Fluorescent Dyes for Small Molecule Labeling

Fluorescent dyes for small molecule labelling are increasingly used in modern biological research and drug discovery. The conjugation of fluorescent dyes to small molecules confers visualisation capability on the molecules, enabling scientists to measure their distribution, metabolism and interaction over time, in vitro and in vivo. Small molecule fluorescent labelling is widely used in drug screening, cell imaging, molecular interaction studies, diagnosis and much more. Because the fluorescent dyes are a broad variety with different fluorescent properties, experimenters can choose the best dye for their experiment, which in turn makes experiments even more sensitive and precise. As the technology matures, fluorescently marked tiny molecules will become more central to disease research and personalized medicine.

Fluorescent Labeling

Fluorescent labeling is a technique that utilizes fluorescent molecules (typically fluorescent dyes or probes) to conjugate with target molecules (such as small molecule drugs, proteins, nucleic acids, cells, or tissues). Through this labeling, the target molecule can emit detectable fluorescent signals when exposed to specific excitation conditions, enabling real-time tracking and observation of the molecule's location, quantity, distribution, or dynamic changes. This technology is highly sensitive, non-invasive, and quantitative, and is widely applied in biology, medicine, and chemistry, especially in cell biology, drug development, disease diagnosis, and treatment monitoring.

Fig. 1. Small molecule fluorescent probes (Org Biomol Chem. 2020, 18(30): 5747-5763).

Fluorescent Labeling Principle

The basic principle of fluorescent labelling involves fluorescent molecules absorbing very short wavelength light (UV or blue light) and emitting fluorescence with a long wavelength (Stokes shift) over time. Here, the fluorescent molecule moves from a low energy state (ground state) to a high energy state (excited state), before returning to the ground state and emitting energy as fluorescence. Due to the nature of fluorescent dyes, individual fluorescent molecules are able to be selectively induced by excitation light so that the fluorescent signal can be separated from different labels on other molecules, which allows multiplex labelling and multicolour imaging. By labelling different molecules (drugs, small molecules, biomacromolecules, cells) with fluorescent dyes, scientists can observe drug metabolism at a fine level, see how cells interact with molecules, follow disease progression or monitor response to treatments. Fluorescent labelling not only enhances the accuracy of molecular tracking but also allows for real-time, dynamic, quantitative experimental analysis. The use of fluorescent labels is safer and more straightforward than conventional radioactive labelling, making it a necessary adjunct to biomedical research today.

Small Molecule Drugs

Small molecule drugs are drug molecules with a molecular weight typically below 900 Dalton (Da), usually obtained through chemical synthesis or natural extraction. Small molecule drugs typically have good bioavailability, strong permeability, and long half-lives, allowing them to be effectively distributed in the body and produce therapeutic effects. Compared to large molecule drugs (such as monoclonal antibodies and vaccines), small molecule drugs have simpler molecular structures, generally low molecular weight, good oral bioavailability, and easier chemical synthesis or production processes. One of the main characteristics of small molecule drugs is their ability to cross the lipid bilayer of cell membranes, enabling them to target intracellular molecules such as enzymes, receptors, and nucleic acids. The mechanism of action of small molecule drugs typically involves binding to specific molecular targets, altering their functions or the biological processes in which they participate to achieve therapeutic effects. These targets can include proteins, nucleic acids, or small molecular chemicals. Common small molecule drugs include:

Small Molecule vs Large Molecule

Small molecules and large molecules exhibit significant differences in biology, chemistry, and drug development. While they differ in molecular structure and function, both play important roles in scientific research and clinical applications. Small molecules typically refer to compounds with low molecular weight and simple structures, while large molecules are those with higher molecular weights and complex structures, such as proteins and nucleic acids. Understanding the comparison between small and large molecules helps to better comprehend their distinct roles in drug development and disease treatment.

Fluorescent Labeling of Small Molecules

Fluorescent labeling of small molecules is a technique that involves attaching a fluorescent dye or fluorophore to small molecule drugs or other compounds, granting them fluorescence properties. This labeling method enables the direct detection and observation of small molecules in various experimental settings, providing a high-sensitivity, high-selectivity analytical tool for research. Fluorescent labeling is used not only in basic research but also in drug development, clinical testing, disease diagnosis, and other fields. Fluorescent labeling of small molecules typically involves chemically linking the fluorescent dye to the target small molecule. The fluorescent-labeled small molecules can emit fluorescence signals at specific excitation wavelengths, allowing researchers to detect them using equipment such as fluorescence microscopes, flow cytometers, and fluorescence spectrometers. Different types of fluorescent dyes have different emission wavelengths and fluorescence intensities, making the choice of an appropriate fluorescent dye crucial. Common fluorescent dyes include fluorescein, rhodamine, chloromethine, and BODIPY, each with different chemical structures, emission characteristics, and application scenarios.

