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

We developed a new fluorescent analog of cytosine, the 4-amino-1H-benzo[g]q uinazoline-2-one, which constitute a probe sensitive to pH, The 2'-O-Me rib onucleoside derivative of this heterocycle was synthesized and exhibited a fluorescence emission centered at 456 nm, characterized by four major excit ation maxima (250, 300, 320 and 370 nm) and a fluorescence quantum yield of Phi = 0.62 at pH 7.1. The fluorescence emission maximum shifted from 456 t o 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 tripler forming oligonucleotide s this new nucleoside can be used to reveal the protonation state of triple ts in triple-stranded structures, Complex formation was detected by a signi ficant quenching of fluorescence emission (similar to 88%) and the N-3 prot onation of the quinazoline ring by a shift of the emission maximum from 485 to 465 nm, Using this probe we unambiguously showed that tripler formation of the pyrimidine motif does not require the protonation of all 4-amino-2- one pyrimidine rings.