Nγ -Hydroxyasparagine: A Multifunctional Unnatural Amino Acid That is a Good P1 Substrate of Asparaginyl Peptide Ligases.

Peptidyl Asparaginyl Ligases (PALs) are powerful tools for peptide macrocyclization. Herein, we report that a derivative of Asn, namely N γ -hydroxyl-asparagine or Asn(OH), is an unnatural P1 substrate of PALs. Using Asn(OH)-mediated cyclization, we prepared cyclic peptides as new matrix metalloproteinase 2 (MMP2) inhibitors displaying the hydroxamic acid moiety of Asn(OH) as the key pharmacophore. Cyclic peptide 17 , the most potent of these (K i = 2.8 ± 0.5 nM), was built on the hyper-stable tetracyclic scaffold of rhesus theta defensin-1. The Asn(OH) residue in the cyclized peptides can also be readily oxidized to Asp. Using this approach, we synthesized several bioactive Asp-containing cyclic peptides (MCoTI-II, kB2, SFTI, and integrin-targeting RGD peptides) that are otherwise difficult targets for PAL-catalyzed cyclization due to unfavorable kinetics of the P1-Asp substrates. This study demonstrates that substrate engineering is a useful strategy to expand the application of PAL ligation in the synthesis of therapeutic cyclic peptides.