Reductive Cleavage Of Azobenzene Derivatives And Application In Dumbbell Antisense Oligonucleotides

As a reactive site,azo bonds can be widely applied to various fields.Through the introduction of azo bond,multiple functions can be organically combined on the basis of the single function of the original system.In the early stage,there was a lot of focus on the research in the photoisomerization and application of azobenzenes,but considering that long-term light irradiation has certain toxic and side effects on tissues and cells,such light-responsive substances are restricted in biological systems.In recent years,the reduction cleavage of azo bond by biological substances has attracted attention.We designed and synthesized an azobenzene linkers with different electronegativity,4,4?-dihydroxymethylazobenzene(Azo)and 2,2?-dimethoxyl-4,4?-dihydroxymethyl-azobenzene(mAzo).The two types of azobenzene linkers are further linked to the antisense oligonucleotide with a covalent bond.First,the azobenzene compound was sensitive to GSH comparing with HLM /NADPH and NADPH.The two azobenzene compounds exhibited higher reducing activity,and particularly,GSH showed an efficiently reduction cleavage ability to mAzo.UV-Vis spectra,HPLC(high performance liquid chromatography),and fluorescence spectroscopy indicated that the azo bond of mAzo is reductive cleavage by GSH.Upon incubation of mAzo with GSH for 3 h,the new main peak appeared at 6.3 min and increasing incubation time caused a concomitant increase in the amount of main product and furtherly by MS and GC-MS analysis for the main cleaved product showed 4-amino-3-methoxyphenylmethanol was produced by azo bond cleavage.Next,the Gaussian 09 software calculations results showed that the energy barrier between trans-mAzo and cis-mAzo is very low,which reasonably explains that the percentage of trans-mAzo and cis-mAzo peaks in HPLC.To further study the velocity of response of mAzo to GSH by visible light and ultraviolet light.According to the fitting curve of peak area,the reaction rate of cis-mAzo and GSH is relatively faster than that of trans-mAzo.The results of GSH reduction cleavage of azobenzene-modified antisense oligonucleotides showed that mAzo-modified antisense nucleotides were sensitive to GSH.With the reaction time reached to 24 h,about 87.8%(AZ18O-4),88.0%(AZ18O-5)and 88.9%(AZ18O-6)sequences were cleaved by GSH,while for Azo modified antisense oligonucleotide,only 12.2%(AZ18P-4),12.0%(AZ18P-5)and 11.1%(AZ18P-6)were cleaved by GSH.The results of the photoisomerization experiment of the AZ18O-6 further show that the AZ18 O series almost exists in the form of dumbbells.The results of the hybridization of dumbbell antisense oligodeoxynucleotides to MB-7 in absence or presence of GSH showed that the background of the sequence itself decreased with the extension of the binding arms.Compared with control,the hybrid strands formed by the MB-7 with AZ18 O series increased by 1.5(AZ18O-4),4.8(AZ18O-5)and 28.8(AZ18O-6)fold,respectively.The current research on the ortho-methoxyl substituted azobenzene derivatives provided a novel strategy on GSH-induced molecular responses,and its attachment to a wide variety of targets can be extended the versatility of azobenzene derivatesfor in vivo use.