Synthesis and Optical Properties of Two-Photon Absorbing GFP-type Probes

Two-photon active green fluorescent protein -type chromophores were successfully synthesized following investigations directed toward a modified version of zFP538 chromophore, a structural analogue to the GFP-chromophore. A generalized approach for the chromophore synthesis via a well-studied cycloaddition reaction combining an iminoglycine methyl ester and a substituted benzaldehyde was developed allowing for flexibility in the incorporation of functional groups such as donor−acceptor substituents and for additional groups to provide extended conjugation. Steady-state spectroscopy, fluorescence quantum yields, and time-resolved fluorescence lifetimes for synthesized chromophores were extensively investigated for the functionalized chromophores. Time-resolved fluorescence lifetimes were found to be biexponential generally with subpicosecond and picosecond components. The individual effects of substitution position of functional groups and relative bulk size were evaluated and found to be rather significant in changing the fluorescence-decay characteristics in the case of positioning, but ambiguous with respect to relative bulk. The GFP-type chromophores were found to possess modest to low two-photon absorption cross sections with the dimethylamino-substituted analogue possessing the largest value at nearly 40 GM. These molecules show promise as biological markers for application in the study of conformation changes and aggregation of amyloid peptides, known to play an important role in many neurodegenerative diseases.