Suppressing the epimerization of endothioamide peptides during Fmoc/t-Bu-based solid phase peptide synthesis

Area of Science:

• Peptide Chemistry
• Chemical Biology
• Biophysics

Background:

• Thioamide-containing peptides and proteins have potential applications in biophysics, pharmacology, and chemical biology.
• However, their synthesis, particularly for long peptides, is challenging due to epimerization and side reactions, limiting their use.
• The thioamide moiety's sensitivity to standard peptide synthesis conditions (Fmoc/t-Bu chemistry) causes stereochemical loss.

Purpose of the Study:

• To develop an efficient and easy-to-implement protocol for synthesizing long endothioamide peptides.
• To significantly suppress epimerization and other undesired side reactions during synthesis.
• To enable the creation of novel thiopeptide and thioprotein-based chemical tools.

Main Methods:

• Utilized Fmoc/t-Bu chemistry with a modified deprotection step.
• Employed a brief treatment with 10% piperidine/dimethylformamide for 1 minute during chain elongation.
• Demonstrated the protocol's efficacy on synthesizing 10-12-mer peptides and a modified protein G peptide.

Main Results:

• Successfully synthesized long (>10-mer) endothioamide peptides with minimal epimerization and side reactions.
• Achieved efficient synthesis of mono- to multiply thioamide-substituted peptides with broad substrate scope.
• Produced a thioamide version of a 16-mer peptide from the B1 domain of protein G, showcasing utility in protein engineering.

Conclusions:

• The developed protocol overcomes significant synthetic hurdles in producing long thioamide peptides.
• This advancement opens new possibilities for engineering and accessing novel thiopeptide and thioprotein chemical tools.
• Facilitates broader applications of thioamide-containing molecules in biological studies and drug development.