Interaction Forces between Tetramethyluric Acid and Aromatic Molecules. A Proton Nuclear Magnetic Resonance Study Antonlo Donesi, Llvio Paollllo, and Plero Andrea Ternus&* lstituto Chimico. University of Neples, Naples, hay (Received August 4, 1975)
IH NMR is used to study the type of forces stabilizing complexes between 1,3,7,9-tetramethyluric acid (TMU) and aromatic hydrocarbons. Equilibrium quotients for complexes of TMU and benzene, naphthalene, phenanthrene, pyrene, and benzopyrene are calculated through chemical shift data. These data are used to evaluate a geometric model which demonstrates the predominance of dipole-induced dipole forces and excludes the existence of charge-transfer complexes in solution.
Introduction Molecular complexes between purines and aromatic hydrocarbons have been investigated rather intensively during the last years, both in solution1-l1 and in the solid state.12-15 The motivation of all these studies is twofold: physicochemical with respect to the forces responsible for the stabilization of molecular complexes in general and biophysical with respect to the role that some aromatic hydrocarbons are suspected to play in carcinogenesis.16 In spite of all this effort no clear-cut conclusion on the nature of the forces responsible for complex formation has been reached. Charge-transfer forces have been invoked by many authors either on the basis of theoretical con~iderationsl~~ or as an interpretation of diffraction14 and ir15 studies in the solid state. It is significant however that other authors on the basis of similar (or even the same) experimental data discard the existence of charge transfer in favor of dipole-induced dipole interactions12 or of dispersion force^.^ The whole issue is complicated by the difficulty of comparing solid-state and solution studies, since most of the latter fail to give conclusive information on the geometry of the complexes. The best technique for studying molecular geometries in solution is probably NMR spectroscopy, but the only NMR study reported in the literature on purine-aromatic hydrocarbons complexesll deals with an unfavorable system and reaches no definite conclusion either on the geometry of the two complexes studied or on the forces responsible of complexation. Here we report a systematic NMR investigation on complexes of 1,3,7,9-tetramethyluric acid (henceforth called TMU) with aromatic hydrocarbons of increasing size, from benzene to 3,4-benzopyrene. These systems were chosen mainly for two reasons: (i) the existence of refined crystal structures of TMU complexesl2J3 can lead to a detailed comparison of solid-state and solution studies; (ii) the presence on TMU of four isolated methyl groups makes this molecule an ideal probe for the determination of the geometry of the complexes. On the other hand, TMU is substantially different from the purinic bases of nucleic acids, which makes all results of this study not directly transferable to the biological problem of carcinogenesis. It is fair to assume, however, that any new piece of information on the nature of the forces between purines and aromatic hydrocarbons may be significant in connection with all biological problems dealing with the action of aromatic hydrocarbons on nucleic acids. Experimental Section Materials. 1,3,7,9-Tetramethyluric acid was purchased
from Fluka AG (Buchs, Switzerland) and used without further purification. Spectrograde benzene was purchased from Merck (Darmstadt), naphthalene and phenanthrene were purchased from Merck (Darmstadt), and pyrene and 3,4-benzopyrene were from Fluka AG (Buchs, Switzerland). Naphthalene, phenanthrene, and pyrene were purified by means of repeated sublimations. All other reagents were used without further purification. Procedure. All solutions were prepared in such a way that the molar concentration of TMU remained approximately constent in each series examined, while the molality of the hydrocarbon increased up to the highest possible molar ratio (moles of hydrocarbon per mole of TMU) compatible with hydrocarbon solubility. A standard solution of TMU, 0.0302 M, in CDCl3 (with 1%TMS) was