(207a) Mechanistic Study of Diketopiperazine Formation during Solid Phase Peptide Synthesis of Tirzepatide

This study focused on investigating diketopiperazine (DKP) and the formation of associated double amino acid deletion impurities during linear solid phase peptide synthesis (SPPS) of tirzepatide (TZP), a 39 amino acid synthetic peptide. The C terminus desProPro double amino acid deletion impurity was observed at significant levels during the synthesis of TZP intermediate. In this study, we initially conducted root cause analysis and identified the DKP formation primarily occurred during the Fmoc deprotection reaction and post-coupling ageing of the unstable API intermediate. Similar phenomena have also been observed for other TZP intermediates which contain penultimate proline and other C Terminus intermediates for both hybrid and linear synthetic approaches. During post-coupling hold times, it is found that Fmoc- deprotection can proceed auto catalytically in various solvents without any piperidine addition, along with subsequent DKP formation on the deprotected peptide. Detailed Fmoc- self-deprotection and subsequent DKP formation mechanisms are proposed and account for intramolecular pathways triggered by the amine group of the penultimate proline and intermolecular pathways catalysed by the deprotected amine group in the peptide and DKP by product. Reaction kinetics parameters are solved for various peptide API intermediates under different solvents and temperature conditions to describe rate of impurity formation. As general synthesis practice described in past literature, we also found use of oxyma additives and lower storage temperature can greatly stabilize the peptide intermediated to DKP by-products.