ordered according to the IUPAC39 rules: δ11 g δ22 g δ33. To minimize the error arising from analysis and to estimate the standard deviation, chemical shift tensor parameters δiso, CSA, and η were determined at three or four different MAS rates, and obtained principal components were averaged. Values for individual MAS rates, averaged values, and standard deviations can be found in the Supporting Information (Tables S1 and S2). Effects of Tautomerism and Substitution - DFT Calculations for Isolated Molecules. Hypoxanthine and 6-mercaptopurine · H2O samples used in this work differ structurally not

J. Phys. Chem. A, Vol. 114, No. 4, 2010 1987 TABLE 1: Anglesa that the 13C CST Principal Components δ11 Assume with the Bonds Depicted in Figure 1

a

atom

1a

1b

2b

C2 C4 C5 C6 C8

17.8 5.0 69.5 22.4 25.9

20.5 25.0 2.1 26.0 22.1

19.6 11.5 20.3 23.9 21.8

Values are given in deg.

TABLE 2: DFT Calculateda Isotropic Chemical Shifts and Principal Components of the 13C CSTb for 1a, 1b, and 2b, and Differences between the Values for Individual Structures atom C2

C4

C5

C6

C8

δ11 δ22 δ33 δiso δ11 δ22 δ33 δiso δ11 δ22 δ33 δiso δ11 δ22 δ33 δiso δ11 δ22 δ33 δiso

1a

∆(1af1b)

1b

∆(1bf2b)

2b

∑∆

227 135 61 140.9 219 155 63 145.6 164 156 51 123.9 251 104 93 149.4 211 117 63 130.2

2 -9 1 -1.8 -5 23 11 9.9 0 -17 -15 -10.7 -7 8 -7 -1.9 -1 15 0 4.6

229 126 62 139.1 214 178 74 155.5 164 139 36 113.2 244 112 86 147.5 210 132 63 134.8

0 3 -2 0.1 -16 3 -7 -6.8 33 4 1 12.5 63 38 -30 23.2 4 5 0 3.2

229 129 60 139.2 198 181 67 148.7 197 143 37 125.7 307 150 56 170.7 214 137 63 138.0

2 -6 -1 -1.7 -21 26 4 3.1 33 -13 -14 1.8 56 46 -37 21.3 3 20 0 7.8

a Geometries of all atoms were optimized by using the B3LYP/ 6-31G* method. Nuclear shieldings were calculated by B3LYP/ 6-311G**. Subsequently, chemical shifts were calculated using the following equation: δi ) σst - σi + δst. The shielding of R-glycine cluster (σC13 of the CdO group; σst ) 0.5 ppm) was calculated by the same method as used for 1a, and the experimental chemical shift δst ) 176.03 ppm for carbonyl carbon in glycine was employed. b Values are given in ppm.

only in the substituent bound to carbon C6, but also in the position of proton (see Chart 1). Hypoxanthine (1a), containing oxygen in position 6, crystallizes as N9-H tautomer,12 while 6-mercaptopurine (2b), containing sulfur, tends to crystallize as monohydrate with proton attached to nitrogen N7.14,15 To separate the effects of the tautomerism (N9-H vs N7-H) and the substitution change (CdO f CdS) on the 13C CSTs within the purine ring, the N7-H tautomer of hypoxanthine (1b) was calculated and analyzed in addition to 1a and 2b. To eliminate the effect of the crystal packing, principal components of the 13 C CSTs were calculated by DFT for isolated molecules of 1a, 1b, and 2b with fully optimized geometries. For all of the carbon atoms, δ11 and δ22 components lay within the plane of the purine ring, and δ33 is oriented perpendicular to the plane. The angles that δ11 components assume with the selected bonds are given in Table 1. Calculated chemical shifts are summarized in Table 2, and orientations of the CST principal components are depicted in Figure 1. Carbon C2 of the purine ring seems to be influenced neither by the position of proton nor by the substituent in position 6, when isotropic chemical shifts are compared among the three molecules. When CST principal components are compared, only

1988

J. Phys. Chem. A, Vol. 114, No. 4, 2010

Figure 1. DFT calculated orientations of the in-plane 13C CST principal components (δ11 in red, δ22 in blue, δ33 perpendicular to the plane; see Table 1 for depicted angles).

the effect of tautomerism is remarkable; δ22 decreases when proton moves from N9 to N7. Bridgehead atoms C4 and C5 are significantly influenced by both structural changes. The effect of tautomerism (1a f 1b) amounts to 10 ppm for δiso, with larger shielding in the case, when proton is attached to nitrogen next to particular carbon. This is in agreement with previous findings on the prototropic tautomerism of purines and purine analogues.40 The substituent effect (CdO f CdS) is more pronounced for C5, which can be understood as a matter of distance to the place of substitution. For C4, the effect of substitution is similar in size to the effect of tautomerism; δiso(C4) in 2b is about 7 ppm smaller than in 1b. Because the first structural change results in deshielding and the other in shielding, δiso(C4) in 1a and 2b differs only slightly. More detailed analysis can be done from the CST principal components. δ33(C4) shows behavior similar to the isotropic chemical shift; it increases with 1a f 1b formal transfer and decreases again with 1b f 2b structural change, with magnitudes nearly matching those observed for δiso. In contrast to that, δ22 reflects almost entirely the position of proton (effect of tautomerism), while δ11 is predominantly influenced by the substitution change in position 6 (CdO f CdS). Opposite to C4, the isotropic chemical shift of carbon C5 decreases from 1a to 1b and increases when the oxygen bound to carbon C6 is replaced by sulfur (CdO f CdS). Among CST principal components of the carbon C5, only δ33 behaves differently from that of carbon C4. Its change is more dramatic between the tautomers, while the substitution effect seems unimportant. Therefore, a decrease of the δ33(C5) value is observed when the N9-H tautomer of hypoxanthine (1a) is compared to the N7-H tautomer of 6-mercaptopurine (2b), unlike δ33(C4) values, which are rather similar. The intermediate principal component δ22(C5) is influenced in the same manner as the smallest one. It decreases significantly when the proton moves to neighboring nitrogen and stays nearly the same for both substituents. This behavior is fully analogous to that observed for δ22(C4). Therefore, the substituent effect is found entirely in δ11(C5); its value is more than 30 ppm smaller in both hypoxanthine tautomers (1a, 1b) than in 6-mercaptopurine (2b). The increase in δ11 component due to substitution is almost equal to the overall decrease in δ22 and δ33 components due to tautomerism. Therefore, δiso(C5) in 1a and 2b is very similar. For this carbon, different orientations of the in-plane components in 1a when compared to 1b and 2b should be noted (Figure 1). Carbon in position 6 is the place of substitution (CdO or CdS). Its isotropic chemical shift is almost the same for both hypoxanthine tautomers, but a more than 20 ppm increase is observed when the oxygen attached to it is replaced by sulfur

