Site-Selective Bioconjugation via Interception of Free-Radical-Induced Dechalcogenation

Griffiths, Rhys (2021) Site-Selective Bioconjugation via Interception of Free-Radical-Induced Dechalcogenation. PhD thesis, University of Nottingham.

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Peptides and proteins play numerous vital roles within cellular biochemistry, as a result they are central to our understanding of the molecular pathways essential to life and the development and progression of disease. The native biological influence of this class of biomolecule has led to the application of polypeptides as effective therapeutics, interest in which has steadily grown over the last four decades. This class of ‘biologic’ drug is now at the forefront of modern medicine. In order to exploit the biological properties of peptides and proteins to develop tools to investigate fundamental science and as therapeutic/diagnostic agents, synthetic methods that enable the site-selective installation of biologically relevant groups are required. There are a wide range of established techniques available which selectively target the canonical amino acids. The limitations of these methods include efficiency, operational simplicity, chemoselectivity, versatility and conjugate stability. There have been many recent advances that attempt to address these issues, but novel methods are still in high demand.

A commonly targeted residue is cysteine (Cys), due to its enhanced nucleophilicity and low abundance across the proteome (approximately 2%). An under-utilised residue is the 21st amino acid, selenocysteine (Sec). This residue has a lower abundance than Cys (< 0.1%) and further enhanced nucleophilicity. This thesis describes the development and application of a novel method for the site-selective modification of peptide and proteins by exploiting the process of free-radical-induced dechalcogenation of these two residues. Chapter 1 gives an introduction to site-selective chemistry, focussing mainly on chemistry that targets Cys. It also describes other uses of peptides, including peptide for radiotherapy and the importance of macrocyclic peptides. Chapter 2 describes the generation of a peptide radical (alanyl radical) from Sec and trapping of this intermediate with a persistent radical (TEMPO) bearing biologically relevant functionality. This method is high yielding and operationally simple, allowing the installation of a range of groups. A one-pot procedure was developed, whereby two peptide fragments were ligated via diselenide-selenoester ligation (DSL), followed by direct conjugation of a range of TEMPO probes in high to excellent yields.

Chapter 3 focusses on the application of the developed TEMPO conjugation method to Cys. The method was optimised and applied to a range of Cys containing peptides. This was further extended to the conjugation of a Cys mutant (K48C) of recombinantly expressed ubiquitin (Ub) in high yield. The method was also applied to the dual-functionalisation of a peptide bearing both Sec and Cys residues, thus allowing the site-selective installation of two different biologically useful moieties.

Chapter 4 demonstrates the utilisation of free-radical induced dechalcogenation to install native and non proteinogenic functionality via C(sp3)-C(sp3) bond formation. Lysine post translational modifications (PTMs) are a widely studied and highly relevant area of science. This chapter describes the development of a novel method to site-selectively install known Lys PTMs. The method is operationally simple and widely applicable to a variety of PTMs. The developed method uses allyl groups to react with the alanyl radical. Using this chemistry, native Lys PTMs were successfully installed into a peptide model in moderate to good yield. The chemistry was then used to modify ubiquitin, installing an acetylated lysine into its native position.

The scope of Chapter 4 was also expanded to an alternate, more stable alkene, for the installation of Lys PTM mimetics and biologically relevant functionality in higher conversions. The alkenes used are structurally similar to the allyl group, but a methyl group on the alkene results in the formation of a stable tertiary radical intermediate. A range of functionality was installed into an 11-mer peptide in good to excellent yield. Using this chemistry, two potential fluorination methods were also developed. A high- yielding, two-step process was developed using known and novel chemistry. A second, one-step method using a novel approach was also developed, albeit with lower isolated yields. This chemistry was also applied to the protein model Ub, installing a trimethylated lysine mimetic and biotin group in excellent yield for a protein model. Additionally, a novel desulfurative arylation reaction is described; while unoptimised, this method has the potential to be developed into a powerful technique for the ate-stage functionalisation of peptides. The data from this chapter shows the scope of this chemistry and the potential for wide-ranging application across numerous fields.

Chapter 5 applies the chemistry developed in Chapter 4 to the macrocyclisation of peptides. The commercially available Fmoc/Boc-allylGlycine-OH was used as the alkene bearing amino acid. The conditions required minor optimisation but were successfully applied to the cyclisation of peptides of various lengths and amino acid sequences. The data gathered in this chapter shows the successful development of a peptide macrocyclisation technique and a novel method of C(sp3)-C(sp3) bond formation.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Mitchell, Nicholas
Keywords: peptides, polypeptides, enzymes, glycosylation, macrocyclisation
Subjects: Q Science > QD Chemistry > QD241 Organic chemistry > QD415 Biochemistry
Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools: UK Campuses > Faculty of Science > School of Chemistry
Item ID: 65646
Depositing User: Griffiths, Rhys
Date Deposited: 12 Oct 2023 14:09
Last Modified: 12 Oct 2023 14:09

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