Publication
The goal of my PhD was the development of a new method for unselective surface labeling, not substrate-specific, that would give access to direct visualization of any surface interactions. In order to achieve this challenging goal, we wanted to develop a general non-specific method for biomolecule surface modification. With this in mind, azide was especially good due to its biorthogonal post-functionalization potential. Due to the precedence in using hypervalent iodine reagents for azide transfer, they were the perfect entry point to this strategy. However, Zhdankin reagent, known for almost 20 years, turned out to be more hazardous than commonly accepted. After few incidents caused by small detonations, an accident occurred in our lab. At this point, the goal became to synthesize first a fine-tuned family of analogues in order to obtain the best compromise between stability and reactivity. Seventeen analogues were synthesized and complete safety studies as well as reactivity comparison were conducted on the most relevant among them. Two analogues were identified as good substitute for the Zhdankin reagent. Then, this library was applied to our main project: obtain a homogeneous amino acid azidation on protein surface. Co-working with Dr. Daniel Abegg and Anton Shuster, the more efficient compound was determined and reaction conditions were screened for both in vitro and ex vivo reactions. Unexpectedly, the best reagent was not the same for in vitro and ex vivo experiments. Our reagents applied for proteomics experiments uncovered the full potential of this new method. We were able to achieve and excellent surface coverage of proteins, allowing us to access numerous useful applications such as: protein dynamics visualization, quantitative protein-protein binding determination, protein-protein dynamics visualization, protein-antibody active site determination. Investigations on azidation site determination on single amino acids and small peptides, combined with proteomic experiments on native and digested proteins confirmed that our method was not suitable for small substrates. Therefore, precise azidation site determination on each amino acid has been inaccessible so far. In summary, the development and applications of a broader family of fine-tuned azide- containing hypervalent iodine reagents are described. Biomolecule labeling was successfully performed via a new process of unselective surface azidation and post-functionalization with clickable fluorophores, or linkers for enrichment and proteomic applications with mass spectrometry analyses. Therefore, our method has been called Protein Surface Azidation coupled with Mass Spectrometry: ProSurA-MS. The great strength of our method is the non-selectivity of the surface azidation, allowing a "coating" of proteins giving straightforward access to the sequences masked by protein-protein, small molecule-protein or even conformational changes. As this method has been demonstrated to be efficient for