A One‐Pot “Triple‐C” Multicyclization Methodology for the Synthesis of Highly Constrained Isomerically Pure Tetracyclic Peptides

Abstract

A broadly applicable one-pot methodology for the facile transformation of linear peptides into tetracyclic peptides through a chemoenzymatic peptide synthesis/chemical ligation of peptides onto scaffolds/copper(I)-catalyzed reaction (CEPS/CLIPS/CuAAC; "triple-C") locking methodology is reported. Linear peptides with varying lengths (≥14 amino acids), comprising two cysteines and two azidohomoalanines (Aha), were efficiently cyclized head-to-tail by using the peptiligase variant omniligase-1 (CEPS). Subsequent ligation-cyclization with tetravalent (T4 ) scaffolds containing two bromomethyl groups (CLIPS) and two alkyne functionalities (CuAAC) yielded isomerically pure tetracyclic peptides. Sixteen different functional tetracycles, derived from bicyclic inhibitors against urokinase plasminogen activator (uPA) and coagulation factor XIIa (FXIIa), were successfully synthesized and their bioactivities evaluated. Two of these (FF-T4 ) exhibited increased inhibitory activity against FXIIa, compared with a bicyclic control peptide. The corresponding hetero-bifunctional variants (UF/FU-T4 ), with a single copy of each inhibitory sequence, exhibited micromolar activities against both uPA and FXIIa; thus illustrating the potential of the "bifunctional tetracyclic peptide" inhibitor concept.

Natural Occurring and Engineered Enzymes for Peptide Ligation and Cyclization

“…It was shown that due to the broad substrate scope and traceless ligation, different sites could be used to synthesize the cyclic peptides. Most recently, the cyclization technology was combined with small organic scaffolds and other ligation technologies such as oxime ligation and click chemistry (Richelle et al, 2018; Streefkerk et al, 2019). Combining enzymatic and chemical ligation technologies, tetracyclic peptides could be synthesized in a one pot fashion that poses two distinct biological activities.…”

Efficient Enzymatic Cyclization of Disulfide‐Rich Peptides by Using Peptide Ligases

“…We have recently employed the peptide ligase (peptiligase) variant omniligase‐1 to efficiently prepare several mono‐, tri‐, and tetracyclic peptides, as well as the naturally occurring cyclotide MCoTI‐II . Omniligase‐1 is a broad‐specificity subtilisin variant, which efficiently and tracelessly catalyzes inter‐ or intramolecular peptide bond formation of unprotected peptides in aqueous solution.…”

Synthesis of Constrained Tetracyclic Peptides by Consecutive CEPS, CLIPS, and Oxime Ligation

“…CEPS cyclization followed by CLIPS and subsequently oxime ligation was envisaged the most straightforward approach, based on previous experience. 21 For oxime ligation, the peptide contains either (a) the aminooxy or (b) the ketone moiety. Therefore, we set out to investigate two different strategies (Figure 1).…”

“…This can likely be attributed to hindered rotation around either the aryl-C(=O) or the HN-C=O bond in the scaffolds, as a result of the relatively small peptide ring size (Figures 2a and 2d), matching earlier observations. 21 Separation of the products was not attempted and was deemed impossible, because the isomerism is considered conformational, rather than configurational, assuming both products are in thermodynamic equilibrium. This assumption was confirmed by the fact that, for c 1 3333 - hS (ONH 2 ) ·T4-3 (C=O) c/o , a third isomer was initially observed at t R = 0.73 min, that disappeared overnight at rt.…”

“…This is consistent with previous findings that the number of product isomers obtained is linearly correlated to the difference in peptide lengths and attributable to conformational/rotational rather than configurational/structural isomers. 21…”