Alkyldiazirine Warheads for G-quadruplex Photoaffinity Labelling by N,O-alkylation on Thymine and Guanine Nucleobases

G-rich repetitive nucleic acid sequences can fold into G-quadruplexes (G4s) upon coordination with potassium ions (K+). Small natural and synthetic molecules (G4 ligands) can bind to and stabilize G4 structures in vitro, thereby interfering with biological processes involving G4-containing genes. Therefore, G4 ligands represent highly valuable tools for unveiling G4 biological functions. Together with non-covalent reversible G4 ligands, reactive compounds capable of forming covalent bonds with G4s upon thermal and photochemical activation represent a new class of promising G4 ligands with the potential to be harnessed for the identification and isolation of G4s from a biological environment. In particular, photoaffinity labelling (PAL) encompasses the potential to obtain spatial and temporal control over the alkylation event. However, to date, only a few examples of covalent G4 ligands for PAL approaches have been described, and very low alkylation yields plague all of them. Herein, the synthesis of new families of photoactivatable G4 ligands functionalized with different warheads (e.g., benzophenones and diazirines) is described, as well as their photoreactivity in the presence of three different G4s: 22AG, cMyc, and 25CEBwt. Among the different compounds, diazirine derivatives displayed the highest G4 selectivity and unprecedented high alkylation yields (up to 70%) together with a remarkable selective N- and O-alkylation reaction only on thymine and guanine nucleobases as identified by using a prototype ligand. These new compounds pave the way for the development of new methodologies, allowing for functional investigation and druggability of G4 in cells.