Synthesis And Application Of Nitrogen-substituted Heptamethine Cyanine Dyes

With the development of fluorescence detecting technology, many research work were focused on its application of fluorescent sensors, since near infrared ?NIR:650 nm-800 nm? detection can avoid effectively self-absorbence in vivo and autofluorescent background. Heptamethine cyanine dyes employed as fluorescent sensors of biomolecules have attracted immense interest because their spectra of emission and absorption reach the NIR region and high values of molar absorptivity.Two heptamethine indocyanine dyes used as precursor compounds in this thesis were synthesized firstly. Two sulphonic groups were introduced to the two N atoms at the indo rings of dyes in order to achieve higher water-solubility. Cyclohexene group was introduced to the middle of conjugated methine chain to improve stability and offer a reactive site. Then eight derived dyes including p-Cy7-A, p-Cy7.5-A, PAP-Cy7-G, PAP-Cy7, Cy7-AA-Tyr, Cy7-AA-Tyr-Me, Cy7-A and Cy7.5-A were obtained by a nucleophilic reaction at the central chlorine atom of the precursor dyes. The maximum absorption and emission wavelength of the derived dyes is longer than 620 nm and 720 nm. The most prominent characteristic of them was the large Stokes shift ?100 nm? because of excited-state intramolecular charge transfer. However, these dyes have low quantum yield ??f<0.001?.Recently, nanomaterials of rare earths exhibit their advantages including good chemical stability and narrow emission peaks, which have made people interested. The nanoparticles ?NPs? Y_0.6Eu_0.4VO₄ described as being about 56.8 nm in diameter by TEM and DLS were prepared from Y?NO3?3, Eu?NO3?3 and Na3VO4 as the starting materials by a simple stirring and centrifuging process. The luminescence of NPs is attributed to energy transition between VO₄^3- groups and Eu³⁺. The VO₄^3- groups absorb the ultraviolet photons and then pass most of the energy to Eu³⁺, and induced Eu³⁺radiative transition and appeared the Eu³⁺characteristic emission peak including 596 nm ?⁵D₀?7F1?,620 nm ?⁵D₀?7F2?,652 nm ?⁵D₀?7F3?,700 nm ?⁵D₀?7F4?Then, we have done the three following work using the heptamethine cyanine dyes and the nanoparticles ?NPs? Yo.6Euo.4VO₄.1. The p-Cy7-A, p-Cy7.5-A and NPs for detection of Alkaline Phosphatase?ALP? in NIR regionA non-fluorescent self-assemblage of Y_0.6Eu_0.4VO₄ nanoparticle ?NP? and heptamethine cyanine dye bearing phosphate group p-Cy7-A, NP-p-Cy7-A, was the first constructed and used as an alkaline phosphatase ?ALP? sensor. Herein, Y_0.6Eu_0.4VO₄ NP and new synthesized p-Cy7-A were employed as donor and acceptor of Fluorescence Resonance Energy Transfer, respectively. With the help of phosphate group, p-Cy7-A as a dark quencher was captured on Y_0.6Eu_0.4VO₄ NP surface to form in situ non-fluorescent NP-p-Cy7-A. The disintegration of NP-p-Cy7-A was triggered from the removal of phosphate group in p-Cy7-A catalyzed by ALP, and released fluorescent Y_0.6EU_0.4VO₄NP. Thus NP-p-Cy7-A assemblage as a selective and sensitive sensor can very well respond to the amount and activity of ALP by measuring the fluorescent instenty at 620 nm ?excited at 270 nm?. This sensor can not only accurately measure the concentration of ALP ?0.03-1.65 U/mL?, but also be applied to the detection of ALP activity and its inhibitor by a simple mix-and-measure manner.2. The synthesis of PAP-Cy7-G and PAP-Cy7D-Glucose, cetic anhydride, hydrogen bromide solution,4-nitrophenol, Pd/C and IR-783 were selected as starting materials, and then through Koening-Knorr reaction and nucleophilic substitution reaction etc. six steps, the target compounds ?PAP-Cy7-G and PAP-Cy7? were synthesized. The structures of intermediates and target molecules were chararcterized by 1H NMR etc. mothods. And the result was that the PAP-Cy7-G was identified as ?-D-glucoside compound. Two heptamethine cyanine dyes, PAP-Cy7-G and PAP-Cy7 have similar fluorescence properties. But they have different maximum absorption wavelength. ?-Glucosidase can catalyze the hydrolysis of the glycosidic bonds in ?-D-glucosides, with releasing of glucose. However, the hydrolysis of PAP-Cy7-G was not observed in the presense of ?-Glucosidase resulting from enzyme kinetics experiment data.3. The synthesis of Cy7-AA-Tyr-Me and Cy7-AA-TyrL-Tyrosine, dichlorosulfoxide,6-aminocaproic acid, trifluoroacetic acid, EDCHC1 and IR-783 were selected as starting materials, and then through protection and de-protection of amino group and nucleophilic substitution reaction etc. six steps, the target compounds ?Cy7-AA-Tyr-Me and Cy7-AA-Tyr? were synthesized. The EGFR belonging to the tyrosine kinase family is an enzyme that can transfer a phosphate group from ATP to a tyrosine residue of target protein. We want to subject the four synthesized heptamethine cyanine dyes containing the tyrosine analogues group, Cy7-A, Cy7.5-A, Cy7-AA-Tyr-Me and Cy7-AA-Tyr as the substrates of EGFR to the phosphorylation experiment. However, the result of enzyme kinetics experiment indicated that they were not phosphorylated in the presence of EGFR and ATP.