A synthetic three-fluorophore system with two enzymatically cleavable linkers has been developed for the simultaneous detection of two proteases in a mixture. The probe was designed to afford single excitation/triple emission ratiometric detection through a fluorescence change during the cleavage of a peptide linker. The developed assays were verified for trypsin and chymotrypsin as the model enzymes.

An efficient methodology using the Fukuyama-Mitsunobu reaction was successfully applied to prepare various Rhodamine B-based amides with the locked possibility to form a lactam ring. The procedure was developed for solid-phase synthesis, which can be advantageously applied to the synthesis of chemical libraries in a combinatorial fashion. A series of derivatives including aliphatic as well as aromatic rhodamine amides alkylated via a reaction with various alcohols were synthesized, and their spectral properties were investigated. Blocking lactamization via N-alkylation enabled us to prepare rhodamine derivatives with an excellent fluorescence response. In comparison to their non-alkylated counterparts, these derivatives exhibited pH independence and higher quantum yields.

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