Malinˇa´kova´ et al. (CdO f CdS). Chemical shift increases in its δ11 and δ22 principal components, while δ33 gets smaller. δ11 changes most dramatically; the difference between hypoxanthine and 6-mercaptopurine is ca. 60 ppm. δ22 increases from 112 ppm (1b) to 150 ppm (2b), and δ33 for the same pair of structures decreases from 86 to 56 ppm. The effect of tautomerism onto this carbon is much less pronounced, but is nevertheless observed in all three components of the CST. In this case, they are almost equally influenced; a 7 ppm decrease in δ11 as well as in δ33 is partially compensated for by an 8 ppm increase in δ22, when considering the formal transfer of proton from nitrogen N9 to N7. Carbon in position 8 of the purine ring is situated between nitrogen atoms N7 and N9; therefore, there is one protonated nitrogen in its proximity for both N7-H and N9-H tautomers. Nevertheless, its isotropic chemical shift slightly changes when comparing the tautomers 1a and 1b. The difference is more pronounced in the δ22 principal component; for the N9-H tautomer, it is 15 ppm smaller. There is also weak effect of substitution on the component δ22(C8); the value is higher for 6-mercaptopurine. Joint action of these structural effects produces a 20 ppm difference in δ22(C8) between 6-mercaptopurine (2b) and N9-H tautomer of hypoxanthine (1a), which is responsible for the 8 ppm difference in δiso(C8). δ11(C8) and δ33(C8) are not remarkably influenced by the changes in structure. Effects of Intermolecular Interactions - DFT Calculations for Supramolecular Clusters. To evaluate intermolecular effects on the 13C CSTs in 1a and 2b, computed isotropic chemical shifts and principal components for a single molecule were compared to results obtained for various supramolecular clusters. Results of these calculations together with experimental chemical shifts and corresponding root-mean-square deviations (rmsds) are shown in Tables 3 and 4. Atomic coordinates of the single molecule and clusters used in this type of calculations were taken from the Cambridge Structural Database with the exception of hydrogen positions, which were optimized individually for the monomer as well as for each selected cluster on the DFT level of theory (see Experimental Section). Supramolecular clusters were selected to examine two important intermolecular forces, hydrogen bonding (HB) and stacking interaction (S). In Tables 3 and 4, the clusters are designated according to the studied interaction as well as according to the number of molecules involved. Hypoxanthine. Effect of Hydrogen Bonding. 13C chemical shift tensors for the monomer (1a-M1) were compared to results obtained for the following clusters to investigate the effect of hydrogen bonding: 1a-HB2, where another hypoxanthine molecule is added to the central one so that hydrogen bonds N7central · · · H-N9second and C6dOcentral · · · H-C2second are formed; 1a-HB3, where another molecule was added to the dimer so that hydrogen bonds are formed as indicated in Figure 2; 1aHB5, where two molecules were added to the trimer; and 1aHB7, where two molecules were added to the pentamer (all shown in Figure 2). Unless indicated otherwise, orientations of the CST principal components differ less than 3° from those summarized in Table 1 and depicted in Figure 1 (1a). Some interesting differences are observed already in isotropic chemical shifts for the central molecule. Particularly in the case of carbon C2 it is nicely demonstrated how the C2-Hcentral · · · N3 hydrogen bond influences its chemical shift; it is about 5 ppm higher in cluster HB5 and HB7 than in the monomer M1 and clusters, in which this hydrogen bond is not contained. In the case of carbon C8, the differences are even more pronounced.

13C

Chemical Shift Tensors in Hypoxanthine and 6-Mercaptopurine

J. Phys. Chem. A, Vol. 114, No. 4, 2010 1989

TABLE 3: DFT Calculateda Isotropic Chemical Shifts and Principal Components of the 13C CSTb for Selected Clusters of Hypoxanthine (1a), the Experimental Chemical Shifts, and Root-Mean-Square Deviation between the Calculated and Experimental Values atom C2 δiso C4 δiso C5 δiso C6 δiso C8 δiso rmsd (δiso) C2 δ11 δ22 δ33 C4 δ11 δ22 δ33 C5 δ11 δ22 δ33 C6 δ11 δ22 δ33 C8 δ11 δ22 δ33 rmsd (CST)

1a-M1

1a-S2

1a-S5

1a-HB2

1a-HB3

1a-HB5

1a-HB7

1a-HBS11

exp

138.3 142.7 121.2 152.9 130.1 7.4 222 134 60 216 152 60 163 155 45 256 117 85 211 115 64 15.7

139.8 142.7 123.6 153.7 129.7 7.1 226 136 58 216 151 62 167 162 43 255 127 79 212 116 61 13.6

138.6 143.5 124.0 153.8 132.9 6.2 227 134 55 219 155 56 168 165 40 256 131 75 219 124 56 11.9

140.1 143.5 120.0 153.3 132.3 6.2 223 138 59 217 153 61 161 153 46 254 121 85 209 127 61 12.9

137.5 142.8 122.6 152.8 138.9 5.5 223 129 60 214 153 63 161 159 47 251 124 83 214 144 58 11.5

143.6 144.4 121.0 156.0 138.9 3.1 225 148 58 214 156 63 161 155 47 236 149 84 215 144 59 5.0

144.3 145.1 119.9 155.8 138.1 3.2 225 150 58 215 158 63 161 152 47 233 151 84 213 142 60 5.1

144.4 146.7 120.9 156.1 140.4 2.1 228 151 54 219 164 57 166 154 43 234 158 76 217 149 55 3.1

145.3 149.4 122.4 159.2 141.8 231 149 56 225 163 60 167 155 45 239 156 82 218 150 57

a Geometry of C, N, and O atoms for 1a determined by X-ray diffraction12 was fixed, and positions of protons for the individual clusters were optimized by using the B3LYP/6-31G* method. Nuclear shieldings were calculated by B3LYP/6-311G**. Subsequently, chemical shifts were calculated using following equation: δi ) σst - σi + δst. The shielding of R-glycine cluster (σC13 of the CdO group; σst ) 0.5 ppm) was calculated by the same method as used for 1a, and the experimental chemical shift δst ) 176.03 ppm for carbonyl carbon in glycine was employed. b Values are given in ppm.

