Design, Synthesis And Application Of Fluorescent Probes For Detection Of Metabolizing Enzymes Activities

Metabolizing enzymes are not only the biochemical barrier for body's defense and removing exotic matter invasion, but also play a vital role in maintaining endogenous metabolism balance. Many scholars have been committed to the development of specific substrates for metabolic enzymes to detect the activity and function of metabolic enzymes in biological system. Compared with non-fluorescence substrates, fluorescence substrates get more and more attention for the advantage of sensitivity, fast operation, real-time monitoring, high-throughput screening and so on. While there are many different kinds of metabolizing enzymes in body, some of them share high homology in amino acid sequence, broad and overlapping substrate specificity, so very few fluorescent probes with metabolizing enzyme-specificity have been reported at present. In this paper, we develop some specific metabolizing enzyme reaction-based fluorescent probes to detect the activitives of carboxylesterase (subtype 1 and subtype 2), cytochrome P450 1A1 (CYP1A1) and human serum albumin (HSA), which provides tools for further exploring the function in the biological system and clinical individualized medication.(1) Carboxylesterase 1 (CEI), one of the most important carboxylesterase subtype in human body, catalyzes the hydrolysis of various endogenous and xenobiotic easer substrates and thus plays a key role in physiological and biochemical processes. In this part, a two-photon fluorescent probe (BABO) based on benzoxazole has been developed for the measurement of CEI activity. BABO exhibits good sensitivity (LOD= 0.04 ?g/mL) and excellent subtype-selectivity towards CEI over other human hydrolases, and has been successfully applied to the detection of real activity of CEI in complex biological samples such as tissue preparations. Furthermore, as a two-photon fluorescent probe, BABO has also been used for two-photon imaging of intracellular CEI in living cells and tissue slices, and shows increased penetration depth. These superior properties suggest that BABO is a good optical substrate for CEI, which could be applied to CEI-associated xenobiotic metabolism and drug discovery, and also serves as a promising imaging tool for real-time monitoring the expression and function of CEI in complex biological systems.(2) Carboxylesterase 2 (CE2), another important carboxylesterase subtype in human body, catalyzes a variety of precursor of chemotherapy drugs. In this part, a highly selective long-wavelength fluorescent probe TCFB based on 1-(3-cyano-2-dicyanomethylen-5, 5-dimethyl-2,5-dihydrofuran-4-yl)-2-(4-hydroxyl phenyl) ethane (TCF) has been developed for the detection of the activity of CE2. Both reaction phenotyping and chemical inhibition assays demonstrate that this probe is highly subtype-selective for CE2 over other human hydrolases. The probe can be used for real-time monitoring CE2 activity in complex biological systems with emission at long-wavelength (?em= 612 nm) and low autofluorescence from biological matrix.A ratiometric flourescent probe 3-AF derived from 3-hydroxyflavone (3-HF) based on ESIPT mechanism has been developed for the activity detection of CE2. Ratiometric measurements reduce the system error and the influence of the uneven distribution of probe concentration.3-AF is appropriate for detecting CE2 in complex system and provids tools for personalizing medicine in the future.(3) HSA has many physiological functions, its level in serum is a smart biomarker for occurrence and development of many disease. In this part, based on ICT mechanism, a novel near-infrared (?em= 660 nm) fluorescent probe DDAP has been developed for the activity detection of HSA based on its unique pseudo-esterase activity. The probe exhibits high sensitivity (LOD= 5.6 mg/L) and has been successfully applied to the measurement of HSA in diluted plasma and trace HSA secreted from human hepatocytes to cell culture supernatant. In addition, as an activity-based probe, DDAP can distinguish between native HSA and denatured HSA, but the currently used dye-binding method cannot, which is important for the detection of physiological activity of HSA.(4) CYP1A1, a member of the CYP1A subfamily, is of particular importance for human health due to its essential role in the biotransformation of procarcinogenic chemicals and toxins and it is also involved in the metabolism of many drugs. Herein, based on ICT mechanism, we develop a resorufin-derived fluorescent probe CHPO that allowed for activity detection of CYP1A1, CHPO has high subtype-selectivity for CYP1A1, excellent sensitivity (LOD= 0.073 nM) and high affinity with CYP1A1 (Km= 0.274 ± 0.039 ?M), CHPO can be used to evaluate CYP1A1 activity and function in human liver microsomal and is applied to the detection of CYP1A1 activity in a panel of 12 HLMs from different individuals, the difference is about 5.1 fold. This probe can promote to understand the functions of CYP1A1 in the process of physiology and pathology, laid a solid foundation for related theoretical research.