| Objective: Polymethine cyanine dyes, especially of polymethine indocyanine dyes,have a lot of advantages, for example: the maximum absorption and emission in the near infrared region, more widely wavelength tuning, a higher molar extinction coefficient, a higher fluorescence quantum yields, no-radioactivity, high signal to noise and can effectively escape fluorescence interference come from the organism itself. Polymethine indocyanine dyes has become the focus of research in the field of life science and can be used in the biomedical field such as proteins detecting, cell imaging, tumor-targeted therapy and DNA Microarray. But,indocyanine dyes that have been developed so far is difficult to meet the need of application of life sciences, research indocyanine dyes with excellent performance has important significance in life and science field.Indocyanine dyes exist some defects such as difficult in separation and purification, poor water solubility, prone to photobleaching and fluorescent quenching, which can limit the range of application and effect. we introduced a nonionic hydrophilic group which contain PEG ether chain in “N” position of indole ring and synthesized a series of novel indocyanine dyes, studying the following influence in separation and purification, optical property and application in biology after introducing the group for obtain new indocyanine dyes with advantages of convenient synthesis, more excellent in optical performance and a better effect in biological application.Methods: A nonionic hydrophilic group has been synthesized by primary material such as sulfonic acid phenyl hydrazine, p-methyl benzene sulfonic chloride and polyethylene three glycol etc, introducing the group to “N” position of indole ring, one indocyanine dye named Cy3-569 and three indocyanine dyes named Cy5-668, Cy5-669 and Cy5-671 have been synthesized by “one-step method” and “hemicyanine method”, and they were all activited in the last.Using optical instruments such as UV spectrophotometer, florospectroph otometer and iodine-tungsten lamp to detect the optical property and photostability of the f our indocyanine dyes, then contrasting with each other at last. Using the four activated in docyanine dyes to label bovine serum albumin, detecting and calculating the ratio of dye t o protein.Selecting Cy5-669 to stain fixed and living cells in human hepatocellular, observi ng and comparing the effect between fixed cells staining and living cells staining.Results: 12 kinds of dye intermediate and 4 kinds of the object product have been synthesized and confirmed by techniques such as 1H NMR and MS.Optical property and photostability of Cy5-668, Cy5-669, Cy5-671 and Cy3-569 were detected, we have found that the maximum UV absorption wavelength of them in water were 647 nm, 648 nm, 650 nm and 553 nm respectively. The maximum emission wavelength of them in water were 668 nm, 669 nm, 671 nm and 569 nm respectively. The stokes shift of them in water were 21 nm, 21 nm, 21 nm and 16 nm respectively. The molar extinction coefficient of them in water were 1.4×105/M-1cm-1, 1.9×105/M-1cm-1, 1.6×105/M-1cm-1 and 0.9×105/M-1cm-1respectively.The fluorescence quantum yields of them in water were 0.09, 0.13, 0.24 and 0.07 respectively.The photostability of the four kinds of dyes were detected and compared with each other,we found that when lighted by odine-tungsten lamp for 8 h, the order of the photostability in these four dyes is Cy3-567>Cy5-671>Cy5-669>Cy5-668, and the ratios of photodegradation were3.2%, 23.5%, 31.9% and 37.3% respectively.Bovine serum albumin(BSA) was labeled by the four kinds of activated dyes, we found that when using different molar ratio(n(dye):n(BSA)=5, 10 or 20)between dyes and BSA to have the covalent labeling reaction, the ratio of Cy3-569 to protein is 1.15?1.57 and 2.14 respectively,the ratio of Cy5-668 to protein is 1.12, 1.59 and 2.11 respectively, the ratio of Cy5-669 to protein is 1.16, 1.75 and 2.16 respectively, the ratio of Cy5-671 to protein is 1.14, 1.86 and 2.54 respectively.Fixed and living cells in human hepatocellular were stained by Cy5-669, we found that the dye can stain the whole fixed cell by acrossing cell membranes, having the most obvious staining effect on nuclear as well as clearly visibled nucleolus. The dye mostly congregate on the surface of the living cell membrane but a small amount of dyes across the membrane into the interior of the cell, peripheral contour of cells can be clearly seen as well as brightly fluorescence.There exist significant differences in staining effect by the dye between the fixed and living cells,which can distinguish cell shape and survival condition obviously.Conclusions: Introducing a “dendritic nonionic hydrophilic group” to “N” position of indole ring can reduce the difficult of the separation and purification for synthesis of dye molecules, and the target compound can be separated and purified by conventional normal phase silica gel column easily. The fluorescence quantum yields, water-solubility and photostability of the dyes have a greatly improve compared to dyes without of “dendritic nonionic hydrophilic group”, and a positive effect has been found when labeling protein.While, Fixed and living cells in human hepatocellular can be stained by Cy5-669 respectively, a bright fluorescence was found and some differents have been found in staining effect, which can distinguish cell shape and survivalcondition obviously, the results indicated that the dye has potential applications in fields of cell imaging and study of apoptosis. |
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