Ethylthio-1H-tetrazole (ETT) as coupling additive for the solid-phase synthesis (SPS) of hindered amino acid-containing peptides

Abstract

Peptides are finding broad applications in drug discovery as Active Pharmaceutical Ingredients (APIs) and delivery systems, and also in the field of new materials. In this context, the increasing relevance of these molecules has fueled the development of more efficient synthetic strategies at both research and industrial scales. The new generation of peptides in the market contains hindered amino acids, examples include the antidiabetic and antiobesity drugs semaglutide and tirzepatide, which contain α,α-dimethylglycine (Aib), and trofinetide, which contains α-methylproline. Given that peptide synthesis involves the proper combination of protecting groups and coupling reagents, it is important to develop coupling strategies for these hindered amino acid-containing peptides. In this first communication, we report that ethylthio-1H-tetrazole (ETT), a reagent widely used in oligonucleotide synthesis but unknown in peptide synthesis, is well suited to be used in combination with N.N’-diisopropylcarbodiimide (DIC) for the preparation of hindered amino acid-containing peptides. ETT is more acidic than 1-hydroxybenzotriazole (HOBt) and ethyl 2-hydroxyimino-2-cyanoacetate (OxymaPure), and, as such, the active species formed will have an excellent leaving group, thereby facilitating the coupling reaction. To demonstrate this concept, we synthesized analogs of Leu-enkephalin amide, where the two consecutive Gly amino acids were substituted by Aib, NMeGly, and NMeAla. The syntheses carried out with ETT were superior to those done with OxymaPure, HOBt, and 1-hydroxy-7-azabenzotriazole. (HOAt). However, in terms of racemization, ETT showed poorer performance than the other coupling additives, especially in the case of His(Trt). In conclusion, ETT emerges as an important contributor to the toolbox of coupling reagents and additives used for peptide and amide formation.