Serendipitous discovery of nitrogen bridgehead fused pyridines: photophysical properties and live-cell imaging potential

Authors:

Hana Kokot, Aljoša Bolje, Jakob Kljun, Andraž Bevk, Natalija Trunkelj, Lucija Peterlin Mašič, Janez Mravljak, Stane Pajk

Keywords:

Nitrogen bridge; head fused pyridine; Fluorescence; pH sensitive probes; Live-cell imaging; Fluorescence lifetime imaging

Abstract:

A novel synthetic route to fluorescent nitrogen bridgehead fused pyridines (NBFPs) was discovered serendipitously during attempts to synthesize a thiazole derivative via a Hantzsch-type thiazole reaction from 2-cyanoethanethioamide and chloroacetone. A mechanism was proposed to rationalize the formation of NBFPs, which guided the subsequent expansion of the reaction scope and optimization of reaction conditions. The scope of the reaction was extended to include arylacetonitriles and esters of 2-(heteroaryl)acetic acids bearing an ortho-positioned pyridine-type nitrogen as a starting material. The structures of selected compounds were confirmed by single-crystal X-ray diffraction. All synthesized NBFPs exhibited pH-sensitive fluorescence and Stokes shifts exceeding 100 nm in several cases. Two compounds, methylthiazole derivative 1 and quinoline derivative 8, displayed pH-dependent emission suitable for lysosomal labelling. However, live-cell microscopy revealed limited colocalization with lysosomal probe Lysotracker Red, suggesting that these probes may localize to other acidic environments as well. Both compounds showed pronounced spectral responsiveness and fluorescence lifetime variations, indicating potential for use as environment-sensitive intracellular sensors. The methyl thiazole derivative 1 exhibited particularly favourable properties, including high photostability, low background fluorescence, minimal cytotoxicity and polarity sensitivity. These findings position NBFPs as a promising class of fluorophores for live-cell imaging and intracellular sensing applications, and warrant further investigation into their structure–function relationships and environmental responsiveness.

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