Advancing Nitrile-Aminothiol Strategy for Dual and Sequential Bioconjugation.

Nitrile-aminothiol conjugation (NATC) stands out as a promising biocompatible ligation technique due to its high chemo-selectivity. Herein we investigated the reactivity and substrate scope of NAT conjugation chemistry, thus developing a novel pH dependent orthogonal NATC as a valuable tool for chemical biology. The study of reaction kinetics elucidated that the combination of heteroaromatic nitrile and aminothiol groups led to the formation of an optimal bioorthogonal pairing, which is pH dependent. This pairing system was effectively utilized for sequential and dual conjugation. Subsequently, these rapid (≈1 h) and high yield (>90%) conjugation strategies were successfully applied to a broad range of complex biomolecules, including oligonucleotides, chelates, small molecules and peptides. The effectiveness of this conjugation chemistry was demonstrated by synthesizing a fluorescently labelled antimicrobial peptide-oligonucleotide complex as a dual conjugate to imaging in live cells. This first-of-its-kind sequential NATC approach unveils unprecedented opportunities in modern chemical biology, showcasing exceptional adaptability in rapidly creating structurally complex bioconjugates. Furthermore, the results highlight its potential for versatile applications across fundamental and translational biomedical research.

An Efficient Approach for the Design and Synthesis of Antimicrobial Peptide-Peptide Nucleic Acid Conjugates.

Peptide-Peptide Nucleic Acid (PNA) conjugates targeting essential bacterial genes have shown significant potential in developing novel antisense antimicrobials. The majority of efforts in this area are focused on identifying different PNA targets and the selection of peptides to deliver the peptide-PNA conjugates to Gram-negative bacteria. Notably, the selection of a linkage strategy to form peptide-PNA conjugate plays an important role in the effective delivery of PNAs. Recently, a unique Cysteine- 2-Cyanoisonicotinamide (Cys-CINA) click chemistry has been employed for the synthesis of cyclic peptides. Considering the high selectivity of this chemistry, we investigated the efficiency of Cys-CINA conjugation to synthesize novel antimicrobial peptide-PNA conjugates. The PNA targeting acyl carrier protein gene (acpP), when conjugated to the membrane-active antimicrobial peptides (polymyxin), showed improvement in antimicrobial activity against multidrug-resistant Gram-negative Acinetobacter baumannii. Thus, indicating that the Cys-CINA conjugation is an effective strategy to link the antisense oligonucleotides with antimicrobial peptides. Therefore, the Cys-CINA conjugation opens an exciting prospect for antimicrobial drug development.