Optimized synthesis routes and biological application of N-peptide-6-amino-D-luciferin conjugates

In the recent years, numerous in vivo and in vitro analytical methods, based on fluorescence and bioluminescence, have been developed for various biological objectives, including immunoassays, gene expression assays, bioimaging, investigation of infectious diseases etc. Plate based, high-throughput viability assays addressing the detection of protease activity is in the focus of intensive research. The advantage of bioluminescent systems over fluorescent ones lies in their superior sensitivity and easy handling. In the bioluminescent methods, diverse sets of luciferases and their substrates, luciferins have been applied in different cellular and animal models, the most ubiquitous enzyme-substrate system is the American firefly (Photinus pyralis) luciferin-luciferase system. Substituting the 6-position hydroxyl group of native luciferin with an amino group, the resulting 6-amino-D-luciferin (2-(6-aminobenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid, hereinafter: aLuc) can form an amide bond with a peptide, while retaining the transport and bioluminescent properties of the original substrate, resulting in a good substrate for different important proteases.Due to this feature, these conjugates can be used for the determination of the enzymatic activity the following way: the protease enzyme to be measured recognizes the peptide part of the conjugate with the suitable peptide sequence, then cleaves the amide bond between the peptide and the aLuc, thus aLuc is released, which, in the presence of luciferase enzyme, emits light.The activity of the given protease enzyme can be determined from the amount of emitted light, as the emitted light is directly proportional to the activity of the enzyme. Unfortunately, the synthesis methods of these substrates published so far are complicated; and the very few commercially available conjugates are very expensive. The aim of this dissertation was to introduce novel routes for the scalable and economical synthesis of N-peptide-aLuc conjugates. The preparative work focused on preparing a precursor (6-amino-2-cyanobenzothiazole, 3), two conjugates (N-Z-Asp-Glu-Val-Asp-aLuc, 6, N-Fmoc-Gly-Pro-aLuc, 8) and an intermediate (NBoc- aLuc, 10)