TABLE 4: DFT Calculateda Isotropic Chemical Shifts and Principal Components of the 13C CSTb for Selected Clusters of 6-Mercaptopurine (2b), the Experimental Chemical Shifts for 2b · H2O, and Root-Mean-Square Deviation between the Calculated and Experimental Values atom C2 δiso C4 δiso C5 δiso C6 δiso C8 δiso rmsd (δiso) C2 δ11 δ22 δ33 C4 δ11 δ22 δ33 C5 δ11 δ22 δ33 C6 δ11 δ22 δ33 C8 δ11 δ22 δ33 rmsd (CST)

2b-M1

2b-HB2

2b-HB3

2b-HB4

2b-HB8

2b-HBS18

2b-HBS18c

exp

138.8 149.2 123.8 169.1 138.0 4.9 230 129 57 199 183 65 195 139 37 307 148 52 215 138 61 12.3

139.6 148.3 123.8 169.7 138.0 4.7 230 132 57 200 180 65 195 138 38 310 147 52 212 144 59 12.9

138.5 149.4 124.7 169.0 140.4 4.2 229 130 57 200 181 66 194 140 39 305 150 52 217 146 59 11.1

143.0 148.3 125.1 170.1 139.8 3.1 232 144 53 198 180 67 193 141 41 310 148 53 215 145 59 12.7

147.0 150.5 124.4 168.8 141.2 2.7 234 159 48 201 185 66 195 139 39 293 164 50 217 148 58 6.8

145.5 148.5 125.9 171.3 144.8 1.1 234 159 43 201 185 60 198 143 36 302 164 48 218 162 55 8.0

145.1 148.9 126.2 169.9 145.6 0.7 234 159 43 202 186 60 198 144 36 298 165 48 220 162 55 7.0

145.6 149.0 127.7 169.9 145.6

210 175 62 196 148 40 286 173 51

a

Geometry of C, N, and S atoms for 2b determined by X-ray diffraction14,15 was fixed, and positions of protons for the individual clusters were optimized by using the B3LYP/6-31G* method. Nuclear shieldings were calculated by B3LYP/6-311G**. Subsequently, chemical shifts were calculated using following equation: δi ) σst - σi + δst. The shielding of R-glycine cluster (σC13 of the CdO group; σst ) 0.5 ppm) was calculated by the same method as used for 2b, and the experimental chemical shift δst ) 176.03 ppm for carbonyl carbon in glycine was employed. b Values are given in ppm. c Population-weighted chemical shifts for 5% of the thiol tautomer in 2b · H2O.

The isotropic chemical shift changes slightly already in the presence of the N7central · · · H-N9 hydrogen bond (1a-HB2), but the really dramatic change appears when the third molecule is added to the cluster and hydrogen bonds C8-Hcentral · · · OdC6 and N9-Hcentral · · · N7 are taken into account (1a-HB3); a 9 ppm difference between this cluster and the monomer is observed.

From an inspection of magnitudes of the chemical shift tensor principal components, it is immediately clear that it is the component δ22 that is responsible for the change of chemical shift values for carbons C2 and C8. Its value is about 15 ppm higher for C2 in the HB5 and HB7 clusters, when compared to the monomer, while in the other components the effect of

1990

J. Phys. Chem. A, Vol. 114, No. 4, 2010

Malinˇa´kova´ et al.

Figure 3. Hypoxanthine cluster built to examine the effect of stacking.

Figure 2. Hypoxanthine cluster with hydrogen bondings. Numbers indicate the central molecule (1) and the molecules successively added to examine the hydrogen-bonding effects.

forming C2-Hcentral · · · N3 hydrogen bond is vanishingly small. For the C8 carbon, a 10 ppm difference in the δ22 component is found already for the HB2 dimer, and between the monomer and the other oligomers about a 30 ppm difference is observed! In analogy to C2, the other two components are influenced only marginally by the hydrogen-bond formations. Isotropic chemical shifts of the other carbons in 1a are not changed remarkably with hydrogen-bonding interactions. However, significant differences were observed in the magnitudes of the CST principal components of C6, indicating that in this case the constancy of the isotropic values is only due to averaging of the principal components. When the weak hydrogen bond C6dOcentral · · · H-C2 is included in the HB2 dimer, all of the principal components stay almost the same. The major interaction appears to be the C6dOcentral · · · H-N1fifth hydrogen bond, which imposes a 20 ppm difference in δ11 and a 30 ppm difference in δ22 components, when the clusters HB5 and HB7 are compared to the monomer! Similarly to observations for C2 and C8, no remarkable effect on the component δ33 was found. CST principal components of the bridgehead quaternary carbons C4 and C5 stay almost the same for all of the clusters, which indicates that their magnitudes are not influenced by the investigated hydrogen bonds. Nevertheless, significant changes were observed in orientations of the in-plane principal components of C5. For the HB2 dimer, the δ11 component moves more toward the C5-N7 bond, and the angle decreases to 27.9°. When another molecule is added and hydrogen bonds C8-Hcentral · · · OdC6third and N9-Hcentral · · · N7third are taken into account (1a-HB3), orientations of δ11 and δ22 components almost interchange, and the same angle is now 94.0°. For the HB5 and HB7 clusters, the angles are 116.0° and 114.0°, respectively. This relatively large variation in orientations of the in-plane principal components of carbon C5 is due to its nearly symmetric chemical shift tensor; δ11 and δ22 components are similar in size and therefore likely to be interchanged. Effect of Base Stacking. To evaluate the effect of molecular stacking on the 13C chemical shift tensors in 1a, data for the monomer were compared to results obtained for the following supramolecular clusters: 1a-S2 with two molecules and 1a-S5 with five molecules including the central one (Figure 3). Orientations of the CST principal components for the clusters do not differ more than 3° from those summarized in Table 1 and depicted in Figure 1 (1a), with an exception of carbon C5, where the angle between δ11 component and the C5-N7 bond changes from 66.4° in 1a-S2 to 80.3° in 1a-S5.

