Incorporation of the trifunctional reactive group described above will be dependent on the availability of a branching point in the ligand that can easily be modified and that is not crucial for binding to the target protein, which could be a potential downside. Probes can be readily prepared by appending different versions of the reported trifunctional reagents onto the scaffold. However, since each individual library compound is generally purified prior to screening, the throughput of the approach is hampered. Preferably, the probes are prepared in such a way that allows the immediate screening of reaction mixtures on small scales, as this would create opportunities for the rapid synthesis and screening of probe libraries in well plates. The research described in this thesis has been carried out in search of such approaches. The search began with the development of hydrazone-based modular chemical probes, inspired by the work of Hamachi and colleagues (Chapter 2).81 A panel of aldehyde-derived reactive groups could be combined with α-nucleophile-containing ligands to form hydrazone probes. Due to the bioorthogonal and clean nature of the hydrazone chemistry, the probe reaction mixtures could be screened directly on a set of proteins of interest. While the approach worked well, it was reasoned that it could be improved upon by utilizing faster reacting and more reversible hydrazone chemistry. A literature search identified the reaction between α-nucleophiles and 2-formylphenylboronic acid (2-FPBA) to form iminoboronates as meeting both requirements (Chapter 3). 2-FPBA-derived reactive groups were synthesized and it was shown that, indeed, this chemistry met both criteria (Chapter 4).
The modular probes could be formed rapidly and were used successfully to label proteins of interest in cell lysates and on cell membranes. The reversibility of the iminoboronate linker could be exploited to introduce reporter groups via a transimination reaction, showing that the linker served a dual purpose. A careful study of the α-nucleophiles involved in the probe formation and in the transimination reaction then resulted in a further optimized labeling protocol (Chapter 5). The potential of the iminoboronate probes was demonstrated by screening a large set of hydrazone probes in E. coli and HEK293 cell lysate, revealing distinct labeling profiles of several of the probes. Alongside the transimination reaction, the palladium-catalyzed Suzuki cross-coupling reaction was
also investigated as a means to read-out iminoboronate probe labeling (Chapter 6). While successful on a model protein, the Suzuki reaction did not prove to be applicable in the presence of cell lysate. Finally, the research journey described in this thesis is summarized and its place within the field of modular probe approaches, as well as its future potential, is reflected upon. (Chapter 7).
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