Development of peptide-based tools for biological heme monitoring

Abstract

Heme plays a crucial role in cell biology and the dysregulation of heme levels is implicated in a wide range of diseases. Currently however there is a lack of convenient chemical tools that can be used to measure the changes in heme levels in biological media or in live cells. The aim of this work was to explore the development of peptide probe molecules derived from natural heme-binding proteins for monitoring heme levels in live cells by fluorescence.
Tryptophan, a naturally fluorescent amino acid, is present in a peptide sequence termed CP3 that is derived from the heme-binding protein, Bach1. It has been shown that the fluorescence of the tryptophan residue is quenched upon binding of heme to this peptide, indicating its potential as a heme probe. In this work, an efficient asymmetric synthesis of a novel tryptophan analogue with red-shifted fluorescence, 7-azatryptophan, was developed using a phase transfer alkylation approach. The asymmetric phase transfer alkylation approach was also extended to the preparation of 1-methyl-7-azatryptophan, which has a more substantially red-shifted fluorescence, however further modifications are needed to improve the yield of this analogue. An optimized preparation of the CP3 peptide probe incorporating the 7-azatryptophan analogue via solid-phase peptide synthesis was also developed, and novel deprotection strategies were identified to allow efficient incorporation of the key tryptophan and cysteine residues. Investigation of the 7-azatryptophan-containing probe in HaCaT cells was not feasible, as the red shift of this probe was insufficient for effective discrimination from endogenous tryptophan, although its heme-binding ability in solution was successfully confirmed. To address this limitation, an alternative approach to probe design was explored by conjugating the N-termini of peptide probes with the dansyl fluorophores, which has a longer red-shifted emission that avoids fluorescence signal overlap upon heme binding. These dansylated peptide probes demonstrated significant potential for heme detection in both solution and cellular environments. They were able to sensitively measure subtle changes in heme concentration in a cellular setting, such as those induced by UVA radiation in human skin fibroblast FEK4 cells. The dansylated CP3 probe also showed its capability for monitoring dynamic heme levels in cells and responded sensitively to the heme precursor 5-aminolevulinic acid (ALA) in the absence or presence of ferric ammonium citrate (FAC). A Cy5 dye, as an alternative fluorophore, was also successfully synthesized and used to label the CP3 peptide probe. This probe also exhibited very promising results for monitoring dynamic heme levels in cells, showing a sensitive response to ALA in the absence or presence of FAC. In addition, this Cy5-labelled probe showed promising results in detecting heme in vivo in a zebrafish system.

Date of Award	25 Jun 2025
Original language	English
Awarding Institution	University of Bath
Supervisor	Ian Eggleston (Supervisor), Charareh Pourzand (Supervisor), Sofia Pascu (Supervisor) & Charlotte Dodson (Supervisor)