Introduction
In 1967, Huisgen reported the unique photochemical reactivity of tetrazoles, demonstrating their ability to undergo light-induced cycloaddition with alkenes.^[1] Subsequent investigations revealed their capacity to interact with biologically relevant molecules upon photoactivation, opening new avenues in chemical biology.^[2] Building on this foundation, we explored the application of photoactive tetrazoles for protein radiolabelling, leveraging their rapid reaction kinetics and high chemoselectivity to develop an efficient strategy for photoradiosynthesis and bioconjugation.

Methods
The study focused on the synthesis, radiolabelling, and bioconjugation of ¹⁷⁷Lu-DOTAGA to the monoclonal antibody onartuzumab using a PEG-functionalised photoactivatable tetrazole. Radiolabelling was performed at 80 °C for 15 minutes and monitored by radio-iTLC and radio-HPLC. Photoconjugation under mild aqueous conditions (pH 8, room temperature, 15 min) with 365 nm irradiation was first validated using human serum albumin (HSA), then applied to onartuzumab. Thiol-selective labelling was examined by comparing unmodified and Traut’s modified onartuzumab versus a control study using maleimide-blocked HSA. Following purification, the radiolabelled antibodies were evaluated in vivo by γ-scintigraphy and biodistribution studies in mice bearing MKN-45 xenografts. In parallel, PEG-tetrazole was synthesised and characterised by NMR, HPLC, and HR-MS. Its photochemical behavior was studied under 365 nm irradiation in aqueous-organic buffer with isolation of several key products. Reactivity with model nucleophiles was monitored by UV-Vis, fluorescence, HPLC, and HR-MS, highlighting selectivity toward thiol-containing species at pH 10.5.

Results and Discussion

Radiochemical studies showed high conjugation yields (83–86%) of the ¹⁷⁷Lu-labelled tetrazole complex with HSA. In contrast, substantially lower yields (25–30%) were obtained with blocked HSA or with native MetMAb, attributed to the lack of available thiol groups on these proteins. Following thiol functionalisation with Traut’s reagent, a marked increase in conjugation efficiency was observed, underscoring the critical role of sulfhydryl groups in enabling efficient and selective bioconjugation.
To investigate the underlying mechanism, photochemical studies were performed by using the isolated PEG-tetrazole. Upon irradiation at 365 nm, the compound underwent photolysis with nitrogen release, forming a highly nitrile imine intermediate, which, in  photolysis experiments in buffered solution revealed a clear kinetic preference for thiol nucleophiles. Mechanistic insight was supported by UV/Vis, fluorescence, HPLC, and HRMS analyses, confirming the thiol-targeting nature of the reaction.

Conclusion
This work establishes the photoinduced reactivtiy of tetrazols with free sulfhydryl groups a robust and chemoselective platform for site-specific functionalisation and radiolabelling of proteins. This approach significantly enhances radiolabelling efficiency, particularly for monoclonal antibodies where thiol sites can be introduced in a controlled manner using establish chemical or enyzmatic processes . The methodology is compatible with clinically relevant radionuclides such as ⁶⁸Ga, ¹⁶¹Tb, and ¹⁷⁷Lu, and offers a versatile, modular tool for te development of precision radiopharmaceuticals.

[1] J. S. Clovis, A. Eckell, R. Huisgen, R. Sustmann, Chem. Ber. 1967, 100, 60 – 70.

[2] R. Fay, J. P. Holland, Chem. Eur. J. 2021, 27, 4893–4897.