Characterization of Radical-Mediated and [2+2] Cycloaddition Photocrosslinking of Maleimide Monomers and Macromers

The maleimide moiety is remarkable for its versatility, participating in thiol-Michael, Diels-Alder, 1,3-dipolar cycloaddition, and [2+2] cycloaddition reactions, as well as both homo- and copolymerizations. Recently, 2-photon excitation with 800 nm light was demonstrated to catalyze [2+2] cycloaddition of maleimides for photopatterning in poly(ethylene glycol) (PEG) hydrogels. However, this work highlighted that irradiation with 2-photon 720 nm light did not initiate this cycloaddition despite known 1-photon reactivity at 365 nm. Additionally, single-photon and multiphoton cycloadditions of maleimide proceed through distinct mechanisms, with significantly faster reaction rates in the 2-photon condition. Previous studies have indicated that maleimide dimerization occurs after charge separation, which generates radical pairs that precede the rapid formation of the cycloadduct. Adding TEMPO, a common radical scavenger, to maleimide-functionalized multi-arm PEG macromers prevents photocrosslinking of hydrogels, suggesting radical dependence of the maleimide [2+2] cycloaddition. By adding the photoinitiators TPO and LAP to various maleimide formulations, we demonstrate radical-accelerated maleimide crosslinking in small molecules, linear polymers, and crosslinked networks including PEG hydrogels. Using NMR, IR, GPC, and EIS, we identify substantial increases in short poly(maleimide) chains and maleimide dimer products in exogenous radical-initiated compared to catalyst-free conditions under identical illumination. Rheological characterization of catalyst-free photocrosslinking at 365 nm of maleimide-functionalized PEG hydrogels revealed an unexpected response to the presence of ions in a Hofmeister series-dependent manner. Increasing chaotropicity attenuates reaction rates, particularly with respect to anions in solution; adding LAP eliminates this effect, highlighting the role of the charge pairing involved in catalyst-free photocrosslinking of maleimides. Despite differences in reaction mechanism, the plateau moduli of these hydrogels are effectively identical, suggesting highly similar molecular weight between crosslinks. Finally, the improved reaction rates realized by radical-mediated photocrosslinking facilitate previously inaccessible single-photon patterning applications, suggesting new opportunities for radical-mediated maleimide photocrosslinking as a viable tool for high-resolution fabrication of spatiotemporally controlled microscale cellular environments.