Development of analyte-responsive diagnostic and theranostic 1,2-dioxetane-based chemiluminescent probes for cancer cell imaging

A ground-breaking milestone in dioxetane-based chemiluminescent probes was achieved by inventing of triggerable phenoxy 1,2 dioxetane derivatives by Schaap and co-workers in 1987. In the mechanism of chemiluminescence action, initially, a cleavable substrate (also known as a cage group), which is masking the phenol, is released by a biologically relevant analyte such as enzymes, small biomolecules, and unstable phenolate-dioxetane is generated. This intermediate decomposition is utilized through a chemiexcitation process to an excited intermediate benzoate ester, emitting light when the excited intermediate relaxes back to its ground state. Thus, chemiluminescent molecules emit light without external light irradiation. However, Schaap's chemiluminescence probes do not have sufficient emission in an aqueous medium, which is critical for bioimaging applications. The amplification of the chemiluminescent signal was obtained by adding surfactant and conjugation of fluorescent dye in various studies. For the direct enhancement, the intrinsic emissive character of the excited benzoate ester was required to be enhanced. In 2017, Shabat and co-workers obtained this adequate amplification in the chemiluminescent-based signal through the addition of electron-withdrawing (EWG) groups on benzoate esters. They have selected methyl acrylate, acrylonitrile, and acrylic acid as EWG substituents. In the case of luminophore decorated with a methyl acrylate and an ortho chlorine substitution, a 780-fold increase in the emission signal has been obtained. The acrylonitrile substitution on the same core bearing chlorine provided 3043-fold stronger emission than the probe without an EWG unit. Throughout the years, various analyte-responsive chemiluminescent probes have been incorporated for bioimaging. Besides the imaging modalities, the chemiluminescent-based agent has begun to be used in theranostic systems. John Funkhouser introduced the term in 1998 theranostics, and these agents consist of imaging and therapeutic agent with a cleavable linker and targeting group. Thus, these agents facilitate diagnosis and therapy simultaneously to enhance pharmacokinetics and the systematic administration of the therapeutic agents by specifically addressing cancerous cells and decreasing the undesirable outcomes of traditional chemotherapy modalities. In the first part of this thesis work, a leucine aminopeptidase (LAP) activatable chemiluminescent probe (CL-LAP) with ortho chlorine and methyl acrylate substituents has been synthesized. In the cell-free experiments, a chemiluminescence signal with a strong intensity has been detected only in the presence of LAP enzyme (15 ng/mL). After demonstrating selective activation of the probe with LAP, we imaged endogenous LAP activity in cancer cells. Secondly, carboxylesterase 2 (CES2) activatable chemiluminescent probe (CL-CES2) with ortho chlorine and acrylonitrile substituents has been synthesized. In the cell-free experiments, a chemiluminescence signal with strong intensity has been detected only in the presence of CES2 enzyme (55 ng/mL). After demonstrating selective activation of the probe with CES2, we imaged endogenous CES2 activity in cancer cells. In the second part of the thesis, we reported the synthesis of a glutathione (GSH) activatable theranostic probe (CL-SS-CBL), including 1,2 dioxetane-based chemiluminescence reporter and chlorambucil (CBL) chemotherapy drug that are conjugated with a cleavable disulfide bond.