Protein synthesis with conformationally constrained cyclic dipeptides.

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Chemistry

Background:

• Protein engineering aims to create novel proteins with desired functions.
• Incorporating non-canonical amino acids expands the chemical diversity of proteins.
• Cyclic dipeptides offer constrained structures for specific molecular interactions.

Purpose of the Study:

• To synthesize and evaluate conformationally constrained cyclic dipeptide analogues as substrates for protein incorporation.
• To investigate the efficiency of incorporating these analogues into dihydrofolate reductase (DHFR) using a suppressor tRNA system.
• To compare the incorporation efficiency of lactam versus acylhydrazide cyclic dipeptides.

Main Methods:

• Synthesis of Boc-protected cyclic dipeptides with 5-, 6-, and 7-membered lactam rings.
• Preparation of activated suppressor tRNA transcripts via in vitro transcription.
• In vitro protein synthesis using modified E. coli ribosomes for dipeptide incorporation into DHFR.
• Synthesis of 7-membered cyclic acylhydrazides via EDCI-mediated cyclization.

Main Results:

• Cyclic dipeptides were successfully incorporated into DHFR at positions 18 and 49.
• Incorporation yields increased with larger lactam ring sizes (5- to 7-membered), ranging from 3.4% to 8.9%.
• Cyclic acylhydrazides showed higher incorporation efficiencies (8.3% to 11.2%) compared to lactam dipeptides.

Conclusions:

• Conformationally constrained cyclic dipeptides can be effectively incorporated into proteins.
• The size of the lactam ring influences the efficiency of protein incorporation.
• Cyclic acylhydrazides represent a promising class of analogues for enhanced protein engineering via non-canonical amino acid incorporation.