molecules in aqueous solution? Do they associate 

 with each other vertically through hydrophobic 

 and stacking interactions, or do they associate 

 horizontally through hydrogen bonding? These 

 thermodynamic data do not support the hypo- 

 thesis of horizontal association through hydrogen 

 bonding for the following reasons: 



1. Methylation and bromination enhance 

 association. 



2. All these bases and nucleosides as- 

 sociate much more extensively than 

 urea which is one of the best hydrogen 

 bonding agents in water. 



More direct information about the mode of 

 association of the bases and nucleosides in 

 solution can be obtained by the study of nuclear 

 magnetic resonance. It is well known that 

 nuclear magnetic shielding is a very sensitive 

 probe of inter- and intra- molecular interactions. 

 In this case, vertical stacking interactions are 

 easily distinguished from hydrogen bonding 

 interactions and these interactions manifest 

 themselves differently in the NMR. It is there- 

 fore hoped that the concentration dependence 

 of the NMR spectra in aqueous solutions of 

 purine and nucleosides will shed some light 

 on the association mechanism. The NMR spectra 

 of purine have been studied over the concen- 

 tration range of .05 to 1 molar (3). Chemical 



-I lOi 

 -100 



-90 



-80 



-70 



-60 



-50 i 



-40 



0.1 02 3 4 0,5 0.6 07 08 0.9 1.0 

 MOLAL CONCENTRATION 



Fig. 1. 



Concentration dependence of the proton chemical shifts 

 for purine in aqueous solution at 25° (corrected for bulk 

 susceptibility); shifts measured from external chloro- 

 form reference: — o — , experimental values; — X — , 

 calculated values from overall average model; — a — , 

 calculated values from statistical partial-overlapping 

 model. (From Chan, Schweizer, Ts'o and Helmkamp, 

 /. Am. Chem. Soc. 86, 4182, 1964; reproduced with per- 

 mission of the American Chemical Society.) 



shifts of the three protons in purine vs the 

 concentration are shown in Fig. 1. A pronounced 

 concentration effect has been observed. Proton 

 resonances in purine are all shifted to higher 

 fields as the solute concentration is increased. 

 Shifts to high fields with concentration are well 

 known for aromatic systems and are generally 

 attributed to the magnetic anisotropy associated 

 with the ring currents in neighboring mole- 

 cules. Because of the mobile-electrons, a large 

 diamagnetic current is induced in the plane of 

 the ring by an external magnetic field when the 

 field is perpendicular to the plane of the mole- 

 cule. This ring current gives rise to a small 

 secondary magnetic field which reinforces the 

 primary field at the peripheral protons in the 

 plane of the ring. In the region directly above 

 and below the molecular plane, the two fields 

 are opposed, however. As the concentration of 

 a solution of aromatic molecules is increased, 

 the average distance between molecules de- 

 creases and the protons of a given molecule will 

 feel the secondary magnetic fields produced by 

 the ring current of neighboring molecules. Since 

 it is much more probable to find the molecules 

 somewhere above or below the molecular plane 

 of another aromatic molecule due to the dish- 

 shaped nature of the aromatic molecules, this 

 magnetic anisotropy of the ring current effect 

 will lead to a high field shift with concentration 

 or to a low field shift upon dilution. At higher 

 temperature, or when the purine is dissolved 

 in organic solvent such as dimethylsulfoxide 

 and dimethylformamide, such concentration- 

 dependent chemical shifts for the purine pro- 

 tons are greatly reduced. Furthermore, when 

 the purines are protonated by hydrochloride so 

 they cannot associate because of carrying a 

 positive charge, such concentration-dependent 

 chemical shifts are again practically elimi- 

 nated. These data clearly suggest that the mode 

 of association of purine is by the vertical stack- 

 ing of rings in a partial overlapping fashion. 

 As described above, the osmotic coefficients 

 and activity coefficients of purine have been 

 interpreted in terms of multiple equilibria and 

 on this basis, populations of various associate 

 species at varying concentrations were com- 

 puted. Based on these population distributions 

 of the associated species, we can calculate the 

 concentration dependence of the chemical shifts 

 which is also given in Fig. 1 (3). It can be seen 

 that the calculated value and the experimental 

 value are in satisfactory agreement. There- 

 fore, a numerical correlation between the NMR 

 data and osmotic data has been successful in 



186 



