AGEing Peptides: Synthesis and Analysis of Peptides Site-Specifically Modified by Advanced Glycation Endproducts

Abstract

Advanced Glycation Endproducts (AGEs) are a family of modified amino acids that form when proteins react with sugars and sugar degradation products. AGEs are commonly found in processed food as well as in human organs, where they accumulate in tissue proteins, such as collagen, during the normal process of ageing. AGE accumulation dramatically accelerates with the onset of diabetes mellitus and the associated hyperglycaemia, a condition in which levels of sugars and sugar degradation product are above those considered healthy. Relative quantities of AGEs in different organs correlate with the pathophysiologic changes in these organs that occur with both advanced age and long-term hyperglycaemia. Emerging evidence indicates that AGE levels not only correlate with organ damage but may also play a causative role in such damage. Increased chelation of copper ions appears to play an important role in this process. However, the precise impact of AGE formation and accumulation on the biochemical properties of the host proteins is yet to be determined. Given their possible role in the detrimental outcomes of ageing (discussed in Chapter 1), in-depth studies of the AGE biochemistry are necessary. The overall aim of this work was to enable such in-depth studies of the AGE biochemistry by providing access to synthetic peptides that can be site-specifically modified by particular AGEs. Five prominent lysyl AGEs, Nε-carboxymethyllysine (CML), Nε-carboxyethyllysine (CEL), pyrraline, glyoxal lysine dimer (GOLD), and methylglyoxal lysine dimer (MOLD), were selected as targets. Synthesis of AGE building blocks, suitably protected for peptide incorporation, is discussed in Chapter 2. Efficient synthetic strategies were developed to provide access to all five AGE building blocks Nα-protected by 9-Fluorenylmethoxycarbonyl (Fmoc) group. Synthesis of Fmoc-CML and Fmoc-CEL was accomplished using a facile and practical approach that involved the Fukuyama amino alkylation methodology. Fmoc-pyrraline was synthesised using an efficient pyrrole introduction protocol. Cross-linked building blocks, Fmoc2GOLD and Fmoc2MOLD, were prepared in the Debus-type amino-cyclisation procedure. Incorporation of the AGE building blocks into peptides is discussed in Chapter 2. Collagen, since it is a major target of glycation, was used as a source of synthetic peptides. By use of solid phase peptide synthesis, peptides mimicking the quaternary structures of collagen were prepared with and without AGEs. Incorporation of the CML, CEL, and pyrraline building blocks into collagen model peptides and collagen telopeptides was successfully carried out using Fmoc solid phase peptide synthesis protocol. A straightforward and cost-effective synthetic procedure to access CML-containing peptides via on-resin N-alkylation of lysyl amines has also been developed. Importantly, conditions for efficient incorporation of the lysyl AGE cross-links, GOLD and MOLD, have been successfully developed and employed in the first syntheses of crosslinked collagen model peptides and collagen telopeptides. Access to site-specifically glycated peptides has enabled us to probe the biochemical properties of glycated peptides (discussed in Chapter 4). Circular dichroism revealed that the introduction of monolysyl AGEs did not hamper the formation of triple helices by collagen model peptides. However, introduction of cross-linking AGEs effectively prevented the collagen model peptides from forming the triple helical structure. Studies of proteolytic digestion using trypsin have revealed the dramatic effect that introduction of lysyl AGEs had on the relative enzymatic digestion rates of the host peptides. These AGEs effectively prevented trypsin from digesting the host telopeptides. Our potentiometric study of copper binding by collagen telopeptides with and without CML has shown that introduction of CML dramatically increased the host peptides capacity to bind copper. Mass spectrometric analysis effectively confirmed the results of potentiometric measurements and provided the first direct evidence of increased copper binding by an AGE-modified peptide.