Reflecting recent work in the Liu Group

To address the limitations of current methods for peptide macrocyclization, which often rely on nonspecific small-molecule linkers, researchers in the Wenshe Liu group, at Texas A&M University, published in the ACS Chemical Biology, report the development of two novel regioselective linkers: 2-chloro-N-(2-cyanopyrimidin-5-yl)acetamide (pCAmCP) and 2-chloro-N-(2-cyanopyrimidin-4-yl)acetamide (mCAmCP). These linkers enable efficient macrocyclization of phage-displayed peptides containing an N-terminal cysteine and an internal cysteine, avoiding the toxicity and nonspecificity of previous approaches.

In this study, five derivatives of 2-cyanopyrimidine were synthesized and evaluated for their ability to cyclize a protein-fused model peptide under physiological conditions. Among these, pCAmCP and mCAmCP demonstrated the highest regioselectivity and efficiency. By leveraging these linkers to create macrocyclic peptide libraries displayed on phage, the team identified peptide ligands for the ZNRF3 extracellular domain, a promising target in membrane protein modulation. One peptide, Z27S1, exhibited potent binding with a dissociation constant (KD) of 360 nM, underscoring the utility of these linkers in generating high-affinity macrocyclic peptides.

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The Wenshe Liu Group at the Texas A&M University at College Station

The findings revealed distinct enrichment patterns in peptide libraries depending on the linker used, highlighting the significant impact of linker composition on selection outcomes. These results demonstrate not only the importance of having diverse linker chemistries for phage display but also the broader potential of these hydrophilic linkers to improve compatibility with aqueous environments while expanding the range of accessible macrocyclic peptide scaffolds.

By streamlining the macrocyclization process and enhancing library diversity, this study provides an important advancement in peptide engineering, paving the way for more effective therapeutic discovery and the development of innovative tools for targeting challenging protein surfaces.