Design And Synthesis Of Several Near-Infrared PH Probes Based On Oxazine Derivatives For Biological Application

Near-infrared probes have been achieved much attention since they have many advantages, for example, they can penetrate tissues deeply, minimize damage to biological samples, in addition, they can also avoid the influence of background autofluorescence from organisms during experiment, so near-infrared probes are preferred for biological detection and bioimaging by researchers. Oxazine derivatives is a kind of stable long wavelength emission dye, they have bright prospects for that they have many advantages, such as big molar extinction coefficient, water solubility, low cytotoxicity and membrane permeability. It was reported that oxazine derivatives had been used in many areas, such as pharmacy, electrochemistry, third-order nonlinear optical, ion probes.Hydrogen ion is a kind of very important charged species that plays an extremely important role in biological processes, such as proliferation and apoptosis, metabolism, endocytosis, enzymatic activity, ion transport, muscle contraction, nervous system signals, multidrug resistance and so on. Abnormal pH values will cause some common diseases such as inflammation, Alzheimer’s, cancer and so on. Thus, detection of pH in organisms with near-infrared fluorescent probes is significant, because it can provide information to clinical diagnostics.Several near-infrared pH probes based on oxazine derivatives are designed and synthesized, and their optical properties are studied, finally, they are all applied to cells images study. It is divided into three parts in this paper, details as follows:1. Three Darrow red-based near-infrared pH probes 5a-5c were designed and synthesized. They are all OFF-ON type of pH probes, as well as lysosome-targeted pH probes. Among these three probes, the emission intensity of probes 5a is very weak at pH=4.0, while it becomes very strong at pH=1.6, the relative fluorescence intensity is enhance to 39 times, with a pKa of 2.4. Fluorescence confocal images of HeLa and KB cells indicated that the emission of probes 5a is near-infrared, and its fluorescence intensity is very strong, and it overlaps very well with commercial lysosomal marker; while in V79 cells, just very weak background fluorescence can be seen. Therefore, probe 5a can be used as a unique near-infrared lysosome-biomarker for the detection of cancer cells for that it can distinguish the normal cells from cancer cells. The fluorescence emission of probe 5b and probe 5c were strong, and they can used to be lysosome-biomarker for the cancer cells and normal cells, but can not distinction the normal cells from cancer cells.2. Three reversible near-infrared pH probes 7a-7c based on phenoxazine derivatives which were applied in base condition were designed and synthesized, and their optical response to hydrogen ion were studied as well. The optical properties of probes 7a-7c were tested in buffer solutions containing 10% DMSO, and the calculative pKa is 8.7, 9.2 and 8.9 respectively. Maximum emission of probe 7a-7c is 676, 685 and 693 nm respectively, all of them are very well near-infrared pH probes. Fluorescence confocal images indicated that the emission of probes 7a-7c is near-infrared in both HeLa cells and V79 cells, but they can not overlap well with commercial lysosomal marker, that is to say they are just used as a kind of cytoplasmic probes, minimizing the light damage to biological tissue greatly.3. Two phenoxazine derivatives-based lysosomal near-infrared pH probes 9a and 9b were designed and synthesized, and their optical response to hydrogen ion were studied as well. The optical properties of probes 9a-9b were tested in buffer solutions containing 10% DMSO, and their maximum emissionis 664 and 667 nm respectively. In acidic conditions, two N atoms of imide and pyridine group of probes 9a protonize simultaneously, thus, there have two pKa, 3.71 and 9.68 respectively; while the H atom of imide N is replaced by a methyl substituent group, probe 9b has only one pKa, that is 2.75, because protonize site just at the N of pyridine group under acidic conditions. Fluorescence confocal images indicated that the emission of probes 9a is near-infrared in both HeLa and V79 cells, and they can overlap well with commercial lysosomal marker, thus they can be used as a kind of lysosomal marker probe, monitoring the pH change in lysosome. The emission of probe 9b is near-infrared in both HeLa and V79 cells as well, but it can not overlap well with commercial lysosomal marker in neither of that two kinds of cells, that is to say probes 9b is just an ordinary pH probe for monitoring the pH change in cytoplasm of the cells.