Design and synthesis of a novel fluorescent benzo[g]imidazo[4,5-c]quinoline nucleoside for monitoring base-pair-induced protonation with cytosine: distinguishing cytosine via changes in the intensity and wavelength of fluorescence

Abstract

A novel fluorescent benzo[g]imidazo[4,5-c]quinoline nucleoside ^BIQA (1) comprising a 3-deaza-2′-deoxyadenosine skeleton was developed and used to monitor ^BIQA–C base-pair formation in oligodeoxynucleotide (ODN) duplexes. The newly synthesized ^BIQA exhibited distinct photophysical properties associated with its protonated/deprotonated forms (monomer: pK_a 6.2) via dramatic changes in its absorption and fluorescence spectra. In ODN duplexes, the induced protonation of ^BIQA occurred, even under alkalescent conditions when cytosine was the opposite base on the complementary strand; the resulting ^BIQA–C base pairs were stable. By monitoring the protonation of ^BIQA under neutral and alkalescent conditions, we could clearly discriminate cytosine through spectral changes in absorption and fluorescence. Similarly, we found that the demonstrated 3-deaza-2′-deoxyadenosine ^3zA forms a stable base pair with cytosine via N¹ protonation in ODN duplexes under neutral and acidic conditions (pH < 7.0). At lower pH values, ^3zA-containing ODNs could clearly discriminate cytosine through melting temperature (T_m) measurements. Therefore, ODN probes containing indicator nucleosides, such as ^BIQA and ^3zA, exhibit great potential as bioprobes for genetic analysis and structural studies of nucleic acids.