4-amino-1 H-benzo[g] quinazoline-2-one: a fluorescent analog of cytosine to probe protonation sites in triplex forming oligonucleotides

摘要

We developed a new fluorescent analog of cytosine, the 4-amino-1 H-benzo[g] quinazoline-2-one, which constitute a probe sensitive to pH. The 2'-O-Me ribonucleoside derivative of this heterocycle was synthesized and exhibited a fluorescence emission centered at 456 nm, characterized by four major excitation maxima (250, 300, 320 and 370 nm) and a fluorescence quantum yield of #PHI#=0.62 at pH 7.1. The fluorescnece emission maximum shifted from 456 to 492 nm when pH was decreased from 7.1 to 2.1. The pK_a (4) was close to that of cytosine (4.17). When introduced in triplex forming oligonucleotides this new nucleoside can be used to reveal the protonation state of triplets in triple-stranded structures. complex formation was detected by a significant quenching of fluorescence emission (approx 88%) and the N-3 protonation of the quinazoline ring by a shift of the emission maximum from 485 to 465 nm. Using this probe we unambiguously showed that triplex formation of the pyrimidine motif does not require the protonation of all 4-amino-2-one pyrimidine rings.