Towards a Total Chemical Synthesis of Human C-C Motif Chemokine Ligand 2

Abstract

Chemokines are cell signalling proteins that promote cellular migration. C-C motif chemokine ligand 2 (CCL2) plays an important role in the inflammatory response, and is involved in the pathogenesis of diseases such as asthma, atherosclerosis and cancer. The potential for modulation of inflammatory processes via inhibition of chemokine interactions makes CCL2 an attractive target for the development of novel anti-inflammatories. In order to characterise these interactions – and the more general role of chemokines within living systems – labelled chemokines are needed as tools for use in biomedical research. Total chemical synthesis has previously been used to produce site-specifically modified proteins (including chemokines) incorporating a wide range of unnatural alterations such as fluorophores and posttranslational modifications. Progress made towards the total chemical synthesis of human CCL2 via Fmoc solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL) is described.

The microwave-assisted solid-phase synthesis of CCL2 as a single peptide chain was found to be problematic due to hydrophobic aggregation, and attempts to avoid this using the poly(ethylene glycol) (PEG)-based ChemMatrix® support (in combination with pseudoproline dipeptides) are reported. Progress has also been made towards a total chemical synthesis utilizing NCL, and syntheses for two of the required fragments – the C-terminal peptide acid CCL2(52-76) and a peptide thioester CCL2(12-35) – are presented. While SPPS of the thioester was achieved using the sulfonamide safety-catch linker, the use of the Dawson Dbz linker for the synthesis of peptide thioesters was also investigated, and the results given here represent the first reported use of the Dbz linker for microwave-assisted SPPS.

In addition, the structure determination of three analogues of a natural product found to inhibit CCL2-mediated monocyte chemotaxis has been carried out using single crystal X-ray diffraction data, thus contributing to ongoing research aiming to elucidate the role of chemokine interactions in biological systems.