Development of Benzodioxazole-based Fluorescent Probes and Peptide-based Hydrogels for Biological Applications


Student thesis: Doctoral Thesis

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Award date13 Aug 2020


Chapter 1 presents a brief introduction of the research background. It revolves around the development of small-molecule fluorescent dyes, fluorescent probes for detecting various analytes, and enzyme instructed self-assembly (EISA) hydrogel based on peptide. The structures and the properties of different fluorophores and probes are summarized in this chapter. In addition, various enzyme triggered peptide-based self-assembly approach were also discussed.

Inspired by the excellent photophysical properties and the simple structures of benzodioxazole derivatives, we designed, synthesized and characterized a new class of small-molecule fluorescent dyes based on the scaffold of benzodioxazole in Chapter 2. These fluorescent dyes are termed as VBDfluors. We conjugated different electron-drawing vinyl groups to position 7 via Knoevenagel condensation reaction to extend the push-pull electron system. By doing so, both the absorption and the emission wavelength of BD-scaffold were expected to be bathochromic shifted while the low molecular weight of fluorophores is kept. The photophysical properties of VBDfluors were thoroughly investigated, and the results indicated that the VBDfluors displays excellent properties, including fluorescence emission in the red to NIR region, large Stokes shifts, good photostability and cell permeability, and low cytotoxicity. These excellent properties endow the fluorophores with broad applications in bioimaging.

In Chapter 3, a novel fluorescent probe for detecting alkaline amino acids (Arginine and Lysine) was developed based on benzodioxazole derivative. Specifically, we conjugated benzodioxazole moiety with a carbon-carbon double bond activated by adjacent electron-withdrawing carbonyl groups. The probe could undergo an accelerated hydrolysis reaction in alkaline conditions to yield an aldehyde product with strong fluorescence and colorimetric property shift from pink to yellow. Arginine and lysine are the most alkaline amino acids among the twenty natural amino acids. Their isoelectric point (PI) of Arg and Lys values are the highest and determined to be 10.76 and 9.74 respectively, providing an alkaline environment in aqueous solution. Therefore, the probe revealed excellent selectivity toward Arg/Lys over other amino acids. The results indicated that the probe displays excellent sensitivity to Arg and Lys with LOD (limit of detection) values of 1.39 and 1.11 μM respectively. Moreover, this probe provides the first fluorescent platform for detecting -NH2 groups in the solid-phase synthesis of peptides.

In Chapter 4, a new building block for ALP/pH dual triggered self-assembling hydrogel, Fmoc-KKYpYp-OH (P1) was rationally designed and synthesized. The resulting ALP-Gel and pH-Gel displayed distinct self-assembly behaviors. TEM imaging analysis revealed that ALP-Gel showed an interesting helical fibril structure, while pH-Gel exhibited the typical fibrous network. Detailed CD analysis revealed that modulation of surface charges of peptide via different stimuli could change the composition of secondary structure, thereby affecting the self-assembling behavior. The formation of α-helix structure in ALP-Gel was attributed to the electrostatic repulsions in the hydrogelator. Moreover, P1 was used as a nano-carrier to co-assemble with a prodrug etoposide phosphate, which prolonged slow release of the drug and enhanced its anticancer effects. We envision that our study could give new insight into designing supramolecular assembly with predefined secondary structures.

In Chapter 5, a SIRT5 triggered in situ self-assembly of peptide-based hydrogel in mitochondria was described. The precursors were conjugated with nitrobenzodioxzole (NBD) group to facilitate the bioimaging study. Moreover, the nanofibers formed by peptides were found to be selectively formed in the mitochondria. The primary reason is the biocatalysis of a mitochondria located enzyme SIRT5. Due to the environment-sensitive property of NBD, the nanofibers exhibited strong fluorescence to reveal the enzymatic activity of SIRT5. In addition, supramolecular self-assembly spatially restricted to mitochondria by SIRT5 might provide a useful tool for mitochondria-based therapy, as evidenced by its capability to modulate the mitochondrial activity in cell-based experiments. Moreover, the peptide 5 was utilized for deliver three different anti-cancer drugs to significantly enhance therapeutic ability. It provides a facile platform for activity-based imaging of SIRT5 in living cells for the first time and might provide a useful method for mitochondria-based therapy.

Finally, Chapter 6 provides a summary of all the research projects performed in the PhD study. The coming works and the directions based on ELSA hydrogel for biological applications were also discussed. We will next utilize the enzyme instructed self-assembly system with different enzyme triggers. For example, other Sirtuin family enzymes that are closely involved in many physiological and pathological processes. We envision that our strategy will provide useful tools for more biological applications such as drug release and cancer therapy and provide a facile platform for investigating the bioactivity of important enzymes in living cells.