Highly Selective Two-photon Fluorescent Probes For Cysteine Detection And Their Bio-imaging In Living Cells

Following the bio-optical microscope, common and confocal fluorescence microscope, two-photon microscope has become a new generation tool for microscopic imaging in vivo. Compared with the traditional single-photon fluorescence confocal microscope, two-photon fluorescence microscope shows a number of significant advantages in the imaging of cells and tissues:near-infrared (NIR) photons as excitation source, deeper penetration depth, lower tissue autofluorescence and self absorption, and weaker photodamage and photobleaching. In particular, the two-photon fluorescence microscope reflects the unique advantages in observations on activities of living biological samples, which makes biologists be able to observe and study the life processes in living cells and tissues. Fluorescence microscopy technique and fluorescent probes are interdependent:new fluorescent technology requires new probes and new probes will promote the development of the detection technology. But the relative lag of the two-photon fluorescent probes restricts the wide application of two-photon fluorescent probes with two-photon fluorescent microscope. Therefore, the development with a large two-photon active absorption cross section and high fluorescence quantum yield of the two-photon fluorescent probes is of very important scientific value and significance. There are many two-photon fluorescent probes reported with specific uses. These probes include two-photon fluorescent nucleic acid probe, two-photon fluorescent ion probe, two-photon fluorescent pH probe, two-photon fluorescent lipid raft probe, two-photon fluorescent lipid raft probe, two-photon fluorescent Cysteine/Homocysteine probe and so on.Cysteine(Cys) and Homocysteine(Hcy) play crucial roles in maintaining the biological redox homeostasis. Both of them are expressed in eukaryotic cells simultaneously, and there only is a nuance in a methylene. But the tiny difference leads to a significantly different function:Cys is an amino acid which constructs proteins and glutathione. While excess of Hcy is shown to be a risk factor for cardiovascular and Alzheimer's diseases. Due to the juvenility of fluorescent probes which can separately probe and bioimage intracellular Cys with high selectivity, the lack of fluorescent sensors for Hey which possesses not only the high selectivity but also performance imaging intracellular Hey and the infantility of two-photon fluorescent probes for Cys with high selectivity and by which the fluorescent imaging can be given in two-photon fluorescent microscopy, it is very important to develop two-photon fluorescent probes for Cys/Hcy with water-solubility and permeability to cell membrane. The aim to probe Cys and Hey selectively may be carried out by modulating the electronic structures of fluorescent senors containing aldehydes and adding the groups interfering midbody. In this dissertation, the two-photon fluorescent probes possessing the potential imaging intracellular Cys with high selectivity have been designed and synthesized successfully and their fluorescent bio-imaging in living cells have been realized by two-photon fluorescent microscopy.In this thesis, a series of two-photon biological fluorescent probes for Cys detection have been synthesized by simple organic reactions based on triphenylamine and carbazole. The target compounds have been characterized by NMR, elemental analysis, IR and mass spectra.The synthetic route is simple and easy to operate. These probes are represented by AM1, CA1, CA2, CA3, ZA1and ZA2respectively. Because CA2and CA3show the same properties as CA1, we mainly discuss the synthesis and properties of AM1,CA1,ZA1and ZA2in this thesis.From the large number of experiments, we found that AM1and CA1are highly selective red-and green-emitting two-photon fluorescent probes for Cys detection and the fluorescent imaging of them for Cys has been shown by two-photon microscopy in living cells. AMI and CA1are not two-photon excitation fluorescence (TPEF)-active. In presence of Cys, the two-photon absorption cross section (δ) of AM1+Cys reaches1700GM at920nm, while the δ of CA1+Cys is90GM at810nm. Their solvents are acetonitrile-Tris/HCl buffer solution (v/v,4:1, tris:10mM, KCl:100mM, pH:7.27) and methanol-Tris/HCl buffer solution(v/v,4:1, tris:10mM, KCl:100mM, pH:7.00), respectively. Especially, the TPEF intensity of AM1+Cys is11,67and60times as strong as that of AM1+NAC (N-acetyl-cysteine), AM1+Hcy and AM1at800nm, respectively, while the corresponding ratios of CA1+Cys are2.64,77and59times to CA1+NAC, CA1+Hcy and CA1at800nm, respectively. To further study the interaction between AM1or CA1and Cys, three titrations on absorption, the single-photon excitation fluorescence (SPEF) and TPEF have been carried out. When the molar ratio between Cys and AM1or CA1is less than30, the photophysical properties of the mixture do not change. At30-60, absorbance, SPEF and TPEF intensities increase fleetly. At larger than60, these optical properties remain stable. The detect limits of AM1and CA1for Cys are about293-408μM. For an excellent probe, high selectivity is a matter of necessity, the selectivity of AM1and CA1on UV-Vis absorbtion, one-photon fluorescence and two-photon fluorescence spectra has been measured. We find that there are obvious changes of UV-Vis absorbtion, one-photon fluorescence and two-photon fluorescence spectra upon addition of Cys to AM1or CA1. Meanwhile, the solution color indicates remarkable changes, which could be observed by the naked eye. These facts suggests that AM1and CA1are two highly selective two-photon turn-on fluorescent probes and that both of them are "naked-eye" probes for Cys.we also examine the influence of the pH value on the fluorescence intensity of AM1and CA1in the absence or presence of Cys:the pH value over a wide range of4-7.5could not affect their fluorescence and applications in biological cells. Moreover, the recognition mechanism between AM1/CA1and Cys are studied in detail.Compared with AM1and CA1, ZA1and ZA2are excellent one-photon fluorescence Cys probes. ZA1and ZA2are essentially nonfluorescent, but the introduction of Cys causes a dramatic change of the fluorescence intensity with105-and200-fold, respectively. Meanwhile, the ZA1+Cys and ZA2+Cys have very strong blue-emitting fluorescence, while the addition of other bioanalytes did not lead to any obvious variation in absorbtion and fluorescence spectra of ZA1and ZA2. Moreover, we also realized the wide-field images of ZA1and ZA2for Cys in living cells. In summary, a series of highly selective fluorescent probes for Cys detection have been designed and synthesized successfully. Furthermore, red, green, blue three colors of fluorescence have been got. In particular, AM1and CA1are two highly selective two-photon fluorescent probes for Cys detection. Meanwhile, the recognition mechanism between AMl/CA1and Cys and their two-photon fluorescent imaging in living cells have been studied in detail. This paper will lay the foundation to get the commercialization of Cys fluorescent probes.