used for 3,4-benzopyrene; standard solutions, always close to 0.08 M, were used for all other hydrocarbons. Appropriate amounts of the solid hydrocarbons were weighed in stoppered flasks, 1.0-ml aliquots of the standard solutions were added, and the flask was reweighed to determine the exact molality of the hydrocarbon. Benzene solutions were prepared in a slightly different way to account for the volume of added benzene. Variable volumes of standard solutions were used in this case, to keep the benzene:TMU molar ratio high enough for the relationship between shifts and molality to hold (vide infra). l H NMR spectra were recorded on a Varian Associates HA-100-15 spectrometer. Chemical shifts were measured with respect to internal TMS and are accurate to better than 0.2 Hz (the line widths of the methyl resonances being always very sharp). All measurements were made at 29.0 f 0.5OC. Equilibrium Quotients. It has been shown by Hanna et al.17Jg that the upfield shifts induced by aromatic hydrocarbons upon complexation, with N-alkylamides, can be used to estimate both the equilibrium quotient and the shift of the pure complex in systems in which a 1:l complex is formed. The lH NMR data can be analyzed by the equation 1 1 1 1 -E-(Aobsdh
where (Aobs& = 60 - 6, is the observed difference in chemical shift between acceptor (TMU in our systems) protons in a solution with donor (hydrocarbon in our systems) concentration mi and the chemical shift of the same protons in a solution without donor. Ac is the corresponding shift for the pure complex and Q is the equilibrium quotient for association of the complex. It is an implicit assumption of eq The Journal of Physical Chemistry, Vol. BO, No. 3, 1976
Donesi, Paolillo, and Temussi
TABLE I : Maximum Observed Shifts of the TMU Methyl Groups in Several Aromatic Hydrocarbons [TMU I , M 0.089, 0.089, 0.081, 0,089, 0.030,
Donor Benzene Naphthalene Phenanthrene Pyrene 3,4-Benzopyrene
Range of donor concn, m 0.609-7.191 0.453-1.660 0.392-1.976 0.499-1.1 10 0.120-0.2 16
’p I T M L l
_ i _
Flgure 1. The 100-MHz spectra of TMU methyl groups in CDCI3 (A) and in CDC13-C& (B).
1 that the systems examined are ideal solutions, in which case Q coincides with the equilibrium constant, or that the ratio among activity coefficients of the species in solution remains constant over the concentration range studied. Another obvious requirement for eq 1to be valid is that in all solutions mD >> mA.
Results The l H NMR spectrum of TMU, shown in Figure 1A, contains only four sharp peaks, attributable to the N methyl groups. In spite of the simplicity of the spectrum, assignment of the resonances is not trivial since the four groups are isolated and are in very similar chemical environments. On the other hand, a correct assignment is crucial for the subsequent interpretation of donor-induced shifts in terms of possible complexes’ geometries. A tentative assignment based on literature data on variously methylated xanthines11r20and thioxanthinesZ0was confirmed by an NOE experiment. The peak at 6 3.37 can be easily assigned to 1-CH3, since in all methylated xanthines this methyl group gives rise to the highest field peak.20Another characteristic feature of the NMR spectra of methylated xanthines is the marked downfield shift of the 3-CH3 peak induced by the presence of a methyl in the 9 position.20Accordingly we have assigned the peaks a t 6 3.58, 3.62, and 3.72 to 7-CH3, 3-CH3, and 9-CH3 respectively. Such an assignment is also consistent with the line shape of the four resonances. I t is shown by Figure 1A that the two downfield peaks have a slightly larger half-height width, as The Journal of Physical Chemistry, Vol. 80. No. 3, 1976
Max obsd shifts, Hz CH3(9 1 71.7 100.2 172.2 218.0 130.5
CH3(3) 68.4 91.5 159.5 202.3 118.9
CH3(71 12.7 19.1 49.0 65 .O 50.3
17.7 46.9 60.3 48.2
