Pro-fluorophore compounds for selective detection and imaging of porcine liver esterase activity

Background

Fluorescent probes and dyes have become indispensable tools in modern biological research, enabling scientists to visualize and quantify a wide range of cellular processes with high sensitivity and spatial resolution. These technologies are particularly valuable in applications such as live-cell imaging, enzymatic activity assays, and the detection of specific biomolecules or gene expression patterns. The use of enzyme-activated pro-fluorophores—non-fluorescent molecules that become fluorescent upon enzymatic cleavage—offers a powerful strategy for signal amplification and selective detection. This approach is especially useful when target proteins or enzymes are present at low concentrations, as the catalytic activity of the enzyme can generate a strong, localized fluorescent signal. However, the effectiveness of such probes depends on their specificity, stability, and the properties of the released fluorophore, such as brightness and pH sensitivity.

Despite the widespread adoption of enzyme-activated fluorescent probes, current approaches face several significant limitations. Many existing pro-fluorophores rely on ester or glycosidic linkages that are susceptible to non-specific hydrolysis by endogenous cellular enzymes, leading to high background fluorescence and reduced assay specificity. Additionally, some commonly used fluorophores, such as fluorescein, exhibit sensitivity to pH changes, which can compromise quantitative measurements in physiological environments. The lack of highly selective substrates for certain enzymes, as well as the limited availability of probes that release bright and stable fluorophores upon activation, further restricts the utility of current technologies. These challenges highlight the need for new strategies that offer improved selectivity, stability, and signal-to-noise ratio for sensitive and reliable detection in complex biological samples.

Technology

This technology centers on a new class of non-fluorescent pro-fluorophore compounds—N,N’-diacyl rhodamine derivatives—engineered for highly selective activation by porcine liver esterase (PLE). These molecules are built on a 3,3-bis(aryl)-isobenzofuranone (phthalide) core with two amide substituents, maintaining a spirocyclic lactone structure that suppresses fluorescence. Upon exposure to PLE, which exhibits a newly discovered amidase activity, the amide bonds are hydrolyzed, releasing Rhodamine 110—a highly fluorescent and pH-stable dye. The pro-fluorophores are chemically stable and inert in environments lacking PLE, making them ideal for sensitive detection of PLE activity in enzymatic assays or for imaging PLE expression in live cells. Their synthesis is straightforward, involving acylation of Rhodamine 110, and they have been validated in both in vitro and cellular contexts, showing strong, selective fluorescence only in the presence of PLE.

What differentiates this technology is its unique reliance on PLE’s amidase activity, as opposed to the more common esterase-mediated activation found in other pro-fluorophores. This direct amidase cleavage confers exceptional specificity, resulting in minimal background fluorescence in non-target cells or tissues. The released Rhodamine 110 offers superior brightness and pH stability compared to traditional dyes like fluorescein, enhancing performance in physiological and imaging applications. Additionally, the pro-fluorophores’ resistance to endogenous cellular enzymes ensures that fluorescence is only triggered by PLE, enabling precise spatial and temporal control in biological assays. This selectivity, combined with robust chemical stability and the ability to tailor the compounds’ substituents, positions the technology as a powerful tool for targeted cellular imaging, sensitive enzymatic assays, and potentially for cell-type specific labeling in complex biological systems.

Benefits

• Selective activation by porcine liver esterase (PLE) via novel amidase activity enables specific detection of PLE expression or activity.
• Pro-fluorophores are stable and non-fluorescent in biological environments lacking PLE, minimizing background signal.
• Release of Rhodamine 110 provides bright, highly fluorescent, and pH-stable signal suitable for physiological conditions.
• Enzymatic activation amplifies fluorescence signal, allowing sensitive enzymatic assays and imaging even at low target protein levels.
• Effective cellular uptake and selective intracellular activation enable live-cell imaging of PLE-expressing cells.
• Biocompatible with no adverse effects on cell growth or morphology upon PLE overexpression.
• Applicable to various biological samples including live cells, tissues, and potentially whole organisms.
• Simple synthesis with good yields from commercially available Rhodamine 110 derivatives.

Applications

• Live-cell imaging of PLE expression
• Enzymatic activity assays for PLE
• High-throughput screening for PLE inhibitors
• Cell-type specific fluorescence labeling

Additional info

These are N,N’-diacyl rhodamine derivatives, non-fluorescent pro-fluorophores featuring a spirocyclic lactone and amide substituents. They become highly fluorescent Rhodamine 110 upon specific amidase-catalyzed hydrolysis of their amide bonds by porcine liver esterase. This enables sensitive detection and cellular imaging of enzyme activity.