Design and characterisation of photoactivatable and lysine reactive <i>o</i> -nitrobenzyl alcohol-based crosslinkers

Photoreactive groups are invaluable tools in structural proteomics, offering reagent-free activation and temporal control of protein labelling. However, traditional UV-activatable functional groups often produce unstable intermediates and diverse products, making these chemistries difficult to deploy at scale. In this study, we performed a systematic analysis of <i>ortho</i>-nitrobenzyl alcohol (<i>o</i>NBA) reactivity for integration into novel reagents for chemical crosslinking-mass spectrometry. <i>o</i>NBA photochemistry represents a promising alternative to traditional photoactivatable crosslinkers due to its unique specificity towards lysine residues. Here, we synthesised two molecules containing <i>o</i>NBA functional groups with different substituents and assessed their labelling efficiency against a model protein. To ensure high labelling yields while maintaining a short irradiation time, we constructed a high power 365 nm irradiation device which improves the efficiency of <i>o</i>NBA photolysis. Our studies identified an amide-substituted probe that labels proteins with high efficiency. We next incorporated this optimised <i>o</i>NBA moiety into a homo-bifunctional crosslinker and a hetero-bifunctional crosslinker in combination with an NHS ester, which both resulted in high yields of crosslinked products. Our findings highlight that optimised <i>o</i>NBA-based reactive groups are viable UV-activated warheads that can deliver high labelling yields and efficient protein crosslinking, unlocking a wealth of potential structural proteomics applications.