Unveiling aggregation-induced emission in benzimidazole–acrylonitriles for fluorescence live-cell imaging in HeLa cells

Abstract

Cellular imaging is a pivotal strategy for unraveling complex biological processes, disease mechanisms, and drug responses, wherein benzimidazole–acrylonitriles are emerging as promising yet underexplored fluorogenic scaffolds for advanced imaging and therapeutic applications. In the present study, five benzimidazole–acrylonitrile conjugates bearing nitrogen-rich heterocycles were synthesized and systematically investigated for their photophysical characteristics and aggregation-induced emission (AIE) behaviour. The synthetic strategy involves a stepwise condensation of o-phenylenediamine with ethyl cyanoacetate, followed by an L-proline-catalyzed condensation of the resulting intermediate with nitrogen-rich arylaldehydes. The molecular structures of the synthesized compounds were confirmed by ¹H and ¹³C NMR spectroscopy and high-resolution mass spectrometry (HRMS); the structure of compound 5 was further unambiguously established by single-crystal X-ray diffraction analysis. Optical studies revealed distinct absorption and emission features attributable to the hybridized local and charge transfer excited state (HLCT), along with pronounced aggregation-induced emission (AIE) behavior in THF/water mixtures. The observed AIE characteristics were further supported by scanning electron microscopy (SEM) and dynamic light scattering (DLS), which revealed the formation of well-defined aggregated morphologies. All compounds demonstrated classic molecular-rotor-type fluorescence enhancement with increasing viscosity using a DMSO–glycerol mixture and showed distinct pH-responsive emission governed by protonation dynamics within the biological pH range, highlighting their potential as robust probes for visualizing intracellular heterogeneity and acidic microenvironments. Density functional theory (DFT) calculations provided complementary insights into the electronic structure and optical transitions. Remarkably, compound 1 exhibited efficient cytoplasmic localization in live HeLa cells, demonstrating its potential utility as a fluorescent bioimaging probe. Collectively, these findings establish benzimidazole–acrylonitrile conjugates as a new class of AIE-active luminogens with promising applications in precision bioimaging, tumor diagnostics, and theranostic platforms.