Strain-Accelerated β-Thiolactone Native Chemical Ligation with Kinetic Control Enables Rapid and Selective Hydrogelation for Biofabrication.

A central challenge in biomaterials design is developing cross-linking reactions that are fast, selective, synthetically accessible, and compatible with the nucleophile-rich environments required for cell encapsulation. Native chemical ligation (NCL) offers an attractive route to amide-linked hydrogels under mild aqueous conditions, yet its implementation in biomaterials has been constrained by slow kinetics, free-thiol byproducts, and inhibition in complex media. Here, we demonstrate that deliberate electrophile design through incorporation of a strain-encoded β-thiolactone enables rapid and selective NCL-mediated hydrogel formation through rapid recyclization of off-target intermediates. A penicillamine-derived β-thiolactone cross-linker synthesized directly on four-arm polyethylene glycol (PEG, 10 kDa) exhibits fast gelation in complete cell culture media while maintaining orthogonality to embedded human dermal fibroblasts. Relative to a conventional alkyl thioester and a γ-thiolactone analogue, the strained β-thiolactone displays accelerated gelation and enhanced tolerance to competing endogenous thiols. Mechanistically, geminal dimethyl substitution promotes rapid β-thiolactone recyclization, suppressing unproductive thiol exchange while productive NCL proceeds through an irreversible S-to-N acyl shift. Because unreacted β-thiolactones persist under physiological conditions, the network remains chemically addressable after gelation, enabling temporally delayed functionalization with N-Cys-containing molecules. This combination of rapid network formation and postgelation addressability enables direct peptide incorporation, hydrogel microfiber fabrication, and long-term three-dimensional cell encapsulation.