Latent blue and red fluorophores based on the trimethyl lock.

Fluorescent molecules are indispensable tools in modern biochemical and biological research, being used as labels for biomolecules, indicators for ions, stains for organelles, and substrates for enzymes. The major targets of this last class are hydrolases that catalyze the removal of a masking moiety, thereby modulating fluorescence. A critical property of fluorogenic hydrolase substrates is their chemical stability in aqueous solution, as spontaneous hydrolysis can compete deleteriously with enzymatic activity. New substrate classes that exhibit increased stability while maintaining enzymatic reactivity would be highly desirable. Our laboratory recently reported on the use of the “trimethyl lock” strategy in the design of a latent fluorophore. This latent fluorophore consists of a trimethyl lock component inserted between a dye and enzyme-reactive group. The trimethyl lock is an o-hydroxycinnamic acid derivative in which unfavorable steric interactions between three methyl groups encourage rapid lactonization to form a hydrocoumarin and release a leaving group. Our initial latent fluorophore exhibited remarkable stability in aqueous solution, but released a xanthene dye (rhodamine 110) upon hydrolytic cleavage by an esterase. Here, we explore the modularity of our design, probing its applicability to unrelated dyes that absorb at short (blue) and long (red) wavelengths. Coumarin-based compounds comprise an important class of blue dyes with UV or near-UV excitation wavelengths. Acyl esters and acyloxymethyl ethers of 7-hydroxycoumarin (i.e. , umbelliferone) can be substrates for esterases. 7-Amino-4methylcoumarin (AMC) is used widely as the basis for protease substrates. Upon amidation, the excitation and emission wavelengths of AMC are shifted to shorter wavelengths with a concomitant reduction in quantum yield. We reasoned that AMC could be subjected to our latent fluorophore strategy. Accordingly, we condensed AMC with acetylated trimethyl lock 1 to give profluorophore 2 (Scheme 1), which displayed the expected hypsochromic shift of excitation and emission spectra relative to free AMC (Figure 1). The hydrolysis of profluorophore 2 was catalyzed by porcine liver esterase (PLE) with kcat/KM=2.5B10 m 1 s 1 and KM=8.2 mm (Figure 2A). This kcat/KM value is 100-fold greater than the apparent kcat/KM value for the latent fluorophore based on rhodamine 110. (We use the term “apparent” because full fluorescence manifestation requires the lactonization of two trimethyl lock moieties.)