Detection of Proton‐Acceptor Sites of Hydrogen Bonding in Adenine · Uracil Base Pairs by the Use of 15N Magnetic Resonance

2′,3′-5′-Tri-O-acetyl[1,3,7,9,amino-15N]adenosine(ac3Ado) and its 8-2H and 8-bromo derivatives (ac3[8-2H]Ado and ac3br8Ado) were synthesized from 95%15N-enriched adenosine which was obtained by microbial fermentation. The chemical shifts and nuclear Overhauser effects of 15N NMR of the adenosine derivatives were measured by changing the concentration of the mixed 1-cyclohexyluracil (cHxUra) in chloroform. The limiting shift of each 15N resonance was calculated by using the association constants obtained from proton magnetic resonances of the ac3Ado/cHxUra and ac3br8Ado/cHxUra systems. From the quantitative analysis of the 15N chemical shifts of the N-1 and N-7 atoms it could be concluded that ac3Ado · cHxUra dimers prefer the Hoogsteen-type pair while the Watson-Crick-type pairs are predominant in the ac3br8Ado · cHxUra dimers. The N-3 atom, which does not locate at the interaction site, showed a fairly large induced shift. Thus the induced shift of 15N resonance by association does not always mean the involvement of the corresponding nucleus to the interaction site. The effect of intermolecular hydrogen bonding on the nuclear Overhauser enhancement of the 15N-7 resonance was observed in the ac3[8-2H]Ado/cHxUra mixture. The presence of the Hoogsteen-type hydrogen bonding was proposed by the use of 15N NMR spectra. The present information will be useful for the elucidation of non-Watson-Crick-type base-pair interaction in transfer RNA and oligonucleotides.