When another molecule is added to the observed one according to the stacking in crystal (1a-S2), chemical shift changes are found only for carbons in the closest proximity of this molecule, C6 and C5. In both cases, intermediate principal component of the CST is most influenced; its value increases with the stacking. For C6, there is also an apparent change in δ33. This component is oriented perpendicular to the purine plane, so it is not surprising that its value is smaller for the dimer. All of the differences do not exceed 10 ppm and are not reflected in isotropic chemical shifts very clearly. Therefore, a cluster of five molecules was examined (1a-S5), two of those being above and the other two below the central molecule (Figure 3). For this cluster, the smallest principal component of the CST, δ33, remarkably decreases for all of the carbons of the purine ring. Also, the effect on the intermediate component δ22 is more pronounced, and a significant increase in its value is now found also for carbon C8. However, isotropic chemical shifts stayed almost the same. Carbons C2 and C4 seem to be less sensitive to the investigated base stacking. 6-Mercaptopurine. 6-Mercaptopurine (2b) differs from hypoxanthine (1a) in position 6 of the purine ring, where the oxygen atom in 1a is formally replaced by sulfur. Another difference between their crystal structures is found in the position of one proton; it is attached to nitrogen N9 in hypoxanthine, while 6-mercaptopurine crystallizes as monohydrate with nitrogen N7 being protonated.14,15 Therefore, also their crystal packing is different, and clusters used to study hydrogen bonding and stacking interactions differ as well. The following clusters were used to evaluate purely the effect of hydrogen bonding on the 13C chemical shift tensors in 6-mercaptopurine: 2b-HB2, where a single water molecule forming a hydrogen bond with nitrogen N9 was added to single molecule 2b-M1 as found in the crystal structure; 2b-HB3, where two 6-mercaptopurine molecules and the single water form the cluster, and the hydrogen bond N7-Hcentral · · · N3third is newly considered; 2b-HB4, where another 6-mercaptopurine molecule is added to the previous cluster so that also the hydrogen bond N3central · · · H-N7fourth appears; and 2b-HB8, where one more 6-mercaptopurine molecule and three surrounding water molecules are added to the four-molecule cluster as depicted in Figure 4. For this compound, no clusters modeling the stacking interaction alone were constructed. Nevertheless, this interaction was included in the largest cluster built, 2b-HBS18 (see the Supporting Information for Cartesian coordinates). This cluster was chosen to take into account all of the important interactions present in the real sample, including sulfur-sulfur interaction. The central molecule is part of a three-molecule layer, where the molecules are connected by hydrogen bonds across the crystal water. An above layer is represented by two molecules connected in the same way; an underlying molecule is connected

13C

Chemical Shift Tensors in Hypoxanthine and 6-Mercaptopurine

Figure 4. 6-Mercaptopurine · H2O cluster with hydrogen bondings. Numbers indicate the central molecule (1) and the molecules successively added to examine the hydrogen-bonding effects.

to water molecules from the central layer. To this stacked system were added three 6-mercaptopurine molecules according to the 2b-HB8 cluster, to account for the hydrogen bonds formed within this cluster. Furthermore, sulfur-sulfur interaction and C8-Hcentral · · · S hydrogen bond were considered by adding another two 6-mercaptopurine molecules together with two water molecules to the cluster formed. Computed isotropic chemical shifts and CST principal components for the monomer 2b-M1 and for the supramolecular clusters are summarized in Table 4, and orientations of the CST principal components are depicted in Figure 1 (2b). The angles that δ11 components assume with the selected bonds differ from those given in Table 1 less than 3° for C2, C5, and C6 in all of the clusters. For carbons C8 and C4, the orientations slightly change. The angle that the δ11(C8) assumes with the C8-H8 bond is changed to 17.0° in the 2b-HB3 cluster (16.6° and 17.1° in the 2b-HB4 and 2b-HB8 clusters, respectively), and in the largest cluster 2b-HBS18 it increases again to 20.2°. The angle that the δ11(C4) assumes with the C4-N9 bond decreases to 8.1° in the 2b-HB2 cluster and stays almost the same for all of the other clusters with an exception of the largest one, where it increases to 19.1°. Concerning isotropic values, significant effects are found only for C2 and C8, where the difference between monomer and the largest cluster amounts to 7 ppm. However, more interesting changes are observed in the CST principal components. Already the addition of the single water molecule imposes a 6 ppm increase in δ22 component of C8. Considering the N7-Hcentral · · · N3third hydrogen bond in 2b-HB3, an additional slight increase in this component is observed for this carbon. Formation of hydrogen bond N3central · · · H-N7fourth is significantly reflected in the δ22 component of C2; the difference between its value in the monomer and in the 2b-HB4 cluster is 15 ppm! Together with an increase in the δ22 component, a slight decrease in the δ33 component is observed in this case. A comparably strong effect on C2 has a hydrogen bond between the proton attached to this carbon and the adjacent water

J. Phys. Chem. A, Vol. 114, No. 4, 2010 1991 molecule; an additional 15 ppm increase in the δ22 component and 5 ppm decrease in the δ33 component is observed for the 2b-HB8 cluster. Other hydrogen bonds formed within this cluster influence carbon C6; its δ11 component is 14 ppm smaller and its δ22 component is 16 ppm larger for this cluster, when compared to the monomer. Their changes could therefore be considered as self-compensating with vanishingly small effects on the isotropic chemical shift. For the largest cluster 2b-HBS18, the effect of base stacking is apparent; the smallest chemical shift component δ33 decreases for all of the carbons of the purine ring. Also, the effect of considering hydrogen bond C8-Hcentral · · · S is significant and together with the effect of stacking results in a 14 ppm increase in the δ22 component of carbon C8, when compared to the 2bHB8 cluster. Sulfur-sulfur interaction may be responsible for the last remarkable change in chemical shifts between clusters 2b-HB8 and 2b-HBS18, for the 9 ppm increase in the δ11 component of carbon C6. Similarly to trends observed for hypoxanthine, chemical shifts of C4 and C5 remain almost identical for all of the clusters, indicating that they are practically uninfluenced by the intermolecular interactions simulated. Description of Interactions in the Real System - Combination of Experimental and Theoretical Approach. In preceding parts, 13C CSTs were examined for two compounds, hypoxanthine and 6-mercaptopurine, with an attempt to describe theoretically the effect of substituent in position 6 of the purine ring (CdO f CdS), the effect of tautomerism (N9-H f N7H), and the effect of intermolecular interactions present in the crystalline samples on the isotropic chemical shifts and CST principal components. Here, the DFT calculated values are compared to those obtained experimentally. For this purpose, root-mean-square deviations between theoretical and experimental data were collected separately for isotropic chemical shifts [rmsd (δiso)] and for the CST principal components [rmsd (CST)]. Hypoxanthine. DFT calculated 13C chemical shifts for single hypoxanthine molecule and several supramolecular clusters are compared to values obtained from solid-state NMR measurements (Table 3). Because in the real sample both hydrogen bonding and stacking interactions are present, the hydrogenbonded cluster 1a-HB7 was combined with stacked cluster 1aS5. 13C CSTs for the resulting cluster 1a-HBS11 (see the Supporting Information for Cartesian coordinates) were calculated and compared to the experimental values as well. Orientations of the CST principal components for the cluster do not differ more than 5° from those summarized in Table 1 and depicted in Figure 1 (1a), with an exception of carbon C5, where the δ11 and the δ22 components are interchanged and the angle between C5-N7 bond and now the δ22 component is 36.6°. For isotropic chemical shifts in the monomer 1a-M1, the rmsd is more than 7 ppm. In the 1a-S5 cluster, where stacking interactions were simulated, a minor change toward better agreement with experiment is observed, and rmsd decreases to 6 ppm. Hydrogen-bonding effects seem to be more important for this system; the rmsd value is decreased to 6 ppm already in the case of dimer and decreases further with the number of molecules building the hydrogen-bonded network. In the 1aHB7 cluster, the overall rmsd value for isotropic chemical shifts is about 3 ppm. When both hydrogen bonding and stacking effects are combined in the 1a-HBS11 cluster, additional improvement of the result is achieved (rmsd ) 2.1). This comparison confirms already for the isotropic chemical shifts how important it is to include hydrogen bonding into

