Synthesis and characterization of stacked and quenched uridine nucleotide fluorophores

Intramolecular aromatic interactions in aqueous solution often lead to stac ked conformation for model organic molecules. This designing principle was used to develop stacked and folded uridine nucleotide analogs that showed h ighly quenched fluoroscence in aqueous solution by attaching the fluorophor e 1-aminonaphthalene-5-sulfonate (AmNS) to the terminal phosphate via a pho sphoramidate bond. Severalfold enhancement of fluorescence could be observe d by destacking the molecules in organic solvents, such as isopropanol and dimethylsulfoxide or by enzymatic cleavage of the pyrophosphate bond. Stack ing and destacking were confirmed by H1- NMR spectroscopy. The extent of qu enching of the uridine derivatives correlated very well with the extent of stacking. Taking 5-H as the monitor, temperature-variable NMR studies demon strated the presence of a rapid interconversionary equilibrium between the stacked and open forms for uridine-5'-diphosphoro-beta-1-(5-sulfonic acid) naphthylamidate (UDPAmNS) in aqueous solution. Delta H was calculated to be -2.3 Kcal/mol, with 43-50% of the population in stacked conformation. Fluo rescence lifetime for UDPAmNS in water was determined to be 2.5 ns as again st 11 ns in dimethyl sulfoxide or 15 ns for the pyrophosphate adduct of AmN S in water. Such a greatly reduced lifetime for UDPAmNS in water suggests c ollisional interaction between the pyrimidine and the fluorophore moieties to be responsible for quenching. The potential usefulness of such stacked a nd quenched nucleotide fluorophores as probes for protein-ligand interactio n studies has been briefly discussed.