Engineering Central\nSubstitutions in Heptamethine\nDyes for Improved Fluorophore Performance

As a major family\nof red-shifted fluorophores that operate\nbeyond\nvisible light, polymethine dyes are pivotal in light-based biological\ntechniques. However, methods for tuning this kind of fluorophores\nby structural modification remain restricted to bottom-up synthesis\nand modification using coupling or nucleophilic substitutions. In\nthis study, we introduce a two-step, late-stage functionalization\nprocess for heptamethine dyes. This process enables the substitution\nof the central chlorine atom in the commonly used 4′-chloro\nheptamethine scaffold with various aryl groups using aryllithium reagents.\nThis method borrows the building block and designs from the xanthene\ndye community and offers a mild and convenient way for the diversification\nof heptamethine fluorophores. Notably, this efficient conversion allows\nfor the synthesis of heptamethine-X, the heptamethine scaffold with\ntwo ortho-substituents on the 4′-aryl modification, which brings\nenhanced stability and reduced aggregation to the fluorophore. We\nshowcase the utility of this method by a facile synthesis of a fluorogenic,\nmembrane-localizing fluorophore that outperforms its commercial counterparts\nwith a significantly higher brightness and contrast. Overall, this\nmethod establishes the synthetic similarities between polymethine\nand xanthene fluorophores and provides a versatile and feasible toolbox\nfor future optimizing heptamethine fluorophores for their biological\napplications.