Detect Intracellular Thiol Compounds Design And Synthesis Of Fluorescent Probe With The Markers

The mercapto compound plays an important role in maintaining the stability ofthe intracellular environment. Many studies have shown that the mercapto compoundsnot only be able to adjust the oxidation reduction balance in vivo, against oxidativestress, slow the body aging and enhance immunity, but also play an important role indetoxification. The mercapto compound is divided into small molecules mercaptocompound （e.g., glutathione, cysteine, homocysteine, etc.） and macromoleculesmercapto protein （e.g.,thioredoxin protein, metallothionein proteins, etc.）. Cysteine（Cys） consists of reducing mercapto group （-SH） in20species of amino acids. Thedeficiency of Cys can cause slowed growth, hair depigmentation, edema, lethargy,liver damage, muscle and fat loss, skin lesions, and weakness. Homocysteine （Hcy） isan intermediates of the methionine-metabolism in vivo. Elevated Hcy in the blood is awell-known risk factor for a number of diseases, such as Alzheimer’s, cardiovasculardiseases, and osteoporosis. Thioredoxin （Trx） is one of the macromolecules mercaptoprotein, containing a typical dual-mercapto active site （-CGPC-）. Trx not only playsan important role in regulating the cellular redox state, but also be closed withintracellular oxidative damage and apoptosis.In recent years, fluorescent method for detecting the mercapto compounds hasdeveloped rapidly, based on its apparent advantages in sensitivity, specificity andnondestructive imaging. A wide variety of sensing mechanisms have been used in thethiols fluorescent probe design, such as Michael addition, cleavage of sulfonamideand sulfonate ester, metal complexes-displace coordination, and cleavage of disulfide.In general, these methods are usually hampered by interference from structurallyrelated thiols （such as Cys, GSH etc.）.Based on the fluorescence properties change of the fluorescent probe reactingwith the mercapto compound, we develop fluorescent probe to achieve the detectionof a specific mercapto compound. The probe is successfully used in confocalfluorescence imaging of mercapto compound in vivo. In this paper, we have carriedout the following two aspects: First, as known, near-infrared （NIR） dyes, wavelengths of which are located in650-900nm range, have the unique advantages of tracing molecular activity in vivobecause their NIR photons can penetrate relatively deeply into tissues with lowauto-fluorescence background and cause less damage to biological samples. Therefore,it is vital to develop a highly sensitive near-infrared fluorescent probe for detectingCys/Hcy. The probe is synthesised by Cy7and4-hydroxybenzaldehyde for detectingCys/Hcy. The cyclization product with cysteine or homocysteine is verified by massspectrometry. Probe based on PET mechanism shows lower fluorescence intensity.After reaction with cysteine or homocysteine, PET process is interrupted and thefluorescence is recovered. The maximum excitation and emission wavelengths arelocated in λ_ex/λ_ex=730/778nm respectively. There is a good linear relationshipbetween the fluorescence intensity and the concentration of Cys, and the linear rangesfrom0.03to2.0μm. The linear equation is （F-F0）=1043.59+101.83×[Cys](10^-7M), with a correlation coefficient of0.9941. The detection limit is7.9nM, which isthe lowest detection limit in previous report. There is no interference with other aminoacids, glutathione, metal ions, H2S, and Na2SO3on the reaction. The low cytotoxicityof the probe is confirmed by MTT assay, and the light stability of the probe isconfirmed by photobleaching experiments. The probe shows a good solubility, highsensitivity, high selectivity, etc., and it is successfully applied to the confocal imagingof endogenous Cys.Second, inspired by Tsien’s bis-arsenic dye to detect a small peptide sequencewith four cysteine, we design and synthesis of two fluorescent probes Rho-Ebselenand the Rho-benzene-Ebselen based on the Se-N bond cleavage reaction mechanism.The probe is applied for detecting double （multiple） mercapto protein which is in thespatial position matching. The probes contain rhodamine110and Ebselen Se-N bond.The probes exist in the form of the lipid, and the fluorescent intensity are weak. Afterreaction of Ebselen Se-N bond with mercapto group, the compound forms aring-opening structure, achieving the purpose of fluorescence enhancement.