Fluorescent reporter groups are widely used in many areas of macromolecular science. In nucleic acid chemistry, fluorescently labeled oligonucleotides find use in the study of nucleic acid conformations, as reporters of the state of hybridization and as probes of nucleic acid-protein interactions, as well as, probes and primers for the detection and quantification of desired sequences. Often, the fluorophore is a separate and independent entity covalently attached to an otherwise unmodified oligonucleotide. Another class of fluorophore is the nucleobase analog - a chemically
modified nucleobase that is unable to base pair properly. Recently, we have become interested in pyrrolocytosine (pC) as an intrinsically fluorescent, pairing-competent nucleobase. It has previously been shown that incorporation of pyrrolocytidine into oligonucleotides gives a fluorescent molecule that responds to the state of hybridization. We have refined the syntheses of pyrrolocytosine derivatives and the related modified uracil derivatives, furanouracil (fU), from cytosine and uracil respectively via three sequential steps: iodination of the nucleobase; Pd-catalyzed cross-coupling with a terminal alkyne; and metal-catalyzed cyclization. The fluorescence properties of the 5-alkynyl nucleobases and the bicyclic pC and fU analogs have been characterized and depend on: 1) the nature of the substituent on the alkyne, 2) whether the nucleobase is 5-alkynyl or cyclized, and 3) the presence of a substituent on the N1-position of the nucleobase. The chemistry and properties of peptide nucleic acid oligomers incorporating these types of base modifications and their possible use in point-mutation detection is presented.