Fluorescent Protein Chromophore Analogs:Multi-step Synthesis,Fluorescent Probe And Application In Photodynamic Therapy

Green fluorescent protein（GFP）was originally isolated from the jellyfish Aequorea victoria.With its unique optical properties,GFP is often used for fluorescent labeling,and has been widely used in cell biology and molecular biology.Therefore,it has attracted particular attention.It is of great scientific significance to prepare fluorescent dyes or biomaterials with optical properties similar to GFP by chemical synthesis rather than genetic engineering.However,how to realize this process,how to simulate the luminescence principle of GFP,and how to explore its application are still huge problems and challenges.In this thesis,three novel luminescence systems with optical properties similar to GFP were designed and synthesized,systematically studied their optical properties,and preliminarily explored their applications in the field of cell viscosity probe,reactive oxygen species HOCl probe and photodynamic therapy.This paper is divided into five chapters.The specific research content and conclusions are summarized as follows:Chapter 1.Introduction.First,it briefly introduces the scientific background,basic structure,luminescence principle and optical properties of GFP.Secondly,starting from its special optical properties,many applications of its chemical modulation of chromophores are extended.Thirdly,for the markers related to disease such as cell viscosity and reactive oxygen species HOCl,their working mechanism and current research status are introduced.Finally,the mechanism of photodynamic therapy is introduced,and the types and research status of photosensitizers based on organic small molecule fluorescent dyes are reviewed.The problems in these three practical applications are listed,and corresponding solutions based on the GFP chromophore and its derivatives are proposed.Chapter 2:Introduce three common methods of synthesizing GFP chromophore and its derivatives.First,we analyzed their advantages and disadvantages in their synthesis,and chose the universal and convenient[2+3]cycloaddition synthesis method.Then we systematically explained the synthesis strategy of the[2+3]reaction,analyzed some of the drawbacks,and proposed a set of novel optimization strategies.The yield of the GFP chromophore and its derivatives obtained by the[2+3]cycloaddition synthesis method can reach more than 90%,which greatly improves the synthesis conversion rate and provides a prerequisite for future industrial mass production.Therefore,through the optimized[2+3]cycloaddition method,three novel GFP chromophores and their derivatives were synthesized,which were used as cell lysosomal viscosity probe and cell reactive oxygen species HOCl probe and photosensitizer for photodynamic therapy.A series of compounds synthesized in this chapter have been characterized by ¹H NMR,¹³C NMR,and HRMS.Chapter 3:A carbazolyl GFP chromophore derivative was synthesized through the[2+3]cycloaddition reaction,and then connected the morpholine-indole unit through the Knoevenagel condensation reaction to extend the conjugated structure of the molecule,and synthesized a sensitive viscosity probe Lys-Cz FP.In the probe structure,the carbazole and indole part give Lys-Cz FP greater molecular plane and viscosity sensitivity.The introduction of the lysosomal localization group morpholine realize the effect of lysosomal localization.At the same time,the electron-rich morpholine moiety weaken fluorescence of the GFP fluorophore via PET,which endue Lys-Cz FP with an acidic p H-activatable lysosome-targeting fluorescence characteristic that is conducive to eliminate the background fluorescence outside of lysosomes.Lys-Cz FP exhibits a strong viscosity dependence.With the viscosity increases,the fluorescence intensity at 560 nm increases by about 98 times within 5s;Φ_F increases from 0.003 to0.253;and the lifetime increases from 0.25 ns to 1.12 ns.Finally,Lys-Cz FP was used to realize the real-time detection of cell lysosomal changes.Chapter 4:A GFP chromophore was synthesized through the[2+3]cycloaddition reaction,and then used the Knoevenagel condensation reaction to extend the conjugated structure of the molecule,connect the phenothiazine unit,and convert the GFP chromophore.The probe RFP-Ptz was prepared as a small active oxygen molecule HOCl.Due to the special oxygen-chloride（O-Cl）bond formation mechanism,RFP-Ptz has good selectivity and specificity,Stokes shift～148 nm,and detection limit of 6.76×10^-7 M.In the presence of HOCl,the solution is accompanied by a significant change in color from yellow to red,the fluorescence quantum yield increases by 12times,and efficient"naked eye"recognition can be achieved within 5 s.Cell experiments show that RFP-Ptz had good biocompatibility and low cytotoxicity.Finally,RFP-Ptz was successfully applied to the imaging of exogenous HOCl in SGC-7901 cells.Chapter 5:The work of this chapter is based on the work of the previous chapters.We used the optimized synthesis method of Chapter 2 to synthesize the RFP-Ptz in Chapter 4.Then through the substitution reaction,the active hydroxyl site in RFP-Ptz was protected with ethylmorpholine.At the same time,the concept of molecular rotor in Chapter 3 was introduced,and a bifunctional GFP chromophore derivative Lys-Ptz FP for cell photodynamic therapy and lysosomal viscosity probe has been successfully synthesized.The photosensitizer Lys-Ptz FP shows high singlet oxygen quantum yield（Φ_Δ=0.42）and low fluorescence quantum yield（Φ_F=0.002）in Me OH.In addition,Lys-Ptz FP has a sensitive viscosity response with obvious near-infrared emission～672nm in glycerol solution.Lys-Ptz FP has good photostability,lysosomal localization characteristics,negligible dark toxicity.Phototoxicity data results show that the IC₅₀ value of the photosensitizer is 0.93μM,which causes most cells to be in apoptotic state.Finally,through the change of red field fluorescence intensity,we found that with the increase of intracellular reactive oxygen species,the lysosomal viscosity could also change synchronously during PDT-induced cell apoptosis.