Automated high-purity fast-flow synthesis of cyclic peptide-PNA conjugates for drug discovery and nanotechnology

Cyclic peptide nucleic acids (PNAs) combine remarkable nuclease resistance with high binding affinity, making them attractive candidates for applications in immunotherapy, biomedicine, and material science. However, their adoption has been limited by inefficient synthesis strategies. Here, we report an automated on-resin head-to-tail cyclization strategy for PNAs and peptide-conjugated PNAs (PPNAs) using a diaminonicotinic acid (DAN) linker. This methodology ensures precise control over chain elongation and cyclization, achieving 2- to 12-residue sequences with up to 95 % crude purity. Utilizing the iChemAFS platform with high-temperature flow chemistry, synthesis time was reduced by an order of magnitude compared to manual approaches. The approach also supports extensive functionalization, including the incorporation of natural and non-natural amino acids, and enables late-stage modifications. A library of 22 PNA/PPNA structures was constructed and screened for membrane permeability using parallel artificial membrane permeability assays (PAMPA). Additionally, cyclic PPNA nanotubes with high aspect ratios were efficiently synthesized and characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). These advances underscore the potential of this strategy to advance PNA applications in drug development and nanotechnology.