Total Chemical Synthesis of Aggregation‐Prone Disulfide‐Rich Starfish Peptides

• Published in: Chemistry : a European journal Vol. 30; no. 33; pp. e202400933 - n/a
• Main Authors: Wu, Hongkang, Praveen, Praveen, Handley, Thomas N. G., Chandrashekar, Chaitra, Cummins, Scott F., Bathgate, Ross A. D., Hossain, Mohammed Akhter
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 12.06.2024
• Subjects: Amino Acid Sequence; Animals; Chemical synthesis; Chromatography, High Pressure Liquid; Cysteine - chemistry; Disulfides - chemistry; High-performance liquid chromatography; Insulin; Liquid chromatography; Oxidation-Reduction; Peptide synthesis; Peptides; Peptides - chemical synthesis; Peptides - chemistry; Relaxin; RLP; RXFP1; Starfish - chemistry; Thiols; Yeasts; Peptides; Insulin; RXFP1; Relaxin; RLP
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.202400933

A relaxin‐like gonad‐stimulating peptide (RGP), Aso‐RGP, featuring six cysteine residues, was identified in the Crown‐of‐Thorns Starfish (COTS, Acanthaster cf. solaris) and initially produced through recombinant yeast expression. This method yielded a single‐chain peptide with an uncleaved C‐peptide (His Tag) and suboptimal purity. Our objective was to chemically synthesize Aso‐RGP in its mature form, comprising two chains (A and B) and three disulfide bridges, omitting the C‐peptide. Furthermore, we aimed to synthesize a newly identified relaxin‐like peptide, Aso‐RLP2, from COTS, which had not been previously synthesized. This paper reports the first total chemical synthesis of Aso‐RGP and Aso‐RLP2. Aso‐RGP synthesis proceeded without major issues, whereas the A‐chain of Aso‐RLP2, in its reduced and unfolded state with two free thiols, presented considerable challenges. These were initially marked by “messy” RP‐HPLC profiles, typically indicative of synthesis failure. Surprisingly, oxidizing the A‐chain significantly improved the RP‐HPLC profile, revealing the main issue was not synthesis failure but the peptide's aggregation tendency, which initially obscured analysis. This discovery highlights the critical need to account for aggregation in peptide synthesis and analysis. Ultimately, our efforts led to the successful synthesis of both peptides with purities exceeding 95 %. This work presents a comprehensive synthetic strategy for creating relaxin‐like peptides characterized by a two‐chain structure linked by three disulfide bonds. Utilizing Fmoc solid‐phase peptide synthesis, the A‐ and B‐chains are independently assembled with orthogonal cysteine protecting groups (tBu, Acm, Trt) to facilitate the controlled formation of disulfide bonds. The synthesis proceeds through four key steps: (i.) Oxidation of the A‐chain to establish the intramolecular disulfide bond; (ii.) Conversion of Cys(tBu) to a more reactive Cys(SPyr) derivative; (iii.) Thiolysis‐driven combination of A‐ and B‐chain to form the first inter‐chain disulfide bond; and (iv.) Final disulfide bond formation via iodine oxidation, culminating in the production of the fully assembled peptide.