Mechanistic Study of Diketopiperazine Formation during Solid-Phase Peptide Synthesis of Tirzepatide

Abstract

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). We identified that the DKP formation primarily occurred during the Fmoc-deprotection reaction and post-coupling aging of the unstable Fmoc-Pro-Pro-Ser-resin active pharmaceutical ingredient (API) intermediate. Similar phenomena have also been observed for other TZP active pharmaceutical ingredient (API) intermediates that contain a penultimate proline amino acid, such as Fmoc-Ala-Pro-Pro-Pro-Ser-resin, Fmoc-Pro-Pro-Pro-Ser-resin, and Fmoc-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-resin, which are intermediates for both hybrid and linear synthesis approaches. During post-coupling aging, it is found that Fmoc deprotection can proceed in dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and acetonitrile (ACN) solvents without any piperidine addition. Density functional theory (DFT) calculations showed that a peptide that has a penultimate proline stabilizes the transition state through the C−H•••π interaction during Fmoc decomposition, which causes those peptides to be more prone to cascade-deprotection reactions. Pseudo-reaction pathways are then proposed, and a corresponding macrokinetics model is developed to allow accurate prediction of the TZP peptide intermediate self-deprotection and DKP formation rate. Based on those studies, control strategies for minimizing DKP formation were further investigated and an alternative to Fmoc protection was identified (Bsmoc-protected amino acids), which eliminated the formation of the DKP byproducts. In addition, the use of oxyma additives and lower storage temperature was demonstrated to markedly improve the peptide intermediate stability to DKP degradation pathways.

Development of a Mechanistic Amino Acid Activation Reaction Kinetics Model for Safe and Efficient Solid Phase Peptide Synthesis

“…In particular, Histidine has been reported to be the most sensitive toward racemization . In this work, we studied the kinetics associated with the activation and racemization of Histidine using online NMR technique reported in literature. , The activation was conducted using a 1:3:2.5 molar ratio of His/Oxyma/DIC at 20 and 30 °C to ensure 100% activation conversion at the end of the reaction . The rate of the activation reaction was simulated using the rate constants derived from Leucine activation above, and the kinetics parameters for Histidine racemization, k 8 and Ea 8 in eq , were obtained by fitting the experimental data.…”

“…Various attempts have been made previously to understand the role of the factors involved in the amino acid pre-activation reaction in forming such impurities, majorly using conventional full factorial design of experiment approach. ,,− However, given the complicated mechanism of the activation reaction, the outcome of full factorial experimental design has been only utilized in establishing empirical polynomial fit models, and a response surface methodology can be further applied to elucidate the complex relationship of input process parameters with output response. ,, While such approaches are sufficient for explaining the experimental results theoretically, they are confined for broader use due to the limited predictability for reaction conditions outside the original design of experiment . Recent advances in molecular simulation allow designing greener activation solvent system to suppress HCN formation in the activation reaction .…”

Solid‐Phase Synthesis of PROTACs and SNIPERs on Backbone Amide Linked (BAL) Resin

“…We considered that this might be due to a diketopiperazine formation side reaction, whereby the exposed terminal amine undergoes an acyl substitution reaction at the carbonyl of the tBu-Hyp residue. [25] A deletion product was obtained which lacked both the hydroxyproline and the tertleucine residues. Changing the deprotection cocktail to 2 % DBU and 5 % piperazine in NMP for 2×5 min suppressed the side reaction and was followed by immediate washing and acylation of the free amine.…”

Morpholine, a strong contender for Fmoc removal in solid‐phase peptide synthesis

“…However, the steric hindrance around CT resin can minimize but not suppress it completely 42,43 . DKP formation can also occur during the preparation of depsipeptides, where a peptide bond is substituted by a (thio)ester moiety, 44 and even in sequences where the leaving group is an amide from a peptide amide bond 45 . In this study, we investigated the effect of Morph–DMF on DKP formation following the strategy shown in Scheme 3.…”

“…In terms of DKP and aspartimide formation, the two most prominent base‐induced side reactions, the performance of 50%–60% Morph–DMF was superior to 20% PIP–DMF. These new Fmoc removal conditions could be implemented in many cases in which DKP formation can occur 45 . With respect to the suppression of aspartimide formation, Morph outperformed PIP at both rt and under stress conditions.…”