Desing,Synthesis And Biological Applications Of Cysteine And Viscosity Probes

Small molecule fluorescent probe worked as a good detection technique is becoming more popular because of its splendid selectivity and rapid response towards specific species in living cells or organisms.Among them,fluorescent probes emiting in near-infrared region show unique advantages in cells imaging applications,since they do minimal ruin to samples and can effectively avoid the background fluorescence from the biological molecules.Cysteine(Cys)is an indispensable biological thiol,which takes parts in majority of physiological processes.The aberrant changes in intracellular Cys levels will induce many diseases including cancers.Mitochondria are a major production place of Cys,since there are lots of reactive oxygen species(ROS)generated in mitochondria,too much ROS can lead to cell death.To avoid the oxidative damage,Cys can act as an antioxidant by scavenging various ROS in mitochondria.Therefore,designing new Cys probes with good optical performance and mitochondria-targetable property is still highly essential.Besides,cellular viscosity plays pivotal roles in modulating biological processes.It has long been speculated that aberrant mitochondrial viscosity correlates with neurodegenerative diseases,atherosclerosis and diabetes,while anomalous lysosomal viscosity relates to heart disease,aging and lysosomal storage diseases.Intracellular viscosity is inhomogeneous because each organelle has different compositions and needs an optimal viscosity to perform its functions.As abnormal viscosity fluctuations can serve as a versatile indicator in early disease diagnosis,thus the development of reliable methods for monitoring the viscosity variations at subcellular level is in urgently need.The task of this article can be divided into two parts.Four Cys fluorescent probes were designed and prepared based on benzophenoxazine and xanthylene skeleton in the first part.Nine viscosity fluorescent probes were designed and prepared based on hemicyanine in the second part.Their optical properties and the applications in cells were systematically discussed.The details are as follows:1.In the first section of the first part,three benzophenoxazine-based(benzoresorufin and nile-red)Cys detection probes 1a-c had been comparatively synthesized and their optical properties were systematically compared in this paper.The optical experiments exhibited probe lb with a crotonoyl group had no response toward Cys;while probes 1a and 1c had the same reaction site(acryloyl group),their optical responses to Cys were quite different.The benzoresorufin-based probe la showed an OFF-ON fluorescence response(enhanced 118-fold)to Cys at 631 nm and afforded a very low detection limit(DL=19.8 nM).Compared with probe la,the nile-red-based probe lc displayed gradually diminishing fluorescence intensity with increased Cys concentration at 665 nm.And the notable different fluorescence response mechanisms of probes la and 1c toward Cys could be interpreted by time-dependent density functional theory(TDDFT)calculations.Meanwhile,probes la and 1c were successfully applied to detect Cys in HeLa cells.In the second section,a mitochondria-targeting fluorescent probe 7 for efficient ratiometric detection of Cys was designed and investigated.Probe 7 is composed of a xanthylene skeleton and a benzyl group containing an acryloyl moiety.The probe owned excellent water solubility and good selectivity toward Cys,and the detection limit to Cys was as low as 33.7 nM.Moreover,cells experiments demonstrated that probe 7 had excellent mitochondrial-targeting ability(the Pearson's correlation coefficient was 0.96),and was capable of monitoring endogenous Cys in living HeLa cells by dual channel ratiometric bioimaging,demonstrating its significant potential in biological applications.2.In the first section of the second part,six mitochondrial targeting viscosity probes 10a-f were synthesized based on the nitrogen cationic cyanine skeleton.Their optical properties were also studied systematically.The optical experiments indicated that probes 10a-f had obvious OFF-ON type response to viscosity in the near infrared region(652-690 nm),and exhibited good linear relationship with solution viscosity.Test in different solvents and the interfering experiments showed that the solution polarity and competing species could not interfere with the viscosity detection ability of probes 10a-f.Meanwhile,cell imaging experiments showed that probes 10a-f could selectively target mitochondria and be used for intracellular mitochondrial viscosity detection.Based on the prior work,three lysosomes targetable viscosity-sensitive probes 12a-c were developed via simple structure modification on cationic cyanine skeleton of the hemicyanine-derived dyes in the second section.They all showed remarkable OFF-ON fluorescent response to viscosity in near-infrared region(662-690 nm)and exhibited good linear relationship with solution viscosity.The transient absorption spectra were used to evaluate the excited-state lifetime of probe 12a in different viscosity environments;it was found that the excited state lifetime increased with the increase of viscosity,which might be caused by the restriction of C-C single bond rotation in the middle.Furthermore,cellular imaging assays indicated that probes 12a-c could sense lysosomal viscosity fluctuations.In conclusion,three different types of Cys fluorescent probes(1a,1c and 7)and two different organelle targeted viscosity fluorescent probes(10a-f and 12a-c)were designed and synthesized.Each probe demonstrated excellent detection performance,which showed great potential in the application of biological field in the future.