Strain-promoted azide–alkyne cycloaddition (SPAAC) is a widely used reaction for the visualization of glycans through metabolic labelling with azidosugars. However, in this work we aim to enable quantitative analysis of bioorthogonal reactions, such as SPAAC, in cells to gain deeper insight into their efficiency and behaviour under physiological conditions. Incorporating Raman-active vibrational reporters, such as nitriles, into SPAAC probes offers the potential for direct, multiplexed detection in cells.
In this project, we are developing a fluorescent dibenzocyclooctyne (DBCO) probe containing a phenyl nitrile, allowing simultaneous fluorescence microscopy and Raman detection via nitrile stretching in the biologically silent region. The design specifically seeks to enhance nitrile Raman intensity by increasing phenyl ring polarizability, thereby improving sensitivity in cellular environments.
We evaluated a library of naphthalimide-based phenyl-nitrile derivatives in the solid state, in aqueous solution, and on fixed A549 cells to assess spectral visibility and fluorescence interference. Several compounds produced clear nitrile peaks in cells with minimal fluorescence interference. Live-cell Raman imaging was also performed using a selected probe, confirming that nitrile-based probes can be detected within the cellular silent region under physiologically relevant conditions. Ongoing work focuses on incorporating DBCO into the nitrile-tagged naphthalimide probe for bimodal cellular imaging and extending this strategy toward quantitative, minimally invasive tracking of bioorthogonal reactions and metabolically labelled glycans in live cells.