would be the case for a small long-range coupling and/or for a through-space interaction. These effects can be predicted in the case of TMU only for the adjacent 3-CH3 and 9-CH3 groups. In order to confirm the existence of a small albeit detectable interaction between these methyls we performed an NOE experiment. A small intensity enhancement was actually observed for the peak a t 6 3.72 when irradiating a t 6 3.62 and vice versa, while irradiation of either upfield peak had no effect on the intensities of the other resonances. Addition of aromatic hydrocarbons to N-alkylamides, as anticipated in the Experimental Section, induces large upfield shifts in the resonances of the alkyl groups.17J9 Figure 1B shows the displacements of the TMU peaks upon addition of a small amount of benzene. A similar behavior was observed for all systems studied, with very large shifts for 3-CH3 and 9-CH3 and relatively smaller shifts for the other two methyls. The maximum observed shifts are reported in Table I, along with the ranges of hydrocarbon concentrations. In all cases the linear dependence of the reciprocal of the shift (Aobsd-l) vs. llm was very good, so that one can rule out large contributions from complexes different from the 1:l.Figure 2 shows the graphs of the four methyl resonances for the system TMU-phenanthrene. The lines have been calculated by means of a least-squares analysis of the data that gave also the formation quotients and the limiting shifts for “pure” complexes. Table I1 summarizes the values of these parameters for each methyl group of every TMU-hydrocarbon pair. The main trend of the data of Table I1 is the monotonic increase of formation constants and limiting shifts as one goes from benzene to 3,4-benzopyrene. Such a behavior is consistent with the increase of ring current, resonance energy,21 and p ~ l a r i z a b i l i t yone ~ ~expects ~ ~ ~ from an increase in the number of benzene rings. On this basis, however, no clear-cut conclusion can be drawn on the nature of the fortes stabilizing the complexes. In fact all types of forces suggested by various authors are compatible with the qualitative trend of polarizability and of resonance energy. Our data, though, are amenable to a more detailed analysis in terms of molecular geometry which, in turn, can give us accurate information on the nature of the stabilizing forces. As mentioned in the Introduction, our systems are characterized by the presence of four “probes” (the methyl groups) well spaced on the purine molecule. Accordingly the number of relative orientations of the two interacting molecules that are consistent with the experimental shifts is very limited. Trial geometrical models of the complex between TMU and benzene were built on the basis of the atomic coordinates of TMU in the crystal structure24and of standard molecular parameters for benzene. The limiting shifts were calculated for each model by means of the formula of Waugh and FessendeneZ5If we restrict our search to 1:l complexes, there are essentially two limiting configurations we can start with, i.e., one with the molecular planes approximately parallel and another with the two
28 1 TMU -PI-IENANTRENE
Figure 2. Plot of experimental methyl shifts of TMU vs. phenanthrene molality for the phenanthrene-TMU complex.
TABLE 11: Results of a Least-Squares Analysis of Experimental Dataa TMU-benzene TMU-naphthalene TMU-phenanthrene TMU-pyrene TMU-3,4-benzopyrene ____Methyl Q, kg mol-' AAD, Hz Q, kg mol-' AAD, Hz Q, kg mol-' AAD, Hz Q , kg mol-' AAD,Hz Q, kg mol-' AAD, Hz
_ I _
0.29 i 0.02 17 i 1 0.60 i 0.03 35 i 2 1.20 i 0.02 66 i 1 1.56 i 0.06 9 5 i 2 2.2 i 0.1 149 i 6 0.22 i 0.01 22 i 1 0.58 i 0.03 39 i 2 1.28 i 0.02 68 i I 1.77 i 0.07 97 i 2 2.8 i 0.1 132 i 4 0.212 i 0.004 115 f 2 0.58 i 0.02 186 i 8 1.12 i 0.01 229 i 2 1.69 i. 0.10 310 i 7 2.8 i 0.2 314 i. 18 9 0.217 i 0.004 119 i 2 0.59 i 0.02 202 i 8 1.14 i 0.01 246 i 3 1.7, i 0.10 340 i 10 2.7, i 0.17 348 i 1 6 aComputed equilibrium quotients ( Q ) and shifts for pure complexes (AAD)between TMU and aromatic hydrocarbons are reported. 1
