Microcin J25, MccJ25, is a lasso peptide whose mechanical interlocking confers exceptional stability, but that same topology has made precise chemical editing at specific loop positions difficult. Earlier approaches to noncanonical amino acid incorporation relied on amber suppression or total chemical synthesis of the linear precursor followed by cyclization; any modification had to be tolerated by the biosynthetic enzymes McjB and McjC. A disulfide-based workaround produced mechanically interlocked molecules but introduced a bond unstable in the presence of reducing agents. Attempts to open the lasso loop cleanly with common proteases all failed: trypsin and V8 protease gave no cleavage, α-chymotrypsin achieved only approximately 40% single cleavage after 72 hours, and pancreatin and proteinase K each produced complex mixtures or non-specific cleavage, leaving no reliable entry point for site-selective chemical editing.
Researchers in the Chu Lab at National Taiwan University, published in JACS Au, identified thermolysin as the one protease that cleaves MccJ25 site-selectively at the Phe10–Val11 bond, yielding a single-cut [2]rotaxane; prolonged incubation releases the Val-Gly dipeptide to give a double-cut intermediate. Because MccJ25 lacks basic residues, the exposed N-terminus is the sole nucleophilic site, enabling regioselective installation of a preactivated building block, though a large excess of 120 equivalents was required, presumably because restoration of the lasso structure is energetically unfavorable due to its high strain, and multiple rounds of HPLC purification reduced the overall isolated yield to approximately 2.5%. Because the building-block step is purely chemical, it is compatible, in principle, with a nearly unlimited range of noncanonical entities, and the team used it to prepare nine new-to-nature lasso peptides bearing loop-size changes, D-form amino acids, N-methylated amino acids, and a naphthalene moiety.
The authors propose that this chemoenzymatic strategy will enable access to diverse mechanically interlocked molecules tailored for biomedical applications beyond antibacterial use, and note that the workflow is applicable in principle to other sites in MccJ25 and to other lasso peptides wherever a defined incision or excision can be generated, though this broader applicability was not demonstrated in the present work. Why some variants lost antibacterial activity whereas others retained it will require further investigation. Structural data, antibacterial profiling, and activity comparisons across variants are available in the original publication.