1992

J. Phys. Chem. A, Vol. 114, No. 4, 2010

calculations. For example, for carbon C8, the error resulting from neglecting intermolecular interactions can be as large as 10 ppm in its isotropic chemical shift. When calculated CST principal components for C8 of the monomer (1a-M1) are compared to the experimental values, considerable differences are found for all three components, but especially notable is the 35 ppm difference in component δ22. The calculated value is 115 ppm, while experiment shows 150 ppm. When stacking interactions are simulated (cluster 1a-S5), the value increases to 124 ppm; for hydrogen-bonding simulation (1a-HB7), it increases to 142 ppm. Finally, when the joint action of both effects is considered (1a-HBS11), the calculated value of 149 ppm nearly matches the experimental one (150 ppm). Exactly the same effect is observed for the δ22 component of C6, which came out as the most sensitive to the studied interactions; the difference between the calculated value for a single molecule 1a-M1 and the experimental one amounts to 39 ppm, while the difference between the value for the final 1a-HBS11 cluster and experiment is only 2 ppm! Another interesting example of the same result is the δ22 component of C4. It seemed to be only slightly influenced by the stacking interaction (1a-M1 vs 1a-S5: 3 ppm difference) and by the hydrogen bonding as well (1a-M1 vs 1a-HB7: 6 ppm difference). However, both effects together in 1a-HBS11 produce a 12 ppm difference from the monomer, along with a nice fit to the experimental value. Carbon C5 is the only case, where the calculated value of the δ22 principal component for the monomer directly matched the experimental one. For individual hydrogen-bonded clusters (including 1a-HBS11), only minor changes of this value are observed, confirming that it is really not so sensitive to this interaction. δ33 directions are dramatically shielded for C4, C5, and C6 atoms, when solely stacking interactions are simulated, resulting in an increased deviation from experiment. However, the values are brought much closer to the experimental data when hydrogen-bonding interactions are considered as well in cluster 1a-HBS11. Concerning δ11 components, the most significant effect is observed for C6; deviation from experiment changes from 17 ppm in the case of monomer (1a-M1) to 6 ppm for the largest cluster simulating exclusively hydrogen bondings (1a-HB7). Overall comparison of experimental data and CST principal components calculated for the 1a-HBS11 cluster yields an rmsd of 3.1 ppm, which confirms that used models are capable of describing the most important intermolecular forces present in the crystalline samples. 6-Mercaptopurine. DFT calculated 13C chemical shifts for a single molecule and supramolecular clusters of 6-mercaptopurine together with values obtained from solid-state NMR measurements and corresponding rmsd’s are summarized in Table 4. The agreement between experimental isotropic chemical shifts and the data for monomer 2b-M1 is notably better than in the case of hypoxanthine 1a-M1; the rmsd is 4.9 ppm versus 7.4 ppm. Already this result suggests that molecular packing has a much larger effect on nuclear shielding in hypoxanthine than in 6-mercaptopurine. However, some intermolecular forces are important, as observed, for example, for the 2b-HB4 cluster. In this case, the rmsd decreases to 3.1 ppm mainly due to a change in δiso(C2). Finally, for the largest cluster (2b-HBS18), an improvement in δiso(C8) is observed, and an excellent agreement between the calculated and experimental values is achieved (rmsd ) 1.1). Concerning CST principal components, the results cannot be presented for all of the carbons of the purine ring, because C2

Malinˇa´kova´ et al. and C8 signals overlap in the 1D spectrum of 6-mercaptopurine and their CSTs cannot be analyzed in a straightforward way by the approach applied here. Nevertheless, the hydrogen bonds influencing these carbons are easily identified already from isotropic chemical shifts, and from the calculated CST principal components it is clearly observed that it is the intermediate component δ22 that is sensitive to the HB interaction. For the remaining three carbons, the rmsd between the CST principal components determined experimentally and those calculated for monomer 2b-M1 is about 12 ppm. The best agreement was found for C5, where δ11 and δ33 nearly matched the experimental values, and in δ22 only a 9 ppm difference was observed. For the clusters simulating only hydrogen bonding, no remarkable change in the values appeared; for the largest cluster, the difference between the calculated and experimental δ22(C5) decreased to 5 ppm. For carbon C4, the differences between data for the monomer and experimentally determined values are more significant. The 11 ppm difference in δ11(C4) and 8 ppm difference in δ22(C4) stay nearly the same for all of the clusters, including the largest one, indicating that the deviation in this case does not arise from neglecting intermolecular interactions and could indicate an experimental error. As expected, C6 appears to be the most sensitive to intermolecular interactions from the three carbons analyzed. The difference between experimentally determined δ11(C6) and the one calculated for the monomer 2b-M1 amounts to 21 ppm; in the case of δ22(C6), the difference is 25 ppm. For the smaller clusters including 2b-HB4, no remarkable change in magnitudes of the principal components is found; therefore, also the rmsd value is still around 12 ppm. A substantial decrease of the deviation is observed for the 2b-HB8 cluster, where the 21 ppm difference in δ11(C6) is reduced to 7 ppm and the 25 ppm difference in δ22(C6) is reduced to 9 ppm. However, for the largest cluster 2b-HBS18, the rmsd increases again, mainly due to a change in δ11(C6). The difference between calculated and experimental value for this component increases to 16 ppm. Large differences between experimentally determined CST principal components and the values calculated for the 2bHBS18 cluster are in contrast to much more consistent results obtained for hypoxanthine. After checking the experimental and calculated data, a possible explanation for the discrepancy was found in the data from single-crystal X-ray diffraction, which indicated that small amounts of minor components might be present in crystal together with the major form. This was further supported by careful inspection of X-ray powder diffraction data (see the Supporting Information). Because the largest error was found for C6, we anticipated the minor form to be the thiol tautomer of 6-mercaptopurine · H2O, with proton transferred from nitrogen N7. To verify the hypothesis, we repeated the calculation for the 2b-HBS18 cluster for the thiol tautomer as the central molecule (see the Supporting Information for Cartesian coordinates) and compared the results to the data obtained previously for the thione form (see Table S3 in the Supporting Information). The smallest rmsd between experimental and population-weighted calculated isotropic chemical shifts was found for 5% of the thiol form (0.7 ppm; Figure 5), and for the CST principal components the smallest rmsd value corresponds to 20% of this form (5.7 ppm; Table S3 in the Supporting Information). It may be concluded that there is about 5-20% of the thiol tautomer in our 6-mercaptopurine · H2O sample, and perhaps some other minor form, which introduces an error in the case when the calculated data for the thione form only are compared to experiment.

