Synthesis and spectroscopic characterization of drug-fluorescent probe conjugates for live cell imaging

Recent advancements in technology and medicine have brought attention to fluorescent probes due to their non-invasive, highly sensitive capabilities in live cell imaging, especially in biomedical fields. These probes are particularly valuable for visualizing cellular signaling processes and tracking drug activities at the cellular level, making them essential tools for studying cell functions and drug delivery systems. In this study, we focus on the development and characterization of a coumarin-based fluorescent probe conjugated with omaveloxolone (OMA), a drug used to treat rare genetic disorders. The resulting drug-fluorophore conjugate provides a means to explore organelle-specific drug activation and monitor cellular signaling processes. The synthesis begins with the preparation of a fluorescent hydrazone compound, which is then conjugated to OMA to create the final drug-fluorophore construct. The conjugation process was monitored using kinetics and UV-vis spectroscopy, with detailed studies of reaction kinetics, including solvent choice and concentration, aimed at optimizing conditions for further investigations. The drug-fluorophore conjugate was characterized through thin-layer chromatography (TLC), nuclear magnetic resonance (NMR), and UV-vis spectroscopy. Various optimization steps were implemented to enhance the conjugate’s stability and efficiency, ensuring maximum fluorescence while preserving the biological activity of OMA. The fluorescent probe is designed to possess strong fluorescence and stability, making it ideal for cellular labeling when conjugated with the OMA drug. This conjugate is expected to demonstrate high fluorescence efficiency, stability within the cellular environment, low toxicity, and sufficient cell permeability. With the integration of fluorescent imaging technologies and molecular designs, drug visualization will have the potential to redefine our understanding of drug action in cellular systems, enabling the drug-conjugate dynamics in subcellular environment from new perspectives.

My name is Maryam Baig. I am a senior undergraduate at George Mason University, and I am presenting my project on the Synthesis and spectroscopic characterization of drug-fluorophore conjugates for live cell imaging. I had the opportunity to work on this project through Dr.Ozlem Dilek, who is part of the Department of Chemistry and Biochemistry located in the Institute for Advanced Biomedical Research at SciTech campus.

To provide a background for this project, I’d like to begin by explaining what fluorophores are. Fluorophores are chemical molecules that absorb Ultraviolet Visible light and project the emission in the form of light, and they help make up fluorescent probes. Fluorescent probes are molecular tools that allow scientists to visualize and observe live cell processes using highly sensitive, non-invasive and safe detection in biological cells. Omaveloxolone (OMA) is a drug being developed to treat Frederick’s ataxia, a rare and worsening disease that affects the nervous system. The fluorophore we are using for this project is a coumarin, and we have found that coumarin-based fluorophores have low inherent toxicity and can be readily internalized and washed out from cells, making them ideal for cell studies. In this project, we will focus on developing the fluorescently labeled OMA to monitor the delivery of fluorophore-OMA drug probes inside cells.

On this slide, we have two molecules that we will be using for our project. On the left side, you can see the OMA drug. It is a big molecule with multiple ketones present. Those are the double bonds with the oxygen molecules. On the right side, we have our CF3 coumarin. This is a published molecule and it is the coumarin that will we will be synthesizing and then conjugating with the OMA.

On this slide we have the synthesis procedure of our starting material, which is an amine, into the CF3 hydrazine that we will be using to conjugate to the OMA. As you can see, we will be adding an NH2 group, which is in amine group, to the existing amine.

To begin, we started by doing a thin layer chromatography between the drug, the dye and the conjugate after we had made the three. We diluted our samples in methanol, and then our TLC chamber solutions included various ratios of solvents that allowed us to visualize the mobility and composition of molecules on the TLC plates.

These are images from our TLC experiment. The samples on the left on the TLC plates are the CF3 dye, while the samples on the right are the product. As you can see, we observed a slight difference in shift between the lowest dots on the TLC samples. This indicates that we may have something new in our product.

From here we moved onto kinetics experiments, and after doing absorbance and emission data collection and nuclear magnetic resonance tests, we were able to make some conclusion. The data from the absorbance and emission graphs show that the drug-dye conjugate is fluorescent. Our NMR comparison between the CF3, the purified conjugate, and the OMA drug also gave us interesting results which we will see in the further slides.

This slide shows a comparison of the absorbance and emission data graphs that we collected for the conjugate to help us understand the composition of our molecule. As stated in the legend at the bottom of the slide, we can see that the CF3 is shown in red, the OMA is shown in blue, and the conjugate is shown in green. And if we look at the graphs, we can see that the red and green lines were very similar while the blue line was not as high. Because the red and green lines are so similar, we came to question if the dye may be overpowering the drug.

To see a more detailed and more accurate composition of the molecules we had worked with we conducted NMRs for each of the molecules. The purpose of an NMR is to analyze the magnetic properties of atomic nuclei to study the structure, the dynamics and interactions of the molecule. The area outlined here by black lines is where we will zoom in for the next slide.

Enlarging that small area shows us these multiple peaks that appear between the three samples. The yellow highlight indicates peaks that belong to the OMA drug. The blue highlighted peaks indicate the CF3 molecule, and the darker blue highlighted peaks indicate that we may still have some starting material remaining in our conjugate.

Based on the results of our NMR and the other test that we conducted we plan to move forward by trying to figure out where the CF3 is attaching on the OMA drug and how we can predict an NMR for it. Because of the dark blue highlight peaks that were present in our NMR on the previous slide, we decided to re-crystallize the CF3 coumarin to purify it further before we make another conjugate, and to try and get rid of those extra peaks. Finally, we plan to expand our range of molecules that can be conjugated with the OMA drug to see which one will be most efficient. We did a quick experiment in vials using small samples of different types of small molecules which you can see in this bottom image here and you can see were able to fluoresce. We plan to go forward with molecules numbers 2, 5, and 8, and study them further to see if they will be able to conjugate with the OMA drug.

Lastly, I’d like to acknowledge and thank Dr.Ozlem Dilek, Eva-Maria Rudler, and the rest of the Dilek team for their support and guidance throughout this project along with the GMU Department of Chemistry and Biochemistry. Additionally, I would like to express my gratitude to Dr.Karen Lee and the OSCAR team for giving me this unique research opportunity. Thank you for listening to my presentation.