Single-Shot Synthesis of Neuroprotective Hi1a by Automated Fast-Flow Peptide Synthesis

Hi1a is a venom peptide isolated from the Australian funnel-web spider Hadronyche infensa, containing an intricate “double knot” tertiary structure and is currently under investigation for the treatment of ischemic stroke. The recombinant expression and chemical synthesis of Hi1a remains a significant challenge due to poor yields and laborious protocols, delaying progress in the translation to the clinic. Herein, we describe the first single-shot chemical synthesis of the Hi1a peptide (76 amino acids, AA) using automated fast-flow peptide synthesis (AFPS), enabling rapid access (4.3 h total synthesis time) to quantities of linear Hi1a (> 10 mg). Our robust protocol facilitated rapid and efficient synthesis of chemical analogues for structure-activity relationship studies, demonstrating that chemical modification of the N- or C-terminus of Hi1a does not significantly perturb binding to acid sensing ion channel 1a (ASIC1a). The synthesis of fluorescently labelled Hi1a permitted live cell imaging using in vitro confocal microscopy, and RNA sequencing demonstrated that Hi1a did not perturb the genetic state of human neurological tissue. This work highlights AFPS as a technology that might address manufacturing issues associated with peptide production.