molecules approximately perpendicular. A sketch of the model with perpendicular arrangement is shown in Figure 3; the center of the benzene ring is coincident with the origin of the coordinate system and the molecular planes coincide with the xy and yz planes. In both models it is easy to find almost correct shifts for the 3-CH3 and 9-CH3 resonances by placing the center of the benzene ring equidistant from both methyl groups and a t ca. 0.3 nm from the line joining them. Neither model however gives a reasonable agreement for the other two shifts. In fact, for the complex with parallel molecules, the two pairs of calculated shifts (1,7 and 3,9) are of opposite sign. Numerous models with the molecular planes at angles (cp) ranging from 0 and 90° were examined at steps of the order of 5 O , allowing for small displacements of the center of the benzene ring. The line joining methyls 3 and 9 was also slightly tilted to dissymmetrize the shifts of these methyl groups. As reported in Table 111, a satisfactory agreement between calculated and observed shifts can be obtained for angles (9) around 4 5 O and a displacement of 0.05 nm of the benzene ring along the x axis.
T A a E 111: Comparison between Experimental and Calculated Shifts of Pure Benzene-TMU Complex for Various Geometrical Arrangementsa 6,,Hz
113 113 aSee text for definition of cp.
119 119 119 119
Exptl cp = 90" c p = 0" c p = 45"
An even better agreement could be obtained by minor adjustments of the relative orientation. We decided against this refinement because on the basis of the experimental data it is not possible to exclude the presence in solution of very small amounts of other (1:l or 2:l) complexes whose effect on the chemical shift changes might compensate the small discrepancies between calculated and observed shifts of Table 111. Before considering in detail the relationship between the geometry of the complexes and the possible intermolecular The Journal of Physical Chemistry, Vol. 80, No. 3, 1976
Donesi, Paollllo, and Temussi
Figure 3. Model of the TMU-benzene complex with perpendicular
forces it is appropriate to discuss the parameters measured for the other complexes in terms of the model found for the TMU-benzene complex. A quantitative calculation of the ring current shifts induced by the other hydrocarbons is not as easy as that for benzene and was not attempted since all NMR parameters can be interpreted, at least qualitatively, with reference to the benzene calculations. It can be seen from Table I1 that in all complexes the 3 and 9 shifts are always very similar and much larger than those of the pair 1,7. The simplest interpretation of this behavior is that the hydrocarbon is closer to the pair 3,9 and that the planes . . and 90’. It is true that for hydrocarbons with a very large area this interpretation may not be univocal, but the regular trend of the shifts along the series of the five hydrocarbons examined points to a substantial continuity of the geometrical model.
The Journal of Physical Chemistry, Vol. 80, No. 3, 1976
The general features of the geometrical model we propose on the basis of the NMR results are not equally consistent with all types of forces previously proposed for these complexes. The essential feature of any charge-transfer complex is a good superposition of the T orbitals of the interacting molecules. It is clear then that any geometrical arrangement in which the planes of TMU and of the hydrocarbon are not nearly parallel cannot be stabilized by charge-transfer forces to any significant extent. As mentioned above, such is the case for all our complexes and thus we must restrict our analysis to the other two main types of forces, Le., dispersion forces and dipoleinduced dipole forces. The latter are in fact consistent with the directions of total dipole moment and local dipole moments of TMU (vide supra) and we are inclined to attribute to these forces the major role in stabilizing the complexes studied. On the basis of our data, however, it is not possible to assess the contribution of dispersion forces with any degree of accuracy. It is only fair to say that dispersion forces may play an increasingly important role in the systems with larger hydrocarbons, but certainly dipole-induced dipole forces play an important role in all complexes studied and are responsible of the geometric arrangements compatible with the NMR shifts.
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