Efficient hydrazide-mediated total synthesis of Möbius cyclotides from Viola japonica and elucidation of the antibacterial mechanism of Vija 31.

Cyclotides represent a unique family of plant-derived cyclic peptides with high structural stability and promising bioactivities, yet their pharmaceutical application is limited by low natural abundance and synthetic difficulty. Herein, we adopted an efficient hydrazide-mediated total synthesis strategy to prepare nine Möbius subfamily cyclotides from Viola japonica transcriptomic data. Among them, Vija 31 exhibited remarkable antibacterial potency (MIC = 2 μM against Acinetobacter baumannii and Bacillus subtilis, 64 μM against Cryptococcus neoformans), along with low hemolytic activity (3.36% at 128 μM). Circular dichroism spectroscopy and AlphaFold-3 modeling indicate Vija 31 adopts a compact β-sheet/β-turn conformation, which may contribute to its antibacterial activity. Scanning electron microscopy revealed Vija 31-induced bacterial membrane disruption (rupture and shrinkage), while nucleic acid and protein leakage assays confirmed concentration-dependent release of intracellular contents and time-kill kinetics demonstrated its concentration-dependent bactericidal activity. Optimization of hydrazide cyclization conditions using Vija 31 as a model further improved reaction efficiency. This work establishes a versatile and scalable route for Möbius cyclotide synthesis and identifies Vija 31 as a promising lead compound against drug-resistant bacteria.