Peptides, an important class of polymers, mainly used as therapeutics, are generally synthesized using solid phase methods. While solid phase peptide synthesis provides for facile manipulation of intermediates retained in the solid support, this technology faces challenges for large-scale synthesis, where its non-quantitative coupling and deprotection leads to error sequences thus hampering final product purification. In contrast liquid phase peptide synthesis, offers high peptide purity and scalability but its development is hindered by inefficient intermediate separations and solubility issues.

Molecular separation in organic solvents via nanofiltration (OSN) offers a perfect separation method for the in-cycle purification of growing peptide chains. This presentation will provide overview of the most recent developments in the liquid phase peptide synthesis combined with OSN separations.

The PEPSTAR platform - Liquid phase peptide synthesis via nanostar-sieving which synthesises peptides in solution with facile intermediate separations via OSN will be presented. Amino acids (AA) are coupled iteratively onto a n-armed, star-shaped macromolecule, forming peptide-nanostar intermediates. After coupling, the unreacted AA is quenched and subsequently proceeded to N-terminal deprotection. The bulky intermediates are then ‘sieved’ out from the debris and quenched AA all together through a crosslinked polybenzimidazole (PBI) polymer-based membrane thus omitting the post-coupling isolation step. This synthetic cycle is repeated until the desired peptide length is achieved. The use of nanostar greatly enhances the molecular sieving efficiency by the >3-fold mass difference between the nanostar and the unreacted building blocks. Most importantly, real-time reaction monitoring can be undertaken by LC-MS with high accuracy.

In this work we demonstrate the successful synthesis of Enkephalin-type model peptides (~5-10 mers) via nanostar-sieving technology. The products are of higher (or equal) purity than peptides produced by a solid phase vendor, while using less equivalents of AA during coupling thus bringing the cost of materials down.