The design and synthesis of cyclic peptides is a widely established practice in the field of peptide chemistry. This has been further expanded by the development of orthogonal chemical reactions allowing for production of more chemically complex peptides. One such reaction is the use of 1,3-dichloroacetone (DCA) to selectively link free cysteine side-chains via an acetone bridge. We have examined the utility of this reaction to synthesize bicyclic dimeric peptides.

We synthesized six head-to-tail cyclic peptides, each possessing a single cysteine residue, and created bicyclic dimeric peptides by linking two copies of the cyclic peptide together using DCA to form an acetone linker. We systematically investigated a range of reaction conditions, including stoichiometry of reagents, peptide concentration, reaction pH and buffer composition. We were successfully able to identify the optimum conditions for peptide dimerization for our six peptide sequences and have used these results to produce an overall guide for preparing acetone-linked bicyclic peptides. The peptides were subsequently analyzed for proteolytic stability in human serum and were observed to still be fully intact after 48 hours. This study provides valuable insights into the use of DCA as a tool in peptide synthesis. The non-reducible nature of the acetone linker between pairs of cysteine residues makes the DCA dimerization reaction attractive compared to the better-known disulfide bond approach.