Observation of internucleotide NH•••N hydrogen bonds in the absence of directly detectable protons

Several structural motifs found in nucleic acids involve N-H ... N hydrogen bonds in which the donor hydrogens are broadened to extinction due to chemical or conformational exchange. In such situations, it is impossible to use the well-established HNN-COSY or soft HNN-COSY experiments, which report the presence of the hydrogen bond directly on the donor proton(s). We present a pulse sequence, H(CN)N(H), for alleviating this problem in hydrogen bonds of the type NdH ... N-a-CH, in which the donor N-d nitrogen is correlated with the corresponding non-exchangeable C-H proton associated with the acceptor N-a nitrogen. In this way, missing NdH ... N-a correlations in an HNN-COSY spectrum may be recovered from CH-N-d correlations in the H(CN)N(H) spectrum. By correlating a different set of nuclei relative to the HNN-COSY class of experiments, the H(CN)N(H) experiment also serves to remove ambiguities associated with degeneracies in HNN-COSY spectra. The technique is demonstrated on d(GGAGGAG)(4), a quadruplex containing a novel A . (G . G . G . G) . A hexad and on d(GGGCAGGT)(4), containing a G . C . G . C tetrad, in which missing NH2 ... N7 correlations are retrieved via H8-(N2,N6) correlations in the H(CN)N(H) spectrum.