Development of Halomethyl-Triazole reagents for installation of protein post-translational modification mimics
Brewster, Richard Christian
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Triazoles have been widely used as amide bond isosteres in chemical biology as linkers and to enhance proteolytic stability. The use of triazoles has grown exponentially since the discovery of the copper (I) catalysed alkyne azide cycloaddition reaction in 2002 as the reaction is solvent and functional group tolerant, and usually high yielding. The reaction is also orthogonal to reactions used in nature, meaning it has become a powerful coupling tool. In post-translational modification (PTM), proteins are modified by covalent attachment of functional groups to amino acid side chains. These PTM processes are generally thought to be dynamic and highly regulated by cell machinery, controlling protein function in response to stimuli. The ability to control function post protein synthesis allows organisms to have a smaller genome, which is advantageous as it reduces the energy required for DNA replication and repair. Research into the function of PTMs has been limited by the difficulty in generating recombinant proteins that bear a single PTM in a specific location. Although many elegant methods have been proposed that solve this problem, to date cysteine alkylation is one of the most successful techniques. For lysine PTMs, thia-lysine II (sLys) derivatives have been shown to be excellent mimics of lysine, where the only perturbation between the native lysine-containing analogue is the switch of a CH2 for S in the side chain. Biotin is a well-known PTM in biotin dependent carboxylases, where biotin is involved in CO2 transfer. Recently biotinylation has also been shown to be a PTM on many other proteins, however the role of biotinylation is not well understood. Biotin triazole III has been shown to be a good mimic of the biotin amide bond and retains excellent affinity to Avidin (Av). In Chapter 1 the effects of modification to the valeryl side chain, and orientation of the biotin triazole bond affect affinity to Av using ITC are investigated. Compounds III, V and VI are shown to have a KD <120 pM, but further information on the binding affinity of these compounds could not be assessed by ITC. Biotin triazoles III-VI were also shown to be resistant to hydrolysis in serum, unlike the native biotin amide bond, which is hydrolysed by the enzyme biotinidase (BTD). Generation of amide sLys derivatives has been shown to be synthetically challenging. In Chapter 2, the synthesis and applications of chloromethyl-triazole biotin as a sulfhydryl selective alkylation reagent are investigated. The electron withdrawing nature of the triazole was proposed to give a ‘pseudo-benzylic’ halide α to the triazole, thus increasing reactivity. The controlled alkylation of peptides and proteins has shown that chloromethyl-triazole biotin shows enhanced reactivity over many commercial alkylation reagents and also gives good selectivity for cysteine. Alkylation of histone H4K12C gave the singly alkylated product, accompanied by low amounts of double alkylation. Biotinylation was confirmed by Western blot with anti-biotin. Due to the wide range of readily available functional azides, it was envisaged that halomethyltriazoles could be incorporated into other PTM mimics. In Chapter 3, efforts to expand the range of PTMs accessible using halomethyl-triazoles and further enhance the reactivity of chloromethyl triazoles by preparation of bromo- and iodomethyl triazoles are detailed. Synthesis of reagents to mimic malonylation, succinylation and GlcNAcylation PTMs is described and the reactivity of these halomethyl-triazole reagents is assessed. An alternate approach to the development of PTM mimics through cysteine propargylation and subsequent CuAAC coupling is also described in chapter 3. In conclusion, a series of new reagents have been developed to mimic protein PTMs through alkylation of cysteine. The reagents, which include biotin, GlcNAc, succinyl and malonyl mimics, are based on a halomethyl-triazole scaffold and have been successfully reacted with cysteine containing peptides and proteins.