A physical aid to site-selective modification of a peptide: targeting of single residues for conjugation, stapling, and labeling with a nanopore and mechanical positioning

Abstract

A physical device to aid post-synthetic modification of specific single residues in a peptide at the single molecule level is proposed; it is similar to a structure that was recently proposed for peptide synthesis without side-chain protection (doi: 10.26434/chemrxiv-2025-gsw8x-v5). An electrolytic cell with cis and trans chambers bridged by a nanopore is combined with a mechanical positioning system in which peptides with a homopeptide header or header and trailer are anchored to a platform at one or both ends. A peptide can be moved electrophoretically through the pore with an applied voltage and positioned electromechanically by moving the platform(s) in sub-nm steps with piezo-electric sensors and actuators. A specific residue can be targeted for shielding inside the pore and movement thereafter into trans for targeted protection before it is exposed to reactants (catalyst, linker, conjugate) for modification. Peptides can be modified at the free N- or C- end (single platform) or at arbitrary internal positions (single or dual platform) by cyclization (including overlapping and nested cycles), stapling, linking, or conjugation with a desired cargo for drug delivery/tracking while allowing for diffusion effects. Modification procedures are outlined in theory and illustrated with examples. In many cases successful modification can be detected from the blockade current level in the pore. The results of coarse-grained probabilistic computational simulations showing target, catalyst, and conjugate converging in space, a pre-condition for target-conjugate binding while diffusing in trans, are presented; they suggest that with a trans chamber height of 1 micron-1 mm, the molar catalyst/conjugate-to-target ratio required for convergence in under a minute with probability 1 is 103-106. Although this appears excessive virtually all of the former can be recovered and recycled. The method may be implemented in parallel with an array of pores in a synthetic membrane; with a pore separation of 20 nm on a 2 cm × 2 cm chip, the theoretical maximum yield is ~1.6 picomoles, the actual yield can be estimated from the blockade current. In its present form the proposed system does not scale to the bulk level so its potential use is limited to research and preclinical studies.