Isotopic labeling of organic compounds is crucial for advancements in life sciences and is extensively utilized in drug discovery. While isotopic labeling is widely applied to small molecules, research on isotopically labeled biologics such as peptides, antibodies, or enzymes remains less developed, despite the increasing prevalence of these larger biologics in the pharmaceutical industry as transformative treatments for patients. We report the development of an in situ prepared catalyst for Hydrogen Isotope Exchange reactions with deuterium or tritium gas as the isotope source to selectively label peptides in aqueous solutions. The method requires only low amounts of catalyst and provides the radiolabeled peptide in a single reaction step with sufficient specific activity for biological in-vitro experiments, paving the way for future developments and applications in biological based drugs studies. Furthermore, we have identified a substrate-mediated formation of the active catalytic species, leading to the high selectivity in this late-stage functionalization approach of complex peptide compounds. DFT calculations and NMR studies have identified the intermediate species involved. The reported insights accelerate the access to radiolabeled tracers to understand biological processes, as well as offer future opportunities for highly selective C-H activation and C-C or C-X bond formation to achieve highly selective late-stage functionalization products. Acknowledgements We thank Claudia Loewe for assistance with mass spectrometry and liquid scintillation analyses related to 3 H-labeling experiments. The authors want to thank Dr. Anika Tarasewicz (Sanofi Germany) for proofreading of the manuscript. Author contributions E.M. performed the experiments, synthesized and characterized the molecules, analyzed the data and discussed the results. R.W. performed the radioactive experiments. S.G. performed the DFT calculations. V.D. organized the funding, conceptualized and directed the project. All authors prepared the manuscript. E.M. is a participant in the EU ITN ISOBIOTICS consortium. The ISOBIOTICS project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No.101072780. Competing interestsAll authors are employees of Sanofi Germany and may hold shares or options of the company. Additional information Supplementary information The online version contains supplementary material available athttps://doi.org/10.1038/s41467-026-69850-x. Correspondence and requests for materials should be addressed to Volker Derdau.
Martinelli et al. (Wed,) studied this question.