The long-term goals of this project are to develop and demonstrate the synthetic strategies necessary for the synthesis and utilization of 15N labeled nucleic acids. Nitrogen-15 labeled oligonucleotides, ribo or deoxyribo, will be invaluable probes of nucleic acid structure, drug- binding, and protein-nucleic acid interaction. In fact, there are some types of information which may be available only from such 15N probes. Moreover, although modern high-field nmr spectrometers have had for some time the necessary capabilities, the 15N labeled oligonucleotides needed have been unavailable. The potential of these 15N labeled oligonucleotides cannot begin to be realized until practical routes to them are available. The methods and procedures to be developed will have a major impact on the availability of 15N labeled oligonucleotides, allowing them to be widely used for the first time. In addition, the labeled oligonucleotides to be prepared and analyzed by 15N nmr will provide important information about DNA structure, drug-binding, and protein-DNA interactions. The first phase of this research will be to develop routes of 15N labeled purine deoxynucleosides, in particular to 15N labeled 06- methyldeoxyguanosine. The 15 N is to be introduced selectively at each nitrogen except the glycosidic nitrogen. Each of the 15N derivatives of deoxyadenosine, deoxyguanosine, and O6-methyldeoxyguanosine will be prepared. Furthermore, the 2-aminopurine, 2,6-diaminopurine, hypoxanthine and O6-methylhypoxanthine deoxyribosides also will be available by the routes proposed. In order to make these 15N labeled compounds generally available, the synthetic routes will be designed to be as economical as possible. The second phase will be incorporation of these 15N labeled deoxynucleosides into oligonucleotides. A large-scale H-phosphonate method, in which efficient use of the labeled deoxynucleosides is emphasized, will be used for the oligonucleotide synthesis. These procedures would be applicable to any oligonucleotide synthesis in which scale and economy are important. The final phase will be to investigate, by 15N nmr, the base pairing of O6MeG, base pairing of mismatches, novel DNA structures which may include Hoogsteen pairing at the 7-position, drug-DNA interactions which may be monitored by the [3- 15N] and [2-15N] in the minor groove, and protein-DNA interactions which may be monitored by the [7-15N] in the major groove.
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