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Convergent solid-phase synthesis of hirudin.
Spyros Goulas1, Dimitrios Gatos, Kleomenis Barlos
1Department of Chemistry, University of Patras, Patras, Greece.
Summary
Researchers synthesized Hirudin variant 1 (HV1), a protein with 65 amino acids and three disulfide bonds, using advanced Fmoc-based solid-phase peptide synthesis. This study details an efficient method for HV1 production and disulfide bond formation.
Area of Science:
- Biochemistry
- Organic Chemistry
- Peptide Synthesis
Background:
- Hirudin variant 1 (HV1) is a potent anticoagulant protein.
- Efficient synthesis of complex peptides with multiple disulfide bonds remains a challenge.
Purpose of the Study:
- To develop and optimize a robust synthetic strategy for Hirudin variant 1 (HV1).
- To establish a reliable method for the correct formation of three disulfide bonds in HV1.
Main Methods:
- Fmoc-based convergent solid-phase peptide synthesis using 7 protected fragments on 2-chlorotrityl resin.
- Purification of the reduced peptide using reversed-phase high-performance liquid chromatography (RP-HPLC).
- A two-step oxidative folding procedure for disulfide bond formation, confirmed by peptide mapping.
Main Results:
- Successful synthesis of the linear HV1 sequence with high purity after RP-HPLC purification.
- Efficient formation of the three native disulfide bonds in HV1 through a controlled two-step oxidation process.
- Confirmation of correct disulfide bond connectivity using Staphylococcus aureus V8 protease peptide mapping.
Conclusions:
- The developed convergent Fmoc-based synthesis is an efficient method for producing HV1.
- The two-step oxidative folding strategy ensures correct disulfide bond formation in HV1.
- This synthetic approach provides a reliable route to biologically active HV1.