Synthesis of Fluorogenic Probes for CLIP-tag Protein Labeling Based on GFP and RFP Fluorophores & Optimization of RFP Mimic Fluorophore Synthesis
Open Access
- Author:
- Kim, Sojung
- Millennium Scholars Program:
- Chemistry
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisor:
- Xin Zhang, Thesis Supervisor
- Keywords:
- Organic synthesis
Fluorogenic probes
Protein labeling
Protein Aggregation - Abstract:
- Abnormal protein aggregation has been linked to multiple neurodegenerative diseases; however, elucidating the biological roles of these protein aggregates has remained challenging due to a lack of available techniques for visualizing protein aggregates in living cells. This work reports progress towards the development and synthesis of CLIP-tag probes using Green Fluorescent Protein and Red Fluorescent Protein based fluorophores to enable simultaneous labeling of insoluble protein aggregates and soluble protein oligomers in living cells. Five different GFP fluorophore based CLIP-tag probes were synthesized to test a series of different linker moieties in the probe (C2, C4, C6, cyclohexane, and proline). Though purification of these probes via preparatory HPLC proved challenging, the synthesis of these GFP based CLIP-tag probes will enable quantification of their fluorogenic properties and selection of the probe that exhibits the most desirable characteristics for use in in vivo protein aggregation imaging. To enable future design and synthesis of RFP fluorophore based fluorogenic probes, the synthesis of the desired RFP fluorophore was optimized. Anhydrous conditions with 0.1 eq. loading of BF3 • Et2O as a Lewis acid catalyst improved yield of the key enamine condensation from approximately 25% to 55%, with improved product to starting material ratio to also increase ease of purification. These optimized conditions will allow for more efficient future large-scale synthesis of the RFP fluorophore to enable synthesis of a series of RFP based CLIP-tag probes. Ultimately, developing such tools to enable the study of protein aggregation in living cellular environments may help elucidate how such protein aggregation causes biological effects, which is of particular interest in the study of neurodegenerative diseases.