Synthesis of Meso-Substituted Thiazole Orange and Their Biological Application

Author:Guan Li

Supervisor:wang lan ying


Degree Year:2016





The methine chain modification of cyanine dye can greatly change its structure,property and selectivity to biological species,and expand its application scope,which is the development trend of cyanine dye structure modification.There is only a few reports on the methylene functionalization of monocyanines,as a major member of the cyanine dyes family.However,these synthesis process were mainly started from the modification of the active methyl groups of the heterocyclic rings,which are multi-step synthesis,and more difficult to separate and purify.Therefore,in this dissertation,a simple and effective method for the direct synthesis of methylene-substituted thiazole orange analogues was developed,compounds and their structures,spectroscopic properties,stability,and cell-related studies and interactions with G-DNA were studied.The main contents are described as follows:1.The recent advances in the study of the chain modification of cyanine dyes is briefly reviewed.2.A novel method for the direct synthesis of substituted thiazole orange analogues on the methylenecholine chain has been developed,which was using thiazole orange as material by the electrophilic addition-elimination reaction.Twelve novel meso-benzyl substituted thiazole orange analogues(meso-TOs)were direct synthesized from thiazole orange(TOs:2TO,4TO)and benzyl alcohol derivatives under Lewis acid catalyst,the reaction pathway of meso-TOs was also proposed.The method is simple in operation,mild in reaction condition and high in yield.The products were identified by UV-Vis spectroscopy,HRMS,1H NMR,and 13C NMR.X-ray crystal structures showed that meso-TOs were twisted as a "Y"-shaped structure,in contrast to TO,which crystallized in the planar conformation.3.The spectroscopic properties of meso-TOs and parent TOs were tested in seven different solvents.The absorption maxima(λmax)of meso-2TOs and meso-4TOs were located in the range of 517~528 nm and 552~565 nm,respectively,the molar extinction coefficient were all above 104 L mol-1 cm-1.The emission maxima(λem)of meso-2TOs and meso-4TOs were located in the range of 607~621 nm and 637~643 nm,respectively.Compared with the parents,prepared new meso-TOs exhibit red shift of λmax and λem,and a larger Stokes shift.Addition of a benzyl group to the meso position methine of TOs inhibits the aggregation,leading to existence of the meso-TOs in the M form in various solvents.4.The photostability of meso-TOs and parent TOs were investigated in PBS buffer by UV-vis.After irradiatinon for 60 min,meso-2TOs and meso-4TOs exhibited less than 5%and 17%photofading,respectively,while 2TO and 4TO showed more than 15%and 50%photofading,respectively.In the presence of 1O2,after 60 min continuous reaction,meso-2TOs and meso-4TOs showed 6%-15%and 30%-37%fading,respectively,while 2TO and 4TO sharply bleached by 73%and 81%,respectively.From the above,incorporating a benzyl group at the meso position methine of parent TO exhibited superior stability.5.meso-CH3-2TO and two meso-Allyl-2TOs were direct synthesized from 2TO as the raw material,and paraformaldehyde or allyl alcohol derivatives as electrophile.Spectra study showed that the λmax of meso-CH3-2TO and meso-Allyl-2TOs were located in the range of 520~537 nm,the molar extinction coefficient were in the range of 1.75×104~3.58×104 L mol-1 cm-1,and their λem were located in the range of 578~588 nm.Compared with 2TO,the new dyes exhibit red shift of λmax and λem,and lower aggregation,existing in the M form in various solvents.6.The photostability of 2TO,meso-CH3-2TO and meso-Allyl-2TOs were investigated in PBS buffer by UV-vis.After irradiatinon for 60 min,meso-CH3-2TO and meso-Allyl-2TOs exhibited less than 4%photofading,while 2TO showed more than 15%photofading.In the presence of 1O2,after 60 min continuous reaction,meso-CH3-2TO and meso-Allyl-2TOs showed less than 20%fading,while 2TO bleached by 73%.From the above,incorporating a aliphatic chain at the meso position methine of 2TO improved their stability.7.TOs,meso-TOs,and meso-CH3-2TO were subjected to cell study(RCF and MCF-7 cells),including the cytotoxicity,cell staining and confocal images of living cells.To ensure the localization of dyes,costaining assays were conducted using the commercially available cell nuclei marker dye Hoechst 33342.The results indicated that incorporation of a substitute at the meso position methine of parent TOs could reduce cytotoxic effects on cells.Hoechst 33342 gave blue fluorescence,TOs gave green fluorescence,and meso-TOs and meso-CH3-2TO gave red fluorescence.TOs stained both nuclei and cytoplasm,whereas meso-2TOs and meso-CH3-2TO could be accumulated in the cytoplasm,and meso-4TOs stained cytoplasm of normal cells RCF but nuclei of cancer cells MCF-7.The fluorescence from TOs became visually undetectable after 4 h.In contrast,the red fluorescence from meso-TOs remained basically unchanged with time,and the cells have a good morphology throughout the imaging process.8.The interaction of TO analogue(2TO,meso-2TOs,meso-CH3-2TO and meso-allyl-2TOs)with different DNA was studied by fluorescence spectroscopy,absorption spectroscopy,circular dichroism and molecular docking software.Meso-substituted thiazole orange were almost non-fluorescent in solution,but exhibited enhancement fluorescence when bounded to G-DNA,due to the restriction of intramolecular twisting in the groove of G-DNA.meso-Bn-2TO exhibited 162-fold enhancement fluorescence after binding with G-DNA htg22,the binding dissociation constant,Kd,and the stoichiometric ratio,n,were 3.16μM and 2,respectively.2TO could bind with a variety of DNA(ds-DNA,ct-DNA and G-DNA),showed bad selectivity with G-DNA.