Syntheses of [8-carbon-13-1,7,amino radical-nitrogen(15,3)]-adenosine, -guanosine, and their deoxy analogs, their incorporation into DNA and RNA fragments, and nitrogen-15 NMR experiments

Oligonucleotides that contain specifically 15N multilabeled monomers can provide useful NMR information. To avoid ambiguity in signal identification when more than one multilabeled 15N monomer is present, a 13C atom can be introduced as a tag into one of a pair of 15N multilabeled nucleosides. [8-13 C-1,7,NH2-15N3]-Adenosine, -guanosine, and their deoxy analogs were synthesized to make use of the 8- 13C label as an indirect tag for the adjacent, biologically significant 15N7 labeled atoms. Although the very small C8-N7 coupling (<1 Hz) precludes its direct detection in 1D 15N spectra, 2D 1H-15N NMR experiments display the large C8-H8 coupling (>200 Hz) because H8 is coupled to both N7 and C8. The 13C8 atom was introduced by means of a ring closure of two exocyclic amino groups on a pyrimidinone using [13C]-sodium ethyl xanthate. The resulting hypoxanthine was converted to the adenosine N1 oxide, from which all the multilabeled nucleosides were made. The final products were synthesized with overall yields ranging from 24% to 36%. Improvements were made in the protection and phosphitylation reactions necessary for incorporating them into oligonucleotides.; Using the phosphoramidite approach, eight DNA oligonucleotides were synthesized in quantities ranging from 11 to 26 mumol for investigations by collaborators. Four unlabeled DNA fragments were synthesized for spectroscopic, calorimetric, and other physical studies, and four different 15N, 13C multilabeled versions of a DNA G-quadruplex were synthesized to determine proton assignments required for its NMR structural determination.; Specifically 15N multilabeled RNA oligonucleotides were synthesized by the phosphoramidite approach in quantities ranging from 2.6 to 25 mumol for NMR studies of the two separate domains of the hairpin ribozyme. Using 15N NMR, the pKa of the N1 of G8 in the A loop motif was found to maintain a normal value greater than eight. 15N NMR was also used to probe for interactions of Co 3+ and Mg2+ with labeled nitrogens in specific adenosine and/or guanosine bases in both the A and B loops. As was previously reported, the loop A domain was found to have no metal binding sites. Due to the dynamic structure of loop B, a collection of different conformations was detected, none of which appear to have a tight metal binding site.