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.[1] The major targets of this last class are hydrolases that catalyze the removal of a masking moiety, thereby modulating fluorescence.[2] 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.[3] 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.[4, 5] 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 possessing UV or near-UV excitation wavelengths.[6] Acyl esters and acyloxymethyl ethers of 7-hydroxycoumarin (i.e., umbelliferone) can be substrates for esterases.[7–9] 7-Amino-4-methylcoumarin (AMC) is used widely as the basis for protease substrates.[10–12] Upon amidation, the excitation and emission wavelengths of AMC are shifted to shorter wavelengths with concomitant reduction in quantum yield.[10] We reasoned that AMC could be subjected to our latent fluorophore strategy. Accordingly, we condensed AMC with acetylated trimethyl lock 1[13] to give pro-fluorophore 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 = 2.5 × 105 M−1s−1 and KM = 8.2 μM (Figure 2A). This kcat/KM value is 102-fold greater than the apparent kcat/KM value for the latent fluorophore based on rhodamine 110.[3] (We use the term “apparent” because full fluorescence manifestation requires the lactonization of two trimethyl lock moieties.[3]) Figure 1 Normalized fluorescent excitation–emission spectra of pro-fluorophore 2 and AMC. Figure 2 (a) Kinetic traces (λex 365 nm, λem 460 nm) and Michaelis–Menten plot (inset) for the hydrolysis of pro-fluorophore 2 (20 μM→9.8 nM) by PLE (2.5 μg/mL); kcat/KM = 2.5 × 105 M−1s−1 ... Scheme 1 Route for the synthesis of pro-fluorophore 2, and the chemical structure of 4-methylumbelliferyl acetate (3). To evaluate the relative stability of pro-fluorophore 2, we monitored the accretion of fluorescence in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA; 1.0 mg/mL) and either pro-fluorophore 2 or 4-methylumbelliferyl acetate (3, which is a common esterase substrate[9]). Pro-fluorophore 2 proved to be highly stable compared to 4-methylumbelliferyl acetate (Figure 3). An additional advantage of pro-fluorophore 2 is that the product of its hydrolysis, AMC, shows little change in fluorescence at pH ≥ 5.[1] In contrast, the fluorescence of 4-methylumbelliferone is highly variable due to its pKa = 7.40[14] being near the physiological pH. Figure 3 Time course of the generation of fluorescence (λex 365 nm, λem 460 nm) from pro-fluorophore 2 (50 nM) or methylumbelliferyl acetate 3 (50 nM) in PBS containing BSA (1 mg/mL). To access longer wavelengths, we turned to the oxazine class of red dyes. One such dye, cresyl violet (CV) has been used for decades to stain tissues[15, 16] and as a laser dye.[17] Although CV has a maximal absorbance at 586 nm, its fused benzo ring broadens its absorption spectrum[18] and thereby allows excitation with a variety of light sources. Diamide derivatives of CV are virtually nonfluorescent and thus useful in the production of fluorogenic protease substrates.[19–21] The attributes of CV make this dye an attractive candidate for our latent fluorophore strategy. Accordingly, we condensed CV with acetylated trimethyl lock 1[13] to give pro-fluorophore 4 (Scheme 2). Although pro-fluorophore 4 was stable in PBS containing BSA (data not shown), its hydrolysis was catalyzed by PLE with apparent kinetic parameters of kcat/KM = 1.2 × 105 M−1s−1 and KM = 0.14μM (Figure 2B). The kcat/KM value for the hydrolysis of pro-fluorophore 4 is similar to that of pro-fluorophore 2; the KM value is, however, 10-fold lower than that of pro-fluorophore 2. Scheme 2 Route for the synthesis of pro-fluorophore 4. Pro-fluorophore 4 serves as a probe for esterase activity in live mammalian cells. The broad excitation peak allowed confocal microscopy experiments using excitation at 543 nm and emission at 605 nm. Pro-fluorophore 4 was hydrolyzed by esterases endogenous in the cells of a rodent (Figure 4A) or human (Figure 4B) to give a red-stained cytosol within minutes. The somewhat enhanced fluorescence observed in the human cells could be indicative of more efficient internalization or hydrolysis of the latent fluorophore. After longer incubation, the liberated CV became further localized in subcellular compartments (data not shown), which is consistent with previous reports.[21] To date, there have been few reports of long-wavelength esterase substrates that are useful in cell biology.[1] Its evident stability, optical properties, and fast intracellular eduction make pro-fluorophore 4 useful in a wide variety of biological applications, especially in assays involving fluorophores of different wavelengths. Figure 4 Unwashed mammalian cells incubated for 15 min with pro-fluorophore 4 (10 μM) at 37 °C in DMEM and counter-stained with Hoechst 33342 (5% v/v CO2(g), 100% humidity). (a) CHO K1 cells. (b) HeLa cells. In conclusion, we have established that one component of our latent fluorophores—the dye—is modular (Figure 5). Specifically, we have now prepared useful latent fluorophores from three dyes (blue, green,[3] and red), all linked by a trimethyl lock moiety to an esterase-reactive group. In future work, we shall explore the modularity of the other component—the enzyme-reactive group. We anticipate that the end result will be a broad spectrum of stable latent fluorophores with numerous applications in biochemical and biological research. Figure 5 Modules in the latent fluorophores described in this work.