Hydroxylamine Mutagenesis: Observation of Inverted Watson‐Crick Base‐Pairing Between N4‐Methoxycytosine and Adenine with the Aid of Natural‐Abundance High‐Resolution 15N NMR Spectroscopy

Abstract

Natural-abundance high-resolution 15N NMR spectra were recorded for 1-methyl-N4-methoxycytosine (the product of reaction of 1-methylcytosine with the mutagen methoxyamine), 2'',3'',5''-tri-O-methyladenosine, and a 1:1 mixture of the 2, all in chloroform solution. The spectra were obtained by application of a sequence of impulses of the type referred to as insensitive nuclear enhancement by polarization transfer (INEPT). Assignments of all the N resonances were based on N-proton coupling constants and literature data. Analysis of the 15N chemical shifts demonstrated the formation of base pairs with H bonds involving the ring N(1) and an N6-H of adenine, and the N(3)-H and C(2)=O of N4-methoxycytosine, namely, N(1) .cntdot. H-N(3) and N6-H .cntdot. O2 = C. The H-bond-stabilized complex formed, thus involves an inverse type of Watson-Crick base-pairing. The data excluded formation of Hoogsteen-type base pairing. The results are considered in relation to hydroxylamine and methoxyamine mutagenesis, to types of base-pairing at the monomer and polymer levels and to interpretations of 15N NMR spectra of heterocyclic rings in general.