Every project has a moment when the first domino falls. For this story, that moment did not happen in the laboratory. It started with a comment after a scientific presentation. At the time, we were working on covalent drug discovery and discussing different electrophilic warheads used to target cysteine residues in proteins. After one of our presentations, a colleague raised an interesting question about whether a simple oxygen-to-sulfur exchange could be used to tune covalent reactivity. It was a short discussion, but it stayed with us. Could such a small chemical modification have a meaningful impact on covalent ligand design? That question then became the starting point of our recent study.
As often happens in science, our first idea was not the one that succeeded. Initially, we focused on thioacrylamides. Based on chemical principles, they appeared to be promising candidates. However, reality quickly reminded us that good ideas on paper do not always translate into practical chemistry. The oxygen-to-sulfur exchange increased electrophilic reactivity dramatically, making most of the compounds difficult to isolate and therefore to characterize. For a while, it felt like we were turning into a dead end street. The gamechanger point came when we shifted our attention to a different warhead class: haloacetamides. This decision changed everything. Once we started preparing the corresponding thioamide analogues, the picture became much clearer. The compounds remained chemically tractable, but their reactivity increased substantially. One observation was particularly exciting. Fluoroacetamides are generally considered very weak covalent warheads and are often regarded as practically inactive in many contexts. We began to wonder whether oxygen-to-sulfur exchange could effectively "switch on" their reactivity. When the first fluorothioacetamide data arrived, it became obvious that created something really valuable chemical methodology hopefully useful for covalent drug discovery. At that point, the project evolved from a chemistry exercise into a broader question. Instead of asking whether oxygen-to-sulfur exchange changes reactivity, we started asking how broadly this strategy could be applied. Could it improve targeted covalent inhibitors? Could it be useful for bioconjugation? Could it reveal new biological targets through chemoproteomics? The more experiments we performed, the more applications emerged. The first focus area was the field of targeted covalent inhibitors. We demonstrated that oxygen-to-sulfur exchange could be used as a late-stage optimization strategy for covalent ligand development. This enabled the design of new JAK3 inhibitors and modified BTK inhibitors derived from ibrutinib, showing that minor changes in electrophile structure can have major effects on covalent engagement. The second story explored bioconjugation chemistry. Here, we demonstrated efficient antibody modification using thioamide-derived electrophiles, highlighting potential applications in antibody-drug conjugate development and other protein-labeling technologies. The third story moved into chemical biology and chemoproteomics. By developing alkyne-tagged probes and profiling them across the proteome, we discovered that chlorothioacetamides labeled a distinct subset of the proteome compared with traditional iodoacetamide-based approach. Among the newly identified targets was PDE6δ, a protein closely connected to KRAS biology through its role in solubilizing prenylated RAS-proteins. To our knowledge, this study reports the first cysteine-targeting covalent PDE6δ ligand with experimentally confirmed functional consequences resulting from binding within the farnesyl-binding pocket.
Like many projects, however, the published paper is only part of the story. We have to mention that the peer-review process significantly strengthened the work. While revisions are often viewed as an obstacle, in this case they became an opportunity to make the study much more complete. Several reviewer suggestions led to important additions of the scientific content. We expanded our proteomic analyses to investigate residue-level selectivity across the proteome. We performed detailed kinetic characterization of the antibody conjugation strategy and evaluated the stability of the resulting antibody-fluorophore conjugates under physiologically relevant conditions. We also carried out deeper computational and experimental investigations of targeted covalent inhibitor performance, particularly for the ibrutinib-derived compounds. Finally, the PDE6δ story matured considerably through additional validation experiments. Looking back, many of these additions became some of the most valuable parts of the final manuscript.
This project also highlights the importance of scientific collaboration. This study was performed together with colleagues from the Weizmann Institute of Sciences in Rehovot, Israel. Despite the exceptionally difficult period in the region and uncertainty surrounding them, our collaborators remained committed to science, and we are deeply grateful for their efforts.
In the end, the central message of the paper is surprisingly simple. Sometimes a very small chemical change can have a very powerful scientific impact. Replacing a single oxygen atom with sulfur transformed weak or conventional electrophiles into a family of warheads with distinct reactivity profiles and broad applicability. Beyond the examples presented in the paper, we believe oxygen-to-sulfur exchange can become a useful strategy for tuning covalent reactivity during probe and drug development.
What began as a brief conference comment ultimately developed into a multidisciplinary project spanning synthetic chemistry, covalent drug discovery, bioconjugation, proteomics, and chemical biology. Not a bad outcome for a single oxygen-to-sulfur exchange…