Fluorescent Dyes for Small Molecules

Fluorescent dyes are chemical substances used for fluorescent labeling that can absorb energy and emit fluorescence at specific wavelengths when exposed to light. Based on their structure, fluorescent properties, and application fields, fluorescent dyes can be categorized into various types. Different dyes have distinct excitation and emission wavelength ranges, fluorescence intensity, light stability, and other characteristics, making them suitable for various biomedical research and applications. Common types of fluorescent dyes include the following:

• Green Fluorescent Dyes

Green fluorescent dyes are widely used in biomedical and cell biology research and typically have a shorter excitation wavelength and a longer emission wavelength. The most common green fluorescent dyes are Fluorescein Isothiocyanate (FITC) and Green Fluorescent Protein (GFP). FITC is the most commonly used green fluorescent dye, with an excitation wavelength of about 495 nm and an emission wavelength of about 519 nm. It is widely used in immunofluorescence, flow cytometry, and cell labeling, providing high contrast and high sensitivity fluorescent signals. GFP is a natural fluorescent protein originally extracted from jellyfish. It is widely used for cell labeling and gene expression analysis. GFP has an excitation wavelength of about 488 nm and an emission wavelength of 509 nm. Due to its natural fluorescence properties, GFP is widely used in transgenic and cell biology research.

• Red Fluorescent Dyes

Red fluorescent dyes are very useful in multiplex labeling experiments, where they can coexist with other dyes of different colors, allowing researchers to simultaneously track multiple target molecules. Common red fluorescent dyes include Cy3, Alexa Fluor 594, and Tetramethylrhodamine Isothiocyanate (TRITC). Cy3 is a commonly used red fluorescent dye with an excitation wavelength of about 550 nm and an emission wavelength of about 570 nm. It is typically used in immunofluorescent labeling, antibody labeling, and multiplex labeling experiments, providing high fluorescence intensity and good signal stability. Alexa Fluor 594 is a high-performance red fluorescent dye with high light stability and strong fluorescence emission. It has an excitation wavelength of 590 nm and an emission wavelength of 617 nm. The Alexa Fluor series of dyes are widely used in cell biology, immunology, and protein interaction research.

• Blue Fluorescent Dyes

Blue fluorescent dyes are commonly used in multiplex labeling experiments and are used in combination with green and red fluorescent dyes. Common blue fluorescent dyes include DAPI, Hoechst, and Alexa Fluor 350. DAPI is a widely used nucleic acid dye that emits strong blue fluorescence. When it binds to DNA, it emits fluorescence with an emission wavelength of 461 nm. DAPI is widely used for nuclear staining, cell cycle analysis, and DNA research. Hoechst dyes are a class of commonly used DNA dyes that can stain the cell nucleus and are used for imaging cell chromatin. Hoechst 33342, a common variant, emits fluorescence at 461 nm and is suitable for cell morphology studies and flow cytometry.

• Ultraviolet Fluorescent Dyes

Ultraviolet fluorescent dyes typically have higher excitation energy and can be used for applications that require short-wavelength excitation. Common ultraviolet fluorescent dyes include Hoechst and Acridine Orange. Acridine Orange is a UV fluorescent dye used for RNA and DNA staining. It emits different fluorescence in various solution environments and is commonly used to distinguish between the cell nucleus and cytoplasm. It has an excitation wavelength around 490 nm and emits fluorescence in both green and red regions.

• Near-Infrared Fluorescent Dyes

Near-infrared (NIR) fluorescent dyes have longer emission wavelengths, allowing them to penetrate deeper into tissues, making them important for in vivo imaging and deep tissue analysis. Common NIR fluorescent dyes include Cy7 and IRDye 800CW. Cy7 is a commonly used NIR fluorescent dye with high fluorescence intensity, an excitation wavelength of 750 nm, and an emission wavelength of 776 nm. It is typically used in in vivo imaging, drug distribution studies, and cancer diagnosis. IRDye 800CW is another NIR fluorescent dye with an emission wavelength of 800 nm, commonly used in in vivo imaging, drug delivery research, and molecular imaging. It has low background signals in vivo, making it suitable for high-sensitivity imaging analysis.

• Fluorescent Probe Dyes

Fluorescent probe dyes are fluorescent molecules used not only for labeling substances but also for responding to changes in biological molecules or environmental conditions. They are commonly used for real-time monitoring of biochemical changes both inside and outside cells. Common fluorescent probes include Calcein-AM, FITC, and Fura-2. Calcein-AM is a fluorescent probe used for cell viability detection. Once it enters a cell, it is hydrolyzed by intracellular esterases into green-fluorescent Calcein. It is widely used in cell survival rate assessment, cell proliferation analysis, and cell function research. Fura-2 is a calcium ion indicator commonly used for monitoring intracellular calcium changes. Fura-2 binds to calcium ions and emits different fluorescent signals at varying calcium concentrations, making it widely used in cell signal transduction research.

