Solid-phase synthesis of sterically hindered peptides via ribosome-mimicking molecular reactors.

The incorporation of N-methylated or α,α-disubstituted amino acids into peptides can markedly enhance their drug-like properties by improving proteolytic stability and membrane permeability. However, the solid-phase synthesis of such sterically hindered peptides remains a persistent challenge. Here we present a detailed protocol based on our recently developed ribosome-mimicking molecular reactor (RMMR) strategy, which substantially improves the synthetic efficiency of these difficult sequences via solid phase peptide synthesis (SPPS). This protocol provides a comprehensive, step-by-step procedure to facilitate a quick startup by the users, including the synthesis of Oxyma-C as the key activating unit precursor in the RMMR structure, the preparation of RMMR resin through modification of conventional SPPS resin and implementation of RMMR-SPPS in both manual and automated synthesizer formats. Peptides containing N-methylated and/or α,α-disubstituted amino acid(s) are synthesized efficiently using our strategy, with crude purities of up to 98% and respectable isolated yields, greatly surpassing existing methods. It is anticipated that this approach will facilitate broader adoption of RMMR-SPPS, addressing critical technical gaps and accelerating the discovery of orally bioavailable or intracellularly targeted macrocyclic peptide drug candidates.