Green Fluorescent Protein Inspired Emission Systems: Construction And Spectroscopic Study

The green fluorescent protein?GFP?has been widely used in cell and molecular biology because of its unique optical property.By chemical synthesis,the development of fluorescent materials mimicking GFP is of important scientific significance.Over past ten years,a variety of methods for the synthesis of GFP chromophore?GFPc?and its analogs have been successfully developed,resulting in the facile preparation of a series of model compounds.The corresponding optical characterizations promote the understanding of the photophysics of GFP,which further induces the generation of numerous physical and chemical methods to recover their fluorescence.However,the construction of emission systems mimicking GFP is still a great challenge.In this dissertation,inspired by GFP,we have developed a series of novel emission systems mimicking GFP,investigated their optical properties systematically,and studied their potential applications.This dissertation can be divided into seven chapters,and the details and key conclusions are described as follows:1.Self-restricted green fluorescent protein chromophore analogsThe confinement effect of the?-barrel structure provides a confined environment for GFPc,making fluorescence as the primary pathway of energy release.Once the?-barrel is broken,GFPc loses its fluorescence.Thus,enhancing the emission response of GFPc in fluid solvents and achieving multicolor fluorescence is still a great challenge.In this work,inspired by GFP,we try to produce a restricted effect which mimics the confinement effect of the?-barrel by the chromophore itself,termed as self-restricted effect,which would greatly improve the emission performance in solutions at the molecular level.By systematically tailoring the GFP core,a family of self-restricted GFPc analogs are found to be highly emissive and show remarkable solvatofluorochromism,creating a color palette across blue to yellow with a maximum spectral shift of 98 nm.More importantly,the fluorescence QY and lifetime increase gradually with enhancing solvent polarity in aprotic solvents.Theoretical calculations demonstrate that the self-restricted effect originates from the strong electron-donating 2,5-disubstitution,illustrating the mechanism relying on inhibition of free rotation around the exocyclic CC bonds.Moreover,the results of theoretical calculations further reveal that the electrostatic interaction between the solvent and the imidazolinone heterocycle oxygen can contribute to suppress the radiationless decay channel around the exocyclic C=C double bond.2.Synthesis and spectroscopic study of emission enhanced azalactone chromophoresOver past years,the investigation of GFP azalactone chromophores were ignored due to the loss of their fluorescence.Thus,enhancing the emission responses of azalactone chromophores and achieving multicolor fluorescence at the molecular level are of great importance.In this work,by increasing the conjugation length and utilizing self-restricted effect,we have prepared two azalactone chromophores,2,5-MeOBDO and PDO,and further studied their optical properties.Firstly,by increasing the conjugation length with pyrene,PDO is synthesized.In solutions,the fluorescence of PDO is enhanced a little bit and the QY decreases with the increasing of solvent polarity.From Hex to MeOH,the emission peak shift of PDO is relatively small.Thus,2,5-MeOBDO was prepared by constructing of self-restricted effect.Certainly,the self-restricted 2,5-MeOBDO shows enhanced emission and remarkable solvatofluorochromism,creating a color palette across blue to yellow.In aprotic solvents,the QYs all exceed 20%with the largest of 30.7%recorded in DMSO.Definitely,self-restricted 2,5-MeOBDO has much better fluorescence than PDO.So,we have selected 2,5-MeOBDO and further investigated the effect of H-bonding and aggregation on its fluorescent property.Finally,we try to detect dopamine through the absorption and emission spectra of 2,5-MeOBDO.3.Multicolor cell imaging under identical excitation conditions with salicylideneaniline analogue-based fluorescent nanoparticlesThrough the inhibition of chromophore free rotation by the?-barrel structure,GFP emits intense green fluorescence through excited-state proton transfer?ESPT?.However,it is still a great challenge for the facile construction of highly fluorescent emission system by defined chromophores,which exhibit fluorescence due to the inhibition of molecular rotation and ESPT.In this work,salicylideneaniline?SA?analogues were facilely prepared by condensation reaction with high yields?>95%?and achieved enhanced fluorescence through self-assembly into nanoparticles,which were further applied for single-excitation multicolor cell imaging.In mixed solvents,the fluorescence of SA analogs was enhanced by restriction of molecular rotation after the formation of H-or J-aggregates.Benefiting from ESIPT,green,yellow and orange fluorescent nanoparticles were easily obtained with large Stokes shifts.Surprisingly,green fluorescent nanoparticles emitted green-yellow fluorescence with the transformation of H-aggregates to J-aggregates.Connected by C=N bonds,multicolor fluorescent nanoparticles were pH-responsive with rapid loss of fluorescence under acid condition.Moreover,the two-photon fluorescence of these nanoparticles was confirmed for the first time.By combination with phospholipids,multicolor fluorescent nanoparticles showed low cytotoxicity and good water solubility,and were applied for single-excitation multicolor cell imaging.4.Green fluorescent protein inspired fluorescent polymersUntil now,studies about GFP and GFPc analogs all confine to biological and organic fields,ignoring the possible combination with synthetic polymers.GFP is a polypeptide-based biomacromolecule,which shows excellent fluorescence in its unique way.By employing macromolecular assembly strategy,the extension of GFPc study into polymeric research field is of great academic value and scientific significance.Therefore,in this work,two fluorescent polymers mimicking GFP,PEG-c-PMMA and PEG-c-PCL,have been prepared by atom transfer radical polymerization?ATRP?and ring-opening polymerization,respectively.In organic solvents,macromolecular strategy shows little influence on their optical properties.After self-assembly into complex micelles,the emission responses of fluorescent polymers have been greatly enhanced with obvious bathochromic shift,which can be attributed to the inhibition of chromophore's free rotation and the enhanced interactions between chromophores and polymer chains.Finally,the emission enhanced GFP-inspired fluorescent polymers were successfully applied in cell imaging,revealing the potential application of this kind of novel fluorescent polymers in cell biology.5.Multicolor fluorescent polymers inspired from green fluorescent proteinGFP variants,together with other identified fluorescent proteins,form a color palette covering the entire visible spectrum,providing a powerful toolkit for multicolor fluorescence labeling.The design and fabrication of multicolor fluorescent polymers mimicking GFP becomes very attractive and is also a great challenge.In this work,by combination of chemical modification and macromolecular assembly strategy,multicolor fluorescent polymers with enhanced fluorescence were developed and successfully applied for single-excitation cell imaging.Through ATRP and click reaction,fluorescent polymers,PEG-c₀-PMMA,with different PMMA chain length were synthesized.After assembly into micelles,their fluorescence was enhanced and further increased with the elongation of PMMA chain length,attributing to the inhibition of chromophore's free rotation and the segmentation effect of polymer chains.Further chemically tailoring the core chromophore,multicolor fluorescent polymers were fabricated through the elongation of conjugation length and the utilization of excited-state intramolecular proton transfer?ESIPT?.Under the same excitation conditions,multicolor fluorescent polymers exhibit a spectrum of color from blue to orange with the maximum Stokes shift of 202 nm.Moreover,the maximum fluorescence quantum yield?QY?reaches 8%,which is more than 80-fold larger than that of the core chromophore.Besides,fluorescent emission color can be effectively regulated by tuning the coassembling constitution of green and orange fluorescent polymers.Finally,multicolor fluorescent polymers were applied to single-excitation cell imaging,which would improve the accuracy and reliability of biological analysis.