Design And Synthesis Of Several Near-infrared Molecular Fluorescent Probes For Biological Application

Near-infrared probes are especailly preferred for biological detection and bioimaging since near-infrared light could avoid the influence of background autofluorescence from organisms, minimize damage to biological samples and penetrate tissues deeply. Among the near-infrared probes reported so far, vast majority are based on multimethylene cyanine derivatives, and because of the poor photostability of the multimethylene cyanine dyes, development of near-infrared probes based on new fluorophores is significant and valuable for application.Hydrogen ion is widely distributed in organisms, and it plays vital roles in various biological processes, such as proliferation and apoptosis, ion transport, endocytosis, enzymes activity, as well as organizational units such as muscle and nervous system. Abnormal p H values are associated with some common diseases such as cancer and Alzheimer’s. Nitric oxide is an essential transient free radical in oganisms, playing a vital role as a signaling molecule in a variety of physiological and pathological processes that take place in the cardiovascular, nervous, and immune systems. Misregulation of nitric oxide production has a crucial relationship with various diseases such as cancer, neurodegenerative injury, and inflammation. Therefore, detection of p H and nitric oxide in organisms with near-infrared fluorescent probes is significant.In this paper, several p H or nitric oxide fluorescent probes based on benzo[a]phenoxazine or dibenzoxanthenium dyes were designed and synthesized, and their optical properties were investigated. The results were as follows:1. Benzo[a]phenoxazine derivatives containing substituted N-aromatic groups(probes 1a-1b) were evaluated for their p H-dependent absorption and emission properties. Among the compounds exhibiting optical responses under near-neutral and subacidic p H conditions, benzo[a]phenoxazine derivative with an electron-withdrawing aromatic group attached to nitrogen of the imino group(probe 1b) showed potential application as near infrared p H sensor. Based on that, three water-soluble p H probes(probes 3a-3c) based on benzo[a]phenoxazