Synthesis, Photophysical Analysis, and Incorporation of Laurdan-Based Microenvironment-Sensitive Fluorescent Nucleoside Analogs

Abstract

Fluorescent nucleoside analogs (FNAs) are attractive candidates for probing nucleic acid dynamics owing to their potential for minimal perturbation, microenvironment sensitivity, and site-specific incorporation. However, the utility of many FNAs is limited in cellular settings because of their UV excitation. Two-photon (2-P) excitable fluorescent nucleobase analogs can overcome this limitation. Drawing inspiration from polarity-sensitive two-photon-excitable Dimethylaminonaphthalene (DAN) moiety based probes, such as Laurdan and C-Laurdan, our work focuses on the synthesis and photophysical analysis of a C5-DAN-modified pyrimidine nucleoside analog, followed by its incorporation into oligonucleotides (ONs). The DAN-modified nucleosides, with a strong intramolecular charge-transfer (ICT) character, displayed emission properties that showed a pronounced dependence on solvent polarity: a progressive red-shift and fluorescence quenching with increasing solvent polarity, culminating in complete quenching in water. DAN-dU-modified DNA ONs showed negligible emission in aqueous buffer, suggesting efficient quenching despite the anticipated rigidification and partial desolvation of the DAN moiety upon incorporation into the ONs. The absence of emission in bulk water prompted further examination of the probe in AOT reverse micelle systems (AOT RMs), which serve as models for nanoconfined hydration environments, where the enclosed water shows lower polarity and higher viscosity than bulk water. In AOT RMs with low water content (w₀ = 0.8- 2.5), both DAN-dU nucleoside and DAN-dU-modified ON displayed a strong and blue shifted emission band. This observation is attributed to the projection of the modified nucleoside into the interfacial water, where it becomes conformationally rigid and experiences a highly nonpolar microenvironment. However, as the water content increased, the modified nucleoside and ON percolated into the water droplet, which is more polar and less viscous, resulting in progressive quenching of fluorescence intensity and a red-shifted emission maximum. Taken together, these findings suggest a potential application of our DAN probe for monitoring interactions in non-polar environments and in nanoconfined water regions, such as those encountered in the hydrophobic core of nucleic acid-interacting proteins (e.g., tumor suppressor p53 protein interacting with its cognate DNA sequence). Consistent with DAN-moiety based probes, the present nucleoside is also expected to exhibit favourable 2-P properties, suggesting its potential application in 2-P imaging of nucleic acids within cells.