Chemical Synthesis of Peptides Containing Site-Specific Advanced Glycation Endproducts

In nature, proteins, lipids, and nucleic acids can nonenzymatically react with sugars and sugar degradation products to give rise to a diverse range of modifications, known as advanced glycation endproducts (AGEs). These AGEs typically occur at lysine and arginine residues of long-lived proteins, such as collagen, and can modify the structure and function of the native protein. AGEs accumulate during the normal aging process, and AGE formation is dramatically accelerated with diabetes. AGEs have also been implicated in a wide range of debilitating conditions including cardiovascular, renal failure, and neurodegenerative diseases. Thus, there is an ongoing interest in studying the role of AGEs in different aspects of these disorders. Typically, glycated proteins are prepared using nonspecific in vitro incubation techniques. However, this method results in a complex mixture of products which is then employed without further purification. In order to determine the effect of individual AGEs in a peptide sequence, in this Account, we highlight our synthetic methods for site-specifically introducing five frequently occurring AGEs, namely, N^ε-(carboxymethyl)lysine (CML), N^ε-(carboxyethyl)lysine (CEL), pyrraline, glyoxal-lysine dimer (GOLD), and methylglyoxal-lysine dimer (MOLD) into collagen peptides. Both a collagen model peptide (CMP) and the telopeptide region of human type I α1 collagen (CTP) were chosen due to being prone to glycation and cross-linking in vivo. For the preparation of the AGE-modified collagen peptides, we investigated both the initial preparation of AGE building blocks in solution followed by incorporation into Fmoc-SPPS, as well as an on-resin method whereby AGEs were selectively introduced by modification of the side-chain of an unprotected resin-bound lysine. Both of our synthetic methods enabled the site-specifically modified AGE-containing collagen peptides to be obtained in high purity and yield. In addition, the on-resin method had the added advantage of requiring fewer synthetic steps. We then evaluated the impact of the specific AGEs on the properties of the native protein and found that the AGE modifications protected against proteolytic digestion, enhanced copper binding at physiological pH, and, for the cross-linking AGEs, disrupted the triple helical structure of CMPs. Overall these synthetic methods offered a new strategy for preparing peptides site-specifically modified by AGEs, which can be applied to other peptidic systems, thereby enabling further insights into the biochemical consequences of AGEs.