Rapid Chemical Synthesis of Neuroprotective Hi1a

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 remain a significant challenge due to laborious protocols, delaying progress in translation to the clinic. We describe the first single-shot chemical synthesis of Hi1a (76 amino acids, AA) using automated fast-flow peptide synthesis (AFPS), enabling rapid access (<4.5 h total synthesis time) to milligram quantities of linear Hi1a (>10 mg). This work highlights the utility of AFPS as a technology that enables chemical production of biomolecules challenging to obtain by recombinant methods.