Heteronuclear Scalar Couplings in the Bases and Sugar Rings of Nucleic Acids: Their Determination and Application in Assignment and Conformational Analysis

Abstract

The scalar coupling constants in uniformly isotope-enriched [13C, 15N] nucleotide 5′-monophosphates (5′-NMPs) and in various non-labelled cyclic nucleotides were investigated. These model compounds yielded an almost complete set of homonuclear and heteronuclear coupling constants in ribonucleotides, the knowledge of which is useful in designing novel heteronuclear NMR experiments and opens up new possibilities in the structure determination of larger nucleic acids. Three sets of heteronuclear coupling constants were obtained: (1) conformation-independent 1H–13C, 1H–15N, 13C–15N, 13C–13C and 15N–15N coupling constants in the base, knowledge of which is essential in optimizing and designing new NMR experiments, which use the coherent transfer of magnetization via the J-coupling network in the nucleic acid base and sugar; (2) 1H–13C coupling constants, 3JH1′C4/2 and 3JH1′C8/6, monitoring the glycosidic torsion angle χ, give important information on the rotamer distribution around the χ angle; a new parameterization of the Karplus equations is presented; and (3) conformation-dependent one-bond and multiple bond 1H–13C coupling constants in the ribose sugar. Conformationally rigid, cyclic, nucleotides were used to determine multiple bond 1H–13C coupling constants in pure N-type and pure S-type sugar rings. Equations were derived for the determination of the fraction S-type sugar, pS, from the three-bond JCH couplings 3JH3′C1′, 3JH2′C4′, 3JH1′C3′ and 3JH4′C2′. Their values for pure N- and S-type sugar conformations were used to derive Karplus equations, which describe the dependence of these coupling constants on the phase angle, P.