Engineering Central Substitutions in Heptamethine Dyes via Aryl-lithium Addition for Improved Fluorophore Performance

27 March 2024, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


As a major family of red-shifted fluorophores that operate beyond the visible light, polymethine dyes are pivotal in light-based biological techniques. However, the methods for tuning this kind of fluorophores by structural modification remain restricted to bottom-up synthesis and modification using coupling or nucleophilic substitutions. In this study, we introduce a two-step, late-stage functionalization process for heptamethine dyes. This process enables the substitution of the central chloride atom in the commonly used 4'-chloro heptamethine scaffold with various aryl groups using aryllithium reagents. This method borrows the building block and designs from the xanthene dye community, and offers a mild and convenient way for the diversification of heptamethine fluorophores. Notably, this efficient conversion allows for the synthesis of heptamethine-X, the heptamethine scaffold with two ortho-substituents on the 4’-aryl modification, which brings enhanced stability and reduced aggregation to the fluorophore. We showcase the utility of this synthesis by a facile synthesis of a fluorogenic, membrane-localizing fluorophore that outperforms the commercial counterparts with higher brightness and contrast. Overall, this method establishes the synthetic similarities between polymethine and xanthene fluorophores, and provides reference for future optimizing heptamethine fluorophores for their biological applications.


Polymethine dyes
Cyanine dyes
Heptamethine dyes
Late-stage functionalization
Water solubility
Reduced aggregation
Fluorescence imaging

Supplementary materials

Supporting Information
Supporting Figures (Figure S1-S8) and Tables (Table S1-S2), experiment procedures and characterization methods, synthetic procedures and NMR spectra.


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