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

• Published in: Angewandte Chemie International Edition Vol. 60; no. 41; pp. 22207 - 22211
• Main Authors: Xia, Yiyin, To, Janet, Chan, Ning‐Yu, Hu, Side, Liew, Heng Tai, Balamkundu, Seetharamsing, Zhang, Xiaohong, Lescar, Julien, Bhattacharjya, Surajit, Tam, James P., Liu, Chuan‐Fa
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley Subscription Services, Inc 04-10-2021
• Edition: International ed. in English
• Subjects: Amino acids; cyclic peptides; enzymes; Gelatinase A; Hydroxamic acid; inhibitors; macrocyclization; Matrix metalloproteinase; Matrix metalloproteinases; Metalloproteinase; Peptides; Substrates
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.202108125

Peptidyl asparaginyl ligases (PALs) are powerful tools for peptide macrocyclization. Herein, we report that a derivative of Asn, namely Nγ‐hydroxyasparagine or Asn(OH), is an unnatural P1 substrate of PALs. By 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. The most potent cyclic peptide (Ki=2.8±0.5 nM) was built on the hyperstable tetracyclic scaffold of rhesus theta defensin‐1. The Asn(OH) residue in the cyclized peptides can also be readily oxidized to Asp. By 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 owing 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.
An unnatural amino acid, Asn(OH), mimics P1‐Asn in the substrates of asparaginyl ligases, enabling efficient cyclization of P1‐Asn(OH) peptides. The hydroxamic acid functionality in Asn(OH) is a metal‐ion chelator for metalloenzyme inhibition, and Asn(OH) can be converted into native Asp by periodate oxidation (see scheme).