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

Amino acid pre-activation is a critical reaction step during solid phase peptide synthesis due in part to the formation of impurities such as isomer, N,N′-diisopropylcarbodiimide-adduct, single amino acid addition, and the generation of hazardous hydrogen cyanide side product. The conventional design of the experiment approach has been applied previously to elucidate the optimal pre-activation reaction conditions, but it is laborious and time-consuming amid numerous variables involved. In this work, we present a detailed mechanistic amino acid pre-activation kinetics model to correlate the input parameters with the output reaction conversion, isomer formation, and hydrogen cyanide generation response. The model is formulated using elementary mechanistic reaction steps and the kinetics parameters are fitted to literature data and goodness of fit is evaluated for the activation of other amino acids. Single-factor sensitivity analysis and multi-objective optimization are applied to understand the impact of process parameter ranges on impurity levels and the corresponding control strategies are recommended.