• Special Functional Fluorescent Dyes

In addition to basic fluorescent dyes, some fluorescent dyes have special functions, such as targeting specific molecules or cells or possessing unique physicochemical properties. Common special functional fluorescent dyes include Quantum Dots, Rhodamine, and BODIPY. Quantum Dots are nanoscale fluorescent materials with extremely high fluorescence intensity and very narrow spectral characteristics. They can emit light across various wavelength ranges and have good light stability, making them suitable for high-resolution imaging, drug delivery, and biological labeling applications. Rhodamine is a commonly used organic fluorescent dye with a wide range of applications. It has an excitation wavelength of about 540 nm and an emission wavelength of 570 nm. Rhodamine dyes exhibit strong fluorescent signals in biological labeling and cell imaging, commonly used for protein labeling and immunofluorescence experiments.

What are Fluorescently Labeled Small Molecules Used For?

Fluorescently labeled small molecules have widespread and important applications in modern biology, drug development, disease diagnosis, and other fields. These applications rely on the ability of fluorescent molecules to emit detectable fluorescence signals when attached to small molecule drugs, enabling researchers to track the behavior of small molecules in vivo and in vitro under specific conditions. Fluorescently labeled small molecule drugs not only help in gaining deeper insights into drug mechanisms but also provide powerful tools for high-throughput screening, targeted therapy, disease detection, and more.

• Drug Screening and High-Throughput Screening

In drug development, fluorescently labeled small molecules are primarily used in drug screening and high-throughput screening (HTS). With fluorescent labeling, the binding of drugs to target molecules or receptors can be monitored in real-time during the molecular screening process. For example, researchers can observe changes in fluorescence signals to track the binding affinity between small molecule drugs and protein targets, allowing them to screen for more active and specific drug candidates. This method improves screening efficiency and enables the identification of promising new drugs, providing a sensitive and efficient tool for drug discovery and development.

• Pharmacokinetic Studies

In pharmacokinetic studies, fluorescently labeled small molecules help researchers monitor the absorption, distribution, metabolism, and excretion (ADME) of drugs in real-time. By combining the drug with fluorescent dyes, researchers can track the dynamic distribution of small molecule drugs in vivo using fluorescence imaging techniques. This is essential for understanding how a drug behaves within the body, optimizing drug delivery methods, and enhancing therapeutic effects. Fluorescently labeled small molecules help pinpoint the location of the drug in various tissues, assess bioavailability, and predict potential side effects.

• Targeted Therapy and Precision Medicine

Fluorescently labeled small molecules are increasingly used in targeted therapy and precision medicine. By attaching a fluorescent label to small molecule drugs, researchers can assess whether the drug effectively targets specific diseased tissues or cells. For example, in cancer therapy, fluorescently labeled small molecules can be used to detect whether they successfully target tumor tissue, helping evaluate the drug's targeting ability. Fluorescence imaging allows clinicians to observe the drug's effects in real-time during treatment, enabling the development of more precise treatment plans and improving therapeutic outcomes and safety.

• Live Cell Imaging

Live cell imaging is another key application of fluorescently labeled small molecules. The fluorescent labeling of small molecule drugs enables researchers to observe interactions between the drugs and intracellular molecules in living cells in real-time. With fluorescence labeling, small molecule drugs can clearly show their distribution inside cells, revealing how the drug enters the cell, interacts with cell components, and exerts its effects. This application is particularly valuable in cellular signaling studies, providing profound insights into complex biological processes.

• Disease Diagnosis

Fluorescently labeled small molecules also play an important role in disease diagnosis. By combining small molecule drugs with fluorescent dyes, researchers can develop probes to detect specific disease markers. These fluorescently labeled small molecules can be used for early disease diagnosis via in vitro diagnostics or in vivo imaging techniques. For example, some fluorescently labeled small molecules are applied in cancer biomarker detection, helping physicians achieve accurate diagnosis and monitoring in the early stages of the disease by targeting cancer cell-specific molecules. Fluorescently labeled small molecules are also used in the detection of infectious diseases, helping to rapidly identify pathogens and monitor treatment effectiveness.

• Molecular Interaction Studies

In molecular interaction studies, fluorescently labeled small molecules can help reveal interactions between small molecules and other biological molecules. For example, fluorescently labeled enzyme inhibitors can be used to study enzyme activity and reaction mechanisms; similarly, fluorescently labeled small molecule drugs can be used to study receptor-ligand binding, antibody-antigen interactions, and more. These studies provide important information on drug mechanisms, molecular recognition, and other fundamental biological questions.

Reference:

1. Jun, J.V. et al. Rational design of small molecule fluorescent probes for biological applications. Org Biomol Chem. 2020, 18(30): 5747-5763.

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