1 Department of Chemistry, Technical University of Denmark2 Organic Chemistry, Department of Chemistry, Technical University of Denmark3 Department of Organic Chemistry, Technical University of Denmark
The role of histone modifications in the tight regulation of gene expression is emerging as a new area within drug discovery. The misregulation of histone modification levels has been linked with several life-threatening diseases, including cancer and diabetes, and reverting these irregularities may improve the prognosis of patients suffering from these diseases. The development of methodologies to modify peptides for use as peptide-based inhibitors of histone modifying enzymes is presented. Several different approaches for both N and C-terminal modification have been developed. Two broadly useful acid-labile traceless azido handles for the solid-phase synthesis of N H-1,2,3-triazoles are presented. A variety of alkynes were efficiently immobilised on a range of polymeric supports by Cu(I)-mediated azide-alkyne cycloadditions. Supported triazoles showed excellent compatibility with subsequent peptide chemistry. Release of pure material from the solid support was readily achieved by treatment with aqueous TFA. In parallel, an azido donor protection group for the synthesis of N H-1,2,3-triazoles was developed for use in both solid-phase and solution-phase chemistry. The N H-1,2,3-triazoles were cleanly deprotected by treatment with TFA (CH2Cl2). Four different libraries of histone demethylase inhibitor candidates have been synthesised based on metal chelation, cofactor mimicking and radial stabilising inhibition strategies. The libraries have all been synthesised on solid-phase using various handle strategies for the clean release of products. Two cofactor mimicking inhibitor candidates, which were synthesised using a safety-catch benzyl hydrazide handle, were found to inhibit the histone demethylase JMJD2C with IC50-values of 23.5µM and 24µM. Two mild and selective methods for the solid-phase synthesis of thiourea- and guanidinemodified peptides are presented. By activating N,N ’-di-Boc-thiourea with Mukaiyama’s reagent or HgCl2, the N -terminal of solid-supported peptides could be cleanly converted into the corresponding thiourea or guanidine derivative. The reactions were found to be compatible with all 20 naturally occurring amino acids, and were furthermore feasible on several commonly used polymeric supports. By using dilute SnCl4 for N -Boc deprotection, and NaOH for the release of material from the solid support, N -modified peptides were cleanly obtained in excellent yields and purities. Libraries of histone H2B tail pieces were synthesised using both parallel and split-pool synthesis protocols. Changes in the acetylation pattern of the individual library members upon treatment with HDAC3 enzyme were measured using LCMS-MS techniques. An MSMS deconvolution strategy was employed in the identification of individual library members. Changes in the acetylation pattern could also be detecting using a quantitative ninhydrin assay.