Synthesis of AGE-modified biologically relevant peptides for the generation and evaluation of antibodies

Abstract

This thesis focuses on the design, synthesis, and evaluation of advanced glycation endproducts (AGEs) and their incorporation into peptides for the development of novel antigens for the generation of antibodies. AGEs are a group of post-translational modifications derived from the non-enzymatic reaction between lysine and arginine residues with sugar and sugar metabolites. Accumulation of AGEs happens naturally over time but is accelerated in diabetic patients due to chronic hyperglycaemia. Furthermore, increased levels of AGEs has been linked to several diseases, including cardiovascular diseases, Alzheimer’s disease, nephropathy, retinopathy, and osteoporosis. Specific AGEs have been widely used as biomarkers for the progression of different diseases. The qualitative analysis of complex biological samples is crucial and several analytical methods such as reverse phase liquid chromatography (RPLC), have been successfully utilized. In this work, the three AGEs: GOLD 1.23, MOLD 1.24 and pentosidine 1.37, were synthesised and utilized to develop a novel methodology for the purification of AGEs via RPLC. The method utilized a silica hydride column and allows for a rapid and sensitive analysis of the different tested AGE crosslinks. A previously published method for the synthesis and incorporation of the AGE, MOLD 1.24, were used for the synthesis of a MOLD crosslinked collagen model peptide (CMP). CMPs are short biomimetic peptides that form the unique triple helical motif found in natural collagen. Synthesis of the required Fmoc2MOLD building block 3.21 was carried out following a previously published synthesis by our group. The synthesised peptide was conjugated on a keyhole limpet hemocyanin (KLH) carrier protein and the conjugate was used to immunise two NZ white rabbits. The obtained polyclonal serum was analysed via Streptavidin-based indirect ELISA. The required biotinylated peptides were prepared containing the AGE modification or an unmodified lysine-residue. The serum of one of the rabbits displayed the successful generation of against the MOLD crosslink. Based on these results it was proposed that MOLD and GOLD crosslinked amyloid-beta (Aβ) peptides could be utilized to generate antibodies. Aβ-peptides are the main components of senile plaques found in the brains of people suffering from Alzheimer’s disease. Several syntheses of Aβ-peptides have been published, however, to date no AGE crosslinked Aβ- peptides have been synthesised. Screening of different crosslinking methods utilizing the Fmoc2MOLD building block 3.21 were tested. Initial testing following results previously reported by our group and Hutton et al. were unsuccessful and no crosslinked Aβ-peptides were obtained. Due to the hydrophobic nature of the Aβ-peptides and the tendency to form aggregates, it was decided to utilize a previously developed double linker system. This double linker system contains a hexa-lysine solubilizing tag, which significantly improves the aggregation behaviour of the Aβ-peptides. Unfortunately, even with this new linker, no crosslinking could be achieved. Further testing was carried out utilizing an on-resin peptide coupling method, which resulted in the discovery of the organogel behaviour of the C-terminal Aβ-29-42 peptide 5.30. It was decided that the crosslinking of Aβ-peptide via diaminodicarboxylic acid building blocks is not feasible, and other routes to obtain AGE crosslinked Aβ-peptides were proposed. Lastly, a series of troponin peptides carrying AGE-modifications were evaluated as potential antigens. Investigations were carried out to evaluate the structural properties of the synthesised peptides and different method to introduce the respective AGE-modification were evaluated.