Design And Synthesis Of Tyrosinase Fluorescent Probes And Their Biological Effects

Melanoma is one of the most dangerous skin cancer and it is a malignant tumor developed from cells containing melanocytes.Current methods for the diagnosis of melanoma include: histopathological examination,imaging examination(including ultrasound imaging,CT,MRI and PET-CT examination);immunoenzymatic labeling and indirect immunofluorescent labeling of anti-human melanoma serum.The first two methods need to be discovered after the onset of cancer,and the latter two methods are auxiliary methods with low sensitivity.To reduce the threat of melanoma to human health,early diagnosis is very important.The National Comprehensive Cancer Network of the US points out that “early diagnosis is saving lives”.Therefore,early diagnosis of melanoma is of great significance.Tyrosinase is a copper-containing enzyme that can catalyze the conversion of tyrosine or tyrosine analogues to their corresponding catechol and eventually oxidize to quinone.Since tyrosinase is overexpressed in melanoma cells and its activity is positively correlated with the melanoma cell's malignant invasive ability,tyrosinase is considered a biomarker for melanoma cancer cells.The study of highly sensitive and selective tyrosinase detection probes has very important theoretical and potential application value for early detection and diagnosis of melanoma.In this research work,we first explored the relationship between the structure and fluorescence properties of the coumarin-based tyrosinase fluorescent probe and optimized a new type of highly sensitive and selective near-infrared diagnostic and killing integrated tyrosinase probe,and its biological effects research.The main research content is as follows:Based on tyrosinase structure,coumarin fluorescent probes and prodrug design concepts.The 7-amino-4-trifluoromethyl coumarin,which has a smaller molecular weight and a longer emission wavelength,as a fluorescent group.Four tyrosine fluorescent probes(F1~F4)were designed and synthesized by attaching different enzyme recognition structures to the coumarin.This type of probes was screened for its ability to react with tyrosinase at the molecular level and two probes(F2,F4)were filtered.Subsequently,the anti-interference ability,temperature sensitivity,and pH sensitivity of the probes F2 and F4 were studied,and high-sensitivity,highly selective TYR probe F2 was successfully screened.The Gibbs free energy in the spontaneous hydrolysis of a series of urea compounds was studied by molecular thermodynamic calculations.It was found that the free energy released during the spontaneous hydrolysis of the diphenylurea compound was the largest.Based on this,a novel tyrosinase fluorescent probe,Res1,was designed using resorufin as a fluorescent group.The experimental results at the molecular level indicate that Res1 not only has high sensitivity and selectivity but also has a fast tyrosinase reaction rate in aqueous solution.Experimental results at the cellular level indicate that Res1 is able to selectively measure tyrosinase overexpressing melanoma cells but has a longer effect time.To overcome the shortcomings of resorufin TYR probes,phenothiazine fluorescent masters were used to synthesize a TYR probe MB1 with near-infrared excitation/emission and diagnostic and killing functions.The results at the molecular level experiences indicate that MB1 not only has a high sensitivity and selectivity but also has a fast tyrosinase reaction rate in aqueous solution.Cell level experiments showed that MB1 can selectively detect tyrosinase overexpressing melanoma cells with a short reaction time and can generate singlet oxygen selective killing of melanoma cells under 655 nm laser irradiation.