Preparation And Gas Sensing Properties Of Core-substituted Perylenediimide

Objective:Indocyanine dyes are a kind of near infrared fluorescent dyes,which possess thecharacter of high quantum yield?molar extinction coefficient?multi-conjugate structure?high signal-to-noise?wide spectral range and nonradioactive et al.So they had been applied to multiple fields,including gene detection?protein measurement?tumor targeted therapy?photodynamic therapy?fluorescence probe and so on.With the development of biotechnology,the synthesized indocyanine dyes had not meet the needs of the application in terms of quality and quantity.Therefore,the study of the outstanding characteristics of indocyanine dyes was an important link in biomedical field.This paper designed and synthesized a new series of symmetric pentamethine cyanine dyes,whose“N”atoms in the indol were linked by nonionic hydrophilic group with long ether chain?PEG?.Then the efficiency of synthetic route?convenient separation and purification method?optical properties and biological applications were researched.Because of the introduction of nonionic hydrophilic group,we expect that these novel symmetric pentamethine cyanine dyes should be better perfected than the earlier dyes.Methods:With formic acid of benzene methyl bromide,4-sulfonic acid hydrazine,3-methyl-2-butanone,3,5-two hydroxy benzoic acid methyl ester,poly triethylene glycol,methyl benzene sulfonyl chloride as starting materials,using“hemi-cyanine”or“one-step”synthesis route,a pentamethine cyanine dyes?Cy5-668?and a Trimethine cyanine dye were obtained,which were purified by C18 silica gel column separation,and two kinds of symmetrical pentamethine cyanine dyes?Cy5-666-1and Cy5-666-2?were obtained,which were purified by methyl-aether recrystallization.The optical properities of these dyes were measured by fluorescence spectrophotometer and ultraviolet-visible detector.The light stability of the dyes were determinated through iodine-tungsten lamp irradiation.The fluorescence spectra and ultraviolet spectra of the dyes Cy5-666-1 and Cy5-668 were measured under different pH conditions.The dye-protein labeling rate of the four kinds of dyes were calculated though marking bovine serum albumin?BSA?.Respectively,Cy5-666-1 and Cy5-666-2 stained the living cells and the fixed cells,then the images of the cells were observed under fluorescence microscope.Results:The four kinds of novel water-soluble fluorescent dyes were synthesized Via thesynthesis of fifteen intermediates.The structure of the target dyes was identified by NMR and HRMS.The maximum absorption wavelength of Cy5-666-1 was 653 nm,the maximun emission wavelength was 666nm,the molar extinction coefficient was 1.5×10⁵/M^-1cm^-1,the fluorescence quantum yield was 0.093.The maximum absorption wavelength of Cy5-666-2 was 649 nm,the maximun emission wavelength was 666nm,the molar extinction coefficient was 1.4×10⁵/M^-1cm^-1,the fluorescence quantum yield was 0.82.The maximum absorption wavelength of Cy5-668 was 647 nm,the maximun emission wavelength was 668 nm,the molar extinction coefficient was 1.4×10⁵/M^-1cm^-1,the fluorescence quantum yield was 0.09.The maximum absorption wavelength of Cy3-569 was 554nm,the maximun emission wavelength was 569 nm,the molar extinction coefficient were 0.9×10⁵/M^-1cm^-1,the fluorescence quantum yield was 0.07.The four kinds of dyes have higher photostability than the reference dye,whose stability order was Cy3>Cy5-666-1>Cy5-666-2>Cy5-668.The different fluorescence intensity of Cy5-666-1 was present under diverse pH condition.When the pH value was about 7.2,its fluorescence intensity reach maximum,and but the fluorescence intensity decreased in strong acid and strong alkaline.Cy5-668 also present different spectral properties.under the condition of acid and weak base,tis fluorescence intensity was almost unchanged,and the fluorescence decreased under the conditions of alkalines.Especially,when the pH value reached 13,its fluorescence intensity of the dye sharply declined.We marked bovine serum albumin?BSA?using the four activated-dyes ester in different D/P ratio.The research results were shown below.The protein labeling rate of Cy5-666-1?Cy5-666-2?Cy3-569 and Cy5-668 were respectively 1.35?1.26?1.13 and 1.16,when n?dye?:n?BSA?was equal to 5:1.The protein labeling rate of Cy5-666-1?Cy5-666-2?Cy3-569 and Cy5-668 were respectively 1.89?1.76?1.59 and 1.61,when n?dye?:n?BSA?was equal to 10:1.The protein labeling rate of Cy5-666-1?Cy5-666-2?Cy3-569 and Cy5-668 were respectively 2.23?2.21?2.02 and 2.04,when n?dye?:n?BSA?was equal to 15:1.There was a significant difference in the staining of fixed cells and living cells by Cy5-666-1.The image of staining fixed cells showed that the dye passed through the cell membrane,and entered into the cytoplasm and nucleus,then stained the whole cell.On the contrary,the image of staining living cells showed the dye gathered on the cell membrane,and a small amount of which passed into the cytoplasm.So the outline of the periphery of the cell was clearly stained.as a result,living cells and fixed cells could be significantly distinguished through dyed.Conclusions:It was a meaningful work to introduce two nonionic hydrophilic groups with PEG long chain ether in the target dyes,because its synthetic procedure was reduced and its separation andpurification difficulty was decreased.The target dyes had good water solubility,high fluorescence quantum yield,and high extinction coefficient.Compared with the Cy5-668 without the above groups,the noveldyes had better light stability and better D/P rate of labeled bovine serum albumin.They could effectively stain the living cells and the fixed cells,and had significant differences between both cell imaging.Finally,the novel symmetric pentamethine cyanine dyes would have wide application prospect in protein markers and immunofluorescence biomedical research field.