Development of a Polar Tag Platform for Sustainable Tag-Assisted Liquid-Phase Peptide Synthesis (TA-LPPS)

Abstract

Tag-assisted liquid-phase peptide synthesis (TA-LPPS) has emerged as a potential solution to synthetic bottlenecks associated with solid-phase peptide synthesis (SPPS) and long development times required for classical liquid-phase peptide synthesis (LPPS). Modern TA-LPPS utilizes hydrophobic alkyl chains attached to the peptide to assist extraction or precipitation after each synthetic step. Unfortunately, these highly nonpolar structures are also prone to self-aggregation and solubility challenges during synthesis. In contrast, the seminal report of TA-LPPS utilized tags derived from polar polydisperse PEG ∼20,000 Da chains, which demonstrated excellent peptide solubility in the polar organic solvents needed for peptide synthesis. Despite their enhanced solubility, polar tags have been overlooked due to the polydispersity of the PEG polymer, which severely complicates LC-MS analysis of tagged products. Herein, we report a monodisperse polar tag (ADO10), which is derived from 2-[2-(2-aminoethoxy)ethoxy]acetic acid (AEEA)—a commercial and low-cost starting material. ADO10 utilizes conventional Fmoc/t-Bu protecting groups and greatly simplifies LC-MS reaction monitoring. Rather than aqueous extraction or precipitation, ADO10 tags utilize a unique organic–organic liquid–liquid separation in green solvents—balancing phase separation speed and effective impurity removal. The established polar TA-LPPS platform is capable of gram-scale synthesis of multiple biologically active peptides including a fragment of semaglutide. Furthermore, we demonstrate that the enhanced solubility of polar tags can resolve challenges observed for SPPS of an aggregation-prone peptide Aβ(25–35).