Single molecule peptide synthesis without any side-chain protection using a nanopore and a sub-nanometer positioning system

Peptide synthesis without side-chain protection is proposed at the single molecule level. A peptide is synthesized C-to-N or N-to-C as an extension to a homopeptide header with one end bound to a fixed surface and the other free and enclosed by a nanopore. The surface is mounted on a platform that can be moved with 0.1-0.15 nm precision; the pore acts like a ribosome tunnel to protect amino acid (AA) side-chains from unwanted couplings and also prevents aggregation and cyclization. Synthesis occurs in cycles with the following steps: a coupling agent attaches an end-protected AA to the terminal residue of the growing header at the end of the pore; completion of coupling is detected optically; coupling agent, protector, and excess AAs are washed out; the platform is retracted by 3.5 Å; the newly added AA is deprotected and the protector washed out. On completion of synthesis the platform is moved toward the pore by the added peptide’s length, the peptide is separated from the header. A combination of electrical potential and hydraulic pressure keeps the peptide fully stretched at all times. The nanopore plays a secondary role, no measurements are made on it. Parallel synthesis can be done with an array of headers and an array of nanopores, capacities of up to a femtomole may be possible. In principle there is no limit to the length of the peptide that can be synthesized. With no side-chain protection, minimal reagent amounts, reduced washing, and almost no post-synthesis cleanup, the method has a potentially high level of greenness.