Thiol-Ene "Click" A Radical Tool for Chemoselective Modification and Labelling of Biomolecules
Citation:Nolan, Mark Daniel, Thiol-Ene "Click" A Radical Tool for Chemoselective Modification and Labelling of Biomolecules, Trinity College Dublin, School of Chemistry, Chemistry, 2023
Mark Nolan - Thiol-Ene “Click” A Radical Tool for Chemoselective Modification and Labelling of Biomolecules.pdf (Published (publisher's copy)) 25.84Mb
Thiol-Ene Click chemistry represents a venerable reaction in chemical synthesis, allowing the efficient formation of a sulfur-carbon bond from thiol and alkene reaction partners. This reaction has found myriad applications in diverse areas of chemistry, from small-molecule synthesis, to polymer science, and even for modification of large, complex biomolecules. In this work, the development of methodology for modification of peptides and other biomolecules using Thiol-Ene chemistry is explored. In the first part of this thesis, the use of the UV-initiated Thiol-Ene reaction for the macrocyclisation of peptides to yield disulfide mimetics is investigated. A number of analogues of the neuropeptide hormone oxytocin are obtained in good yields through Thiol- Ene mediated peptide macrocyclisation. This approach is then applied to the synthesis of the therapeutic Carbetocin, with quantitative cyclisation observed. To further expand the scope of this methodology, the cyclisation of small peptide macrocycles is then briefly investigated as this has applications in peptide drug discovery. Further, the use of mild blue LED initiation is developed for cyclisation of these short peptide substrates. In the third chapter, the use of Thiol-Ene chemistry in Deep Eutectic Solvents for development of a green bioconjugation approach is investigated. Deep Eutectic Solvents represent a newly emerging class of green solvent, with advantageous characteristics such as non-volatility, non-toxicity and recyclability. First, the screening of a range of DESs using a model small-molecule UV-initiated Thiol-Ene reaction reveals successful reaction in a range of solvents, following which a scope focused on biomolecules is established. A subset of the scope examples are then also obtained via an oxygen-initiated Thiol-Ene reaction. Following this, the goal of green bioconjugation is investigated. An example peptide based on the minimum binding motif of human angiotensin converting enzyme 2 is synthesised, and Thiol-Ene bioconjugation is achieved to yield lipidated, glycosylated and fluorescent tagged analogues. The fourth chapter of this work examines the use of the Thiol-Ene reaction in high- throughput for the efficient diversification of peptidic macrocycles to yield high-purity crudes suitable for Direct-to-Biology applications. Initial investigation discussed covers considerations around the compatibility of Thiol-Ene chemistry within the context of high- throughput experimentation. A suitable UV reactor is designed and a small-molecule model Thiol-Ene reaction is studied. With suitable parameters, the application of the conditions to modification of an alkene-containing peptide macrocycle is then investigated and the reaction conditions optimised. With optimal conditions, a high-throughput approach is envisaged utilising acoustic droplet ejection and automated bulk dispensing. This is then applied to twelve peptide examples and eight thiols for a ninety-six entry scope presented as a heat map. The fifth chapter covers the development of thiol probes for labelling of alkene groups in a biological context. The synthesis of an alkenyl monosaccharide substrate is first presented. For the thiol probe component, a range of fluorescent probes are synthesised including dansyl, napthyl, fluorescein and aminobenzamide thiols, with only the latter showing successful reaction in UV- or blue LED-initiated Thiol-Ene conditions. To then extend this methodology to fluorophores of use for microscopy, an indirect labelling approach is envisaged. For this purpose, a biotin thiol probe is synthesised which shows successful quantitative consumption of the alkene substrate in UV-initiated conditions. This probe was then applied to labelling of glycans on the cell surface via metabolic incorporation of an alkene-containing sugar followed by on-cell TEC. In the sixth chapter, a brief overall summary of the work discussed in this thesis is given. Experimental details and compound characterisation data are presented in the seventh and final chapter.
Author: Nolan, Mark Daniel
Publisher:Trinity College Dublin. School of Chemistry. Discipline of Chemistry
Type of material:Thesis
Availability:Full text available