Construction Of Fluorescent Probes And The Detection Of Amino Acids In The Application

BackgroundWith the deepening of amino acid research,we found that the biological activity of some amino acids and their metabolites in vivo has a regulatory effect on nutrient metabolism and immune function.According to our previous studies,arginine plays an important role in cell division,immune system function and hormone release,especially gene regulation of glycoproteins.Therefore,selective recognition of arginine is crucial in biochemistry and medicine.In addition,normal levels of Cys?30-200?M?are a major source of sulfides for the synthesis of various proteins,antioxidant glutathione?GSH?,and the body's metabolism.However,too much or too little quantities of cysteine in body are associated with diseases,including Parkinson's disease,Alzheimer's disease and etc.Therefore,it is essential to understand the metabolic balance of cysteine in the body.ObjectiveBased on the Schiff base reaction,a new series of molecular probes containing naphthalene,coumarin and other fluorophores were constructed and characterized by the methods of IR,NMR,elemental analysis and mass spectrometry.The interactions between the molecular probes and common amino acids were studied by UV-vis spectroscopy,fluorescence spectroscopy and nuclear magnetic resonance titration.The fluorescence probes were selected which showed high sensitivity and strong selectivity for arginine and cysteine.A new detection method was provided for their detection.To explore the specific binding of fluorescent molecular probes with MCF-7 and Hela cancer cells,and preliminary assessment of their cytotoxicity which can provide scientific basis for the further clinical application of the molecular probes.Methods1.Synthesis of Molecular Probes:In this experiment,fluorescent probes were prepared based on fluorophores such as naphthalene and coumarin,and characterized by IR,¹H NMR,elemental analysis and mass spectrometry.2.In vitro detection of amino acids:The interaction of series of molecular probes and biological important amino acids?Cysteine,Lysine,tryptophan,Methionine,Threonine,Leucine,Isoleucine,Valine,Histidine,Arginine,Tyrosine,Glycine,Alanine,Glutamic Acid,Aspartic Acid,Amino Acid,Serine?were determined in the physiological environment by UV-vis absorption spectroscopy,nuclear magnetic resonance and fluorescence emission spectroscopy.The binding constants of the host and guest were calculated according to the non-linear least squares curve fitting.Then the fluorescent probes were selected in order to show high selectivity and high sensitivity for Arginine or cysteine.The effects of different substituents on the stability of host-guest complexes were compared in order to analyze the possible mechanism of host-guest interaction from the perspective of optics.3.Molecular probes in vitro cytotoxicity experiments:In the experiment,the cells used were cervical cancer cells and breast cancer cells,adjusted cell concentration of 5×10⁶ inoculated into 96-well plates.After cell attachment,samples were diluted to a concentration gradient of 5-200 g·mL^-1 and then added to the medium.After culturing for24 h,the medium was replaced by fresh medium.Then,MTT solution(20?L,5 mg·mL^-1)was added to each well for another 4 h.After that,the old medium was removed and then the DMSO solvent?150?L?was added.Finally,the absorbance of the cells was measured at 490 nm with a blank cell culture medium control with a microplate reader.The cell viability information was conveyed by the ratio of the absorbance of the medium cells in the sample group and the blank group.Studies indicated that the molecular probe had a good inhibitory effect on cancer cells.Results1.The chromophore NO₂,-OH,benzene and naphthalene group were successful introduced in the molecular structures.The selective recognition of amino acids became more convenient and improved the sensitivity of recogntion due to the combination of photophysical and photochemical properties with ultraviolet-visible spectra,fluorescent spectra and nuclear magnetic resonance?NMR?method.In synthesized fluorescent probes,the hydrazine derivatives containing chromophore NO₂ showed high sensitivity and selectivity for Arg.While the coumarin derivatives showed high sensitivity and selectivity for Cys accompanied with the obvious color changes in the process of host-guest interactions.2.The interfered experiments using UV-vis absorption spectra showed that the strong binding ability of some fluorescent probes for Arg or Cys was not influenced by the existence of other amino acids.The application of amines and coumarin molecular procbes was achieved in neutral conditions.The application ranges of amines and coumarin molecular probes were also broadened.3.The fluorescent probes were applied to MCF-7 cells and Hela cells for preliminary evaluation of cytotoxicity.It was found that fluorescein and coumarin fluorescent probes containing the chromophoric group NO₂ had a good inhibitory effect on cancer cells.Conclusion1.Eleven kinds of fluorescent probes were synthesized based on naphthalene and coumarin derivatives.2.Five kinds of fluorescent molecular probes for detecting arginine or cysteine with good binding ability,good fluorescence response and high selectivity were selected by UV-vis absorption spectroscopy,fluorescence emission spectroscopy and nuclear magnetic titration.3.The cytotoxicity assay showed that the above five fluorescent probes had a good inhibitory effect on cancer cells,providing potential value for clinical application.