Synthesis and characterization of stacked and quenched uridine nucleotide fluorophores

Abstract

Intramolecular aromatic interactions in aqueous solution often lead to stacked conformation for model organic molecules. This designing principle was used to develop stacked and folded uridine nucleotide analogs that showed highly quenched fluoroscence in aqueous solution by attaching the fluorophore 1-aminonaphthalene-5-sulfonate (AmNS) to the terminal phosphate via a phosphoramidate bond. Severalfold enhancement of fluorescence could be observed by destacking the molecules in organic solvents, such as isopropanol and dimethylsulfoxide or by enzymatic cleavage of the pyrophosphate bond. Stacking and destacking were confirmed by ¹-H NMR spectroscopy. The extent of quenching of the uridine derivatives correlated very well with the extent of stacking. Taking 5-H as the monitor, temperature-variable NMR studies demonstrated the presence of a rapid interconversionary equilibrium between the stacked and open forms for uridine-5'-diphosphoro-β-1-(5-sulfonic acid) naphthylamidate (UDPAmNS) in aqueous solution. ΔH was calculated to be -2.3 Kcal/mol, with 43-50% of the population in stacked conformation. Fluorescence lifetime for UDPAmNS in water was determined to be 2.5 ns as against 11 ns in dimethyl sulfoxide or 15 ns for the pyrophosphate adduct of AmNS in water. Such a greatly reduced lifetime for UDPAmNS in water suggests collisional interaction between the pyrimidine and thefluorophore moieties to be responsible for quenching. The potential usefulness of such stacked and quenched nucleotide fluorophores as probes for protein-ligand interaction studies has been briefly discussed.