Preparation Of New Organic Two-photon Probes And Their Applications In Biological Imaging

Since W. Denk et al. introduced the two-photon excitation method into fluorescence microscopy in 1990, there is an increasing interest in two-photon excitation fluorescence microscopic imaging (2PM). 2PM has some significant advantages over conventional single-photon fluorescence imaging techniques, including a capacity for a highly confined excitation, intrinsic three-dimensional resolution and the possibility of imaging at an increased penetration depth in tissue with reduced photodamage and background fluorescence.2PM of biological systems will benefit from probes with large two-photon excitation cross-sections and good specificity. Over the past 20 years, numerous organic two-photon absorption (2PA) molecules with good 2PA properties have been reported. However, few efforts have been made on using efficient 2PA molecules as organic probes in practical 2PM. In this thesis, some 2PA probes with good prospects in 2PM application have been designed and synthesized. The main contents and results are summarized as follows:(1) A series of 2-hydroxy-pyrimidine-based mono-substituted D-π-A (D: donor,π:π-conjugation, A: acceptor) and disubstituted D-π-A-π-D type (Y shape) organic 2PA probes (1a-4a and 1b-4b) have been designed and synthesized. Photophysical properties and sing- and two-photon fluorescence Hep G2 Cell imaging of the molecules are investigated systematically. The results indicate that the two-photon absorption cross-sections were found to have significant (up to 12 times) increase when changing from monosubstituted molecules(1a and 1b) to their disubstituted counterparts (2a-4a and 2b-4b). In addition, no significant difference in the single- and two-photon spectroscopic properties of these molecules can be observed when comparing the hydrophobic molecules (ethyl ester, 1a-4a) to the corresponding amphiphilic molecules (carboxylic acid salt, 1b-4b). However, single- and two-photon fluorescence microscopic imaging experiments revealed that the disubstituted amphiphilic molecules can efficiently stain the cytoplasmic region of HepG2 cells, while little or no dye uptake happened when incubating those cells with the hydrophobic molecules under the same experimental conditions. (2) Three new organic 2PA probes (5-7) based on 2-amino-pyrimidine core were designed and greenly synthesized by Aldol condensation in the absence of any organic solvents. Compounds 5-7 could be prepared after short reaction times (1 h, 1.5 h and 0.5 h for compounds 5-7, respectively), high yields (84%, 75% and 94% for compounds 5-7, respectively) and simple separation and purification. Probes 5-7 were found to have large two-photon absorption cross-sections. Probe 7 has the largest two-photon absorption cross-section value (819 GM) in toluene, which is 22 times larger than that of fluorescein standard. In addition, strong modulation of single-photon and two-photon fluorescent spectra of these molecules by (de)protonation makes them promising candidates for two-photon pH sensor.(3) An amine-reactive organic 2PA probe (8) based on 2-pyrimidinyl- isothiocyanate for covalent attachment onto amine-containing biomolecules was prepared. A model probe-adduct (9) was prepared by reacted (8) with D-glucosamine. Although no significant changes in the single-photon spectroscopic properties and the quantum yield could be observed between 8 and the modle adduct, the 2PA cross-sections of molecule 9 sharp increases in polar CHCl3 solvent, which is 3 times larger than that of probe 8. Tf-8 conjugates were produced by coupling molecule 8 to transferrin (Tf). Competirion experiment between Tf-8 and Tf indicate that Tf-8 was recognized by and bound to the transferrin receptor on the membrane, and delivered into cytoplasm.(4) In order to study the photostability of alkynes derivatives under UV light, two asymmetrical (D-π-A) organic 2PA probes with substituted acetylene as central rigid elongated conjugation have been designed and synthesized. Photophysical properties, photostability, antiphotobleaching and sing- and two-photon fluorescence Hep G2 Cell imaging of the molecules 10 and 11 are investigated systematically. The results indicate that the acetylene-substituted molecules (10 and 11) were found to have good photostability under UV light (λirr = 365 nm; I0 = 4 mW/cm2). In addition, these acetylene-substituted molecules were found to have high two-photon absorption cross-sections (for example, 690 GM for molecule 10 in toluene).(5) Four symmetrical organic 2PA molecules (D–π–D) with substituted acetylene as central rigid elongated conjugation have been designed and synthesized. One derivative with great enhancement on the two-photon absorption cross-section in 820 nm, which is among the best output wavelength range of a typical Ti:Sapphire femtosecond laser, could be obtained by inserting an anthracene ring into the rigid elongatedπ-conjugation. Such kind of structure modification is expected to be helpful in designing better organic 2PA probes for biological imaging.