Functionalized Nitrobenzothiadiazoles as Embedded Fluorescent Probes

Described herein is the synthesis and photophysical characterization of novel dipeptidomimetic fluorogenic probes. The new dipeptidomimetic cassette is based on the environment-sensitive fluorophore nitrobenzothiadiazole (NBTD) and is designed to be embedded within a polypeptide backbone, not attached as part of a conformationally unrestrained amino acid side chain. Compounds 1-6 were prepared to investigate the effect of introducing a methyl group at positions 5 and 6 of the nitrobenzothiadiazole scaffold on their photophysical properties. Additionally, we investigated the effect of N-methylation of the 4-amino group on their chemical and photophysical properties. The nitrobenzothiadiazole scaffold was functionalized with amino- and carboxy-termini, resulting in a conformationally restricted dipeptidomimetic scaffold (NBTD-Gly) that can potentially be embedded within peptides or proteins of interest and used as a biophysical tool for studying protein structure and function. Thus, we investigated the synthesis and photophysical properties of functionalized nitrobenzothiadiazoles (1-11). The amino-terminus of the dipeptidomimetic scaffold (NBTD-Gly) was introduced at the benzylic position of the benzothiadiazole core using Gabriel synthesis, followed by nitration. The carboxy terminus was introduced via nucleophilic aromatic substitution as part of the glycine moiety. Subsequent steps involved the removal of a phthaloyl protecting group and a condensation reaction to form protected dipeptidomimetic analogues 7-11.