13C

Chemical Shift Tensors in Hypoxanthine and 6-Mercaptopurine

Figure 5. Plot of the rmsd between experimental and calculated population-weighted isotropic chemical shifts for 6-mercaptopurine · H2O versus the amount of thiol form in the sample.

Differences between Hypoxanthine and 6-Mercaptopurine. In this part, experimentally determined chemical shifts are compared for the hypoxanthine and 6-mercaptopurine · H2O samples. DFT calculated data are used here to get a deeper insight into the origin of differences. In Table 5, experimental differences given in the last column are calculated as differences between the chemical shift value in the 6-mercaptopurine monohydrate sample (2b · H2O) and the value in the hypoxanthine sample (1a); that is, δ(1a) is subtracted from δ(2b · H2O). These values are compared to data obtained from DFT calculations. In the first column, large values indicate isotropic chemical shifts and CST principal components significantly influenced by the position of proton in the hypoxanthine molecule (the effect of N9-H f N7-H tautomerism). In the second column, large values indicate a significant effect of CdO f CdS substitution. The third column summarizes these two differences for isolated hypoxanthine and 6-mercaptopurine molecules and as such can be compared to the values obtained from experiment. In the next two columns, the values are calculated as differences between chemical shift in isolated molecule (DFT optimized geometry) and chemical shift in the largest supramolecular cluster, once for 1a and once for 2b, highlighting the isotropic chemical shifts and CST principal components significantly influenced by topology and intermolecular interactions. Finally, chemical shifts in the largest hypoxanthine cluster (1a-HBS11) were subtracted from the values in the largest 6-mercaptopurine cluster (2b-HBS18). This column contains DFT calculated differences between 1a and 2b, which should correspond to those obtained experimentally. Unfortunately, the experimental data for CST principal components of C2 and C8 are not available, and δ11(C6) together with δ22(C6) are significantly influenced by the presence of the minor form in the 6-mercaptopurine sample. However, the remaining experimental values agree with the calculated data very well. As expected, the largest experimental difference between chemical shifts in hypoxanthine (1a) and 6-mercaptopurine monohydrate (2b · H2O) samples is found for C6. The oxygen atom bound here in 1a is formally replaced by sulfur in 2b · H2O, which induces a 10.7 ppm increase in δiso(C6). As seen from the second column in Table 5, this substitution change predominantly influences the δ11 component. In δ22, the effect is more than one-half that, but it is largely balanced by the differences in crystal packing between 1a and 2b · H2O samples (effects of intermolecular interactions, ∆(isol.fclust.) in Table 5). In the hypoxanthine sample, hydrogen-bonding interactions

J. Phys. Chem. A, Vol. 114, No. 4, 2010 1993 are of great importance for the δ22(C6) component (∆(isol.fclust.) ) 54 ppm), while in 6-mercaptopurine monohydrate the effect is not so large (∆(isol.fclust.) ) 14 ppm). Therefore, the overall change in δ22(C6) is not so dramatic as expected from the results for the substitution change only. The increase in δ11 and δ22 components with the formal CdO f CdS replacement is compensated by a substantial decrease in the δ33 direction. As a result, only a 0.7 ppm increase in δiso(C6) is observed. The effect of tautomerism onto the C6 is in our case overruled by the effect of substitution and can be therefore considered as practically unimportant. Carbon C5 is close to the place of substitution and also close to nitrogen N7, which is involved in tautomeric process. It is therefore anticipated that both the substitution and the tautomerism effects are responsible for the 5.3 ppm difference in its isotropic chemical shift, when 1a and 2b · H2O samples are compared. Indeed, the effect of substitution is strongly pronounced in the δ11 component, and the effect of tautomerism, however, by one-half smaller, is represented in both of the others. Because the formal CdO f CdS replacement induces increase and the formal N9-H f N7-H proton transfer decrease in particular CST principal component(s), the resulting difference in δiso(C5) appears not so remarkable. Intermolecular interactions have only a negligible effect on the CST principal components of this carbon. Carbon C4 is more distant from the place of substitution than carbon C5 and directly bonded to nitrogen N9. In the 1a and 2b · H2O samples, isotropic chemical shift of the carbon is almost the same, indicating that the weaker substitution effect and the effect of tautomerism compensate each other. Similarly to C5, the δ11 component is predominantly influenced by the formal CdO f CdS substitution. However, the effect is approximately by one-half smaller, and the chemical shift in this component decreases. Analogously, an increase in δ22 is observed for the formal N9-H f N7-H proton transfer. Because the substitution effect in δ11 and the effect of tautomerism in δ22 are equally sized for this carbon, and in δ33 the two effects compensate each other, almost no change in isotropic chemical shift is observed. Intermolecular interactions influence this carbon only marginally. The position of carbon C8 between N7 and N9 suggests that the 3.8 ppm difference in δiso(1a) and δiso(2b · H2O) arises mainly due to the effect of tautomerism. Indeed, no substantial change with the substitution is found in the DFT calculated CST principal components. Furthermore, it is observed that tautomerism influences significantly only the intermediate principal component δ22. Therefore, it may be concluded that it is this component only that is responsible for the change in the isotropic chemical shift. According to the ∆(isol.fclust.) column in Table 5, the δ22 component is also highly sensitive to intermolecular interactions. However, the effect for hypoxanthine and 6-mercaptopurine monohydrate samples is similar and modulates the tautomerism-induced difference only slightly. Carbon C2 is equally far from the substituted C6 as is carbon C4. Nevertheless, the formal CdO f CdS replacement has almost no impact on its chemical shift. The effect of tautomerism is also small and is clearly observed only in the δ22 component. Far more important for this carbon are intermolecular interactions. Opposite to C6, hydrogen bonds formed within 6-mercaptopurine sample have a larger impact on the chemical shift of carbon C2 than intermolecular interactions present in the hypoxanthine sample; the differences are 14 ppm in δ22 and 10 ppm in δ33 components. In δ22, this effect compensates for the effect of tautomerism; therefore, the final difference between

1994

J. Phys. Chem. A, Vol. 114, No. 4, 2010

Malinˇa´kova´ et al.

TABLE 5: DFT Calculated Differences in Isotropic Chemical Shifts and Principal Components of the 13C CSTsa between Structures 1a and 1b, 1b and 2b, Differences between Isolated Molecules and Supramolecular Clusters (1a vs 1a-HBS11 and 2b vs 2b-HBS18), Total Differences, and Experimental Differences between 1a and 2b · H2O effect of structure atom C2

C5

C6

C8

a

total effect

experimental difference ∆exp(1af2b · H2O)

∆(1af1b)

∆(1bf2b)

∑∆DFTisol.(1af2b)

∆(1af1a-HBS11)

∆(2bf2b-HBS18)

∆DFTclust.(1a-HBS11f2b-HBS18)

2 -9 1 -1.8 -5 23 11 9.9 0 -17 -15 -10.7 -7 8 -7 -1.9 -1 15 0 4.6

0 3 -2 0.1 -16 3 -7 -6.8 33 4 1 12.5 63 38 -30 23.2 4 5 0 3.2

2 -6 -1 -1.7 -21 26 4 3.1 33 -13 -14 1.8 56 46 -37 21.3 3 20 0 7.8

1 16 -7 3.5 0 9 -6 1.1 2 -2 -8 -3.0 -17 54 -17 6.7 6 32 -8 10.2

5 30 -17 6.3 3 4 -7 -0.2 1 0 -1 0.2 -5 14 -8 0.6 4 25 -8 6.8

6 8 -11 0.7 -17 22 3 2.2 32 -10 -7 5.3 64 7 -28 13.8 3 13 0 5.2

δ11 δ22 δ33 δiso δ11 δ22 δ33 δiso δ11 δ22 δ33 δiso δ11 δ22 δ33 δiso δ11 δ22 δ33 δiso

C4

effect of inter-molecular forces

0.3 -15 12 2 -0.4 29 -7 -5 5.3 47 17 -31 10.7

3.8

Values are given in ppm.

δ22(C2) in 1a and 2b · H2O is only 8 ppm (∆DFTclust.(1af2b) in Table 5). Because the chemical shift increases in δ22 and decreases in the δ33 component with the hydrogen bonds formed, the resulting difference in isotropic chemical shift is negligible. Although the calculated differences in CST principal components for C2 and C8 cannot be correlated with experimental data, differences in isotropic chemical shifts agree with the experimental values very well. Conclusions 13

C chemical shift tensors in purine-based compounds are sensitive to local structural changes like substitution on the purine ring, tautomerism, and intermolecular interactions, as shown by hypoxanthine and 6-mercaptopurine as examples. The effect of substitution is, as expected, highly pronounced at the directly substituted carbon and decreases fast with the number of bonds between the observed nucleus and the place of substitution. Tautomerism significantly influences carbon atoms directly bonded to nitrogens involved in this process. Hydrogen bonding, as the most important intermolecular interaction, has a vanishingly small effect on the quaternary bridgehead carbons, while the protonated ones are very sensitive to it. Carbon C6 is in the present case the place of substitution; the oxygen bound here in hypoxanthine is in 6-mercaptopurine formally replaced by sulfur. Therefore, its chemical shift is primarily influenced by substitution; a 20 ppm difference in isotropic chemical shift is reflected in all three principal components. The 60 ppm increase in δ11 along with the 40 ppm increase in δ22 with the CdO f CdS formal exchange is partially compensated for by a 30 ppm decrease in δ33. In the real sample, the effect of substitution is combined with the effect of intermolecular interactions. Their strength depends on the substituent bonded to the carbon. For the oxygen, 20 and 34 ppm changes in the δ11 and δ22 components were found, respectively, while interactions involving sulfur are not so strong and result only in 14 and 16 ppm changes in the respective components. The bridgehead carbon C4 is much more influenced by the substitution than the protonated C2, although they are equally

far from the substituted carbon C6. The formal substituent exchange results in a 7 ppm difference in the isotropic chemical shift of C4. For C5, situated one bond closer to the place of substitution, the difference is 13 ppm. The principal component most strongly influenced by the structural change is the largest one, δ11. For C4, the substitution-induced change in this component is 20 ppm; for C5 located closer to the substituent it is 30 ppm. Orientations of the in-plane principal components are notably changed with the substitution only for C4 and C5. The effect of tautomerism is in the present case most strongly pronounced in chemical shifts of bridgehead carbons C4 and C5. The presence of proton on the neighboring nitrogen results in a 10 ppm decrease in the isotropic chemical shift of the particular carbon. For C4, this is because of a 20 ppm difference in the δ22 principal component, while for C5 δ22 and δ33 are equally influenced, and a 10 ppm difference in both components is found. The effect onto C8, which is directly bonded to both nitrogens participating in the N7-H/N9-H prototropic tautomerism, is not so large, and a 5 ppm difference in its isotropic chemical shift, originating in a 15 ppm change in the δ22 principal component, is found. Orientations of the in-plane principal components of carbons C4, C5, and C8 are changed slightly with the position of proton. The protonated carbons C2 and C8 are the most sensitive to intermolecular interactions. They are influenced not only by hydrogen bonds that they are directly involved in, but also by interactions of neighboring nitrogens with other molecules. Neglected hydrogen bonding leads in the present case to a 5-10 ppm error in isotropic chemical shift for these carbons. In the intermediate CST principal component δ22, which was found to be the most sensitive to intermolecular interactions, the error induced by neglecting hydrogen bonding can be as large as 30 ppm. The substantial sensitivity of the CSTs of protonated carbons C2 and C8 to intermolecular interactions points to the inevitability of considering these effects in NMR studies of biomolecular dynamics using relaxation data. Although the actual error in calculated dynamics, introduced by neglecting intermolecular forces, is dependent on another parameters like external magnetic field and size and shape of the molecule, the deviation

13C

Chemical Shift Tensors in Hypoxanthine and 6-Mercaptopurine

introduced by incorrect bond distance and chemical shift tensor can be significant in some cases. For this type of analysis, we recommend the evaluation of dynamic dependence of the CSTs, which will represent the topic of our future study. Acknowledgment. We thank Andrzej Wojtczak for the discussions and suggestions concerning X-ray data of compound 2b · H2O, Pavel Kaderˇa´vek and Radovan Fiala for the discussions concerning spin relaxation, and Reijo Kauppinen for his help with NMR measurements during the stay of L.N. at the University of Jyva¨skyla¨ under the Socrates program. This work was supported by the Ministry of Education of the Czech Republic (MSM0021622413 and LC06030). The computational resources were provided by the MetaCentrum, Czech Republic (under the research grant MSM6383917201). Supporting Information Available: Experimental principal components of the 13C chemical shift tensors determined at different MAS frequencies, calculated chemical shifts for the thiol tautomer of 6-mercaptopurine, Cartesian coordinates of all of the supramolecular clusters, image of the R-glycine cluster used for chemical shift referencing, and details of the X-ray powder diffraction. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Wink, D. A.; Kasprzak, K. S.; Maragos, C. M.; Elespuru, R. K.; Misra, M.; Dunams, T. M.; Cebula, T. A.; Koch, W. H.; Andrews, A. W.; Allen, J. S. Science 1991, 254, 1001–1003. (2) Shapiro, R.; Pohl, S. H. Biochemistry 1968, 7, 448–455. (3) Moe, A.; Ringvoll, J.; Nordstrand, L. M.; Eide, L.; Bjoras, M.; Seeberg, E.; Rognes, T.; Klungland, A. Nucleic Acids Res. 2003, 31, 3893– 3900. (4) Paterson, A. R. P.; Tidd, D. M. 6-Thiopurines; Springer Verlag: New York, 1975. (5) Lennard, L. Eur. J. Clin. Pharmacol. 1992, 43, 329–339. (6) Coulthard, S.; Hogarth, L. InVest. New Drugs 2005, 23, 523–532. (7) Somerville, L.; Krynetski, E. Y.; Krynetskaia, N. F.; Beger, R. D.; Zhang, W.; Marhefka, C. A.; Evans, W. E.; Kriwacki, R. W. J. Biol. Chem. 2003, 278, 1005–1011. (8) Amo-Ochoa, P.; Rodrı´guez-Tapiador, M. I.; Castillo, O.; Olea, D.; Guijarro, A.; Alexandre, S. S.; Go´mez-Herrero, J.; Zamora, F. Inorg. Chem. 2006, 45, 7642–7650. (9) Chenon, M. T.; Pugmire, R. J.; Grant, D. M.; Panzica, R. P.; Townsend, L. B. J. Am. Chem. Soc. 1975, 97, 4636–4642. (10) Bartl, T.; Zacharova´, Z.; Secˇka´rˇova´, P.; Kolehmainen, E.; Marek, R. Eur. J. Org. Chem. 2009, 1377–1383. (11) Pazderski, L.; Łakomska, I.; Wojtczak, A.; Szłyk, E.; Sitkowski, J.; Kozerski, L.; Kamien´ski, B.; Koz´min´ski, W.; Tousˇek, J.; Marek, R. J. Mol. Struct. 2006, 785, 205–215. (12) Schmalle, H. W.; Ha¨nggi, G.; Dubler, E. Acta Crystallogr. 1988, C44, 732–736. (13) Yang, R.-Q.; Xie, Y.-R. Acta Crystallogr. 2007, E63, 3309. (14) Sletten, E.; Sletten, J.; Jensen, L. H. Acta Crystallogr. 1969, B25, 1330–1338. (15) Brown, G. M. Acta Crystallogr. 1969, B25, 1338–1353. (16) Facelli, J. C. Concepts Magn. Reson., Part A 2004, 20A, 42–69. (17) Dokalik, A.; Kalchhauser, H.; Mikenda, W.; Schweng, G. Magn. Reson. Chem. 1999, 37, 895–902.

J. Phys. Chem. A, Vol. 114, No. 4, 2010 1995 (18) Reif, B.; Hennig, M.; Griesinger, C. Science 1997, 276, 1230–1233. (19) Duchardt, E.; Richter, C.; Ohlenschlager, O.; Gorlach, M.; Wohnert, J.; Schwalbe, H. J. Am. Chem. Soc. 2004, 126, 1962–1970. (20) Kroenke, C. D.; Rance, M.; Palmer, A. G. J. Am. Chem. Soc. 1999, 121, 10119–10125. (21) Kojima, C.; Ono, A.; Kainosho, M.; James, T. L. J. Magn. Reson. 1998, 135, 310–333. (22) Hall, K. B.; Tang, C. G. Biochemistry 1998, 37, 9323–9332. (23) Stueber, D.; Grant, D. M. J. Am. Chem. Soc. 2002, 124, 10539– 10551. (24) Hu, J. Z.; Facelli, J. C.; Alderman, D. W.; Pugmire, R. J.; Grant, D. M. J. Am. Chem. Soc. 1998, 120, 9863–9869. (25) International Centre for Diffraction Data, ICDD-PDF2, Release 2007; 12 Campus Boulevard, Newton Square, PA, 2007. (26) (a) Macrae, C. F.; Edgington, P. R.; McCabe, P.; Pidcock, E.; Shields, G. P.; Taylor, R.; Towler, M.; van de Streek, J. J. Appl. Crystallogr. 2006, 39, 453–457 (CSD v. 5.30 + 2009 updates). (b) Allen, F. H. Acta Crystallogr. 2002, B58, 380–388 (CSD v. 5.30 + 2009 updates). (27) Le Bail, A.; Duroy, H.; Fourquet, J. L. Mater. Res. Bull. 1988, 23, 447–452. (28) Opella, S. J.; Frey, M. H. J. Am. Chem. Soc. 1979, 101, 5854– 5856. (29) Xiaoling, W.; Shanmin, Z.; Xuewen, W. J. Magn. Reson. 1988, 77, 343–347. (30) Massiot, D.; Fayon, F.; Capron, M.; King, I.; Le Calve´, S.; Alonso, B.; Durand, J.-O.; Bujoli, B.; Gan, Z.; Hoatson, G. Magn. Reson. Chem. 2002, 40, 70–76. (31) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision C.02; Gaussian, Inc.: Wallingford, CT, 2004. (32) Kaupp, M.; Bu¨hl, M.; Malkin V. G. Calculation of NMR and EPR Parameters: Theory and Applications; Wiley-VCH: Weinheim, 2004. (33) (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648–5652. (b) Becke, A. D. Phys. ReV. A 1988, 38, 3098–3100. (c) Stephens, P. J.; Delvin, F. J.; Chabalovski, C. F.; Triech, M. J. J. Phys. Chem. 1994, 98, 11623–11627. (34) Ditchfield, R. Mol. Phys. 1974, 27, 789–807. (35) Malkin, V. G.; Malkina, O. L.; Salahub, D. R. J. Am. Chem. Soc. 1995, 117, 3294–3295. (36) Haeberlen, U. In AdVances in Magnetic Resonance, Suppl. 1; Waugh, J. S., Ed.; Academic Press: New York, 1976. (37) Mehring, M. Principles of High Resolution NMR in Solids, 2nd ed.; Springer Verlag: Berlin, 1983. (38) Antzutkin, O. N. Prog. Nucl. Magn. Reson. Spectrosc. 1999, 35, 203–266. (39) Mason, J. Solid State Nucl. Magn. Reson. 1993, 2, 285–288. (40) (a) Marek, R.; Sklena´rˇ, V. Annu. Rep. NMR Spectrosc. 2005, 54, 201–242. (b) Secˇka´rˇova´, P.; Marek, R.; Malinˇa´kova´, K.; Kolehmainen, E.; Hockova´, D.; Hocek, M.; Sklena´rˇ, V. Tetrahedron Lett. 2004, 45, 6259– 6263. (c) Tsikouris, O.; Bartl, T.; Tousˇek, J.; Lougiakis, N.; Tite, T.; Marakos, P.; Pouli, N.; Mikros, E.; Marek, R. Magn. Reson. Chem. 2008, 46, 643–649.

JP9100619