(121e) Liquid Phase Peptide Synthesis Via Nanostar-Sieving

Peptides, an important class of polymers, mainly used for therapeutic purposes, are generally synthesised using solid phase methods. While the rapid synthesis characteristic of solid phase peptide synthesis (SPPS) benefits the drug discovery, this technology faces challenges for large scale synthesis, where its non-quantitative coupling leads to error sequences. In contrast liquid phase peptide synthesis (LPPS), offers high peptide purity and scalability but its development is hampered by inefficient intermediate separations.

Liquid phase peptide synthesis via nanostar-sieving (LPPS-NS) is a platform which synthesises peptides in solution with facile intermediate separations. Amino acids (AA) are coupled iteratively onto a 3-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 via organic solvent nanofiltration (OSN) thus omitting the post-coupling isolation step. This synthetic cycle is repeated until the desired peptide length is achieved, as shown in Figure 1. Standard Fmoc peptide chemistry is applied throughout the synthetic cycle. 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 because of the monodispersity of the nanostar. OSN plays a pivotal role in synthesis efficiency. A solvent resistant membrane made of crosslinked polybenzimidazole (PBI) polymer was chosen for OSN. The polymer-based membrane was proven to be durable and to have a high separation factor which remained consistent throughout many synthesis cycles. We speculate that this new concept of LPPS is a truly one-pot synthesis where no transfer of liquids between cycles occurs and has a good potential for full-automation.

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 purity than, or at least comparable to, peptides produced by a reliable solid phase vendor, while using less equivalents of AA during coupling. To further validate this technology, high purity linear Octreotide (8AA) was synthesised. Our ambition is to develop this platform into a robust technology for large-scale, fully automated and high purity peptide synthesis.