One-pot multi-substrate screening of ligation reactions using PNA tags

Chemical ligation is an essential tool for constructing complex biomolecular architectures. To accelerate reaction discovery, a one-pot multi-substrate screening (OPMSS) platform was developed, combining peptide nucleic acid (PNA) tagging with direct MALDI analysis. This approach enables the simultaneous evaluation of multiple substrate pairs in a single pot without the need for chromatographic separation. Short PNA tags promote a uniform combinatorial pairing of substrates while the neutral polyamide backbone facilitates MALDI analysis to allow direct readout of ligated products as predominantly singly charged ions. Using this system, we readily detected established ligations, including Huisgen cycloaddition and amide bond formation, validating the platform in pilot screens pairing 8 × 8 substrates (64 possible combinations). Applying the method to discovery-mode screening of 13 × 11 substrates under visible-light photocatalytic conditions identified a previously unexplored ligation between alkyl azides and alkenes, consistent with pathways involving aminyl radicals or aminium radical cations. This work demonstrates the potential of OPMSS with PNA tagging as a practical and discovery-oriented approach for identifying new ligation reactions directly from complex mixtures.