Synthesis and Photophysical Characterization of a pH‐Sensitive Quadracyclic Uridine (qU) Analogue

Herein, we present the synthesis and detailed photophysical characterization of qU – a novel fluorescent base analogue of uridine. qU exhibits interesting properties such as excitation in the visible region with high brightness, dual emission, pH sensitivity, and an ESPT reaction. This work has extended the toolbox of internal RNA labels for future applications such as fluorescence lifetime imaging.

Abstract

Fluorescent base analogues (FBAs) have become useful tools for applications in biophysical chemistry, chemical biology, live-cell imaging, and RNA therapeutics. Herein, two synthetic routes towards a novel FBA of uracil named qU (quadracyclic uracil/uridine) are described. The qU nucleobase bears a tetracyclic fused ring system and is designed to allow for specific Watson-Crick base pairing with adenine. We find that qU absorbs light in the visible region of the spectrum and emits brightly with a quantum yield of 27 % and a dual-band character in a wide pH range. With evidence, among other things, from fluorescence lifetime measurements we suggest that this dual emission feature results from an excited-state proton transfer (ESPT) process. Furthermore, we find that both absorption and emission of qU are highly sensitive to pH. The high brightness in combination with excitation in the visible and pH responsiveness makes qU an interesting native-like nucleic acid label in spectroscopy and microscopy applications in, for example, the field of mRNA and antisense oligonucleotide (ASO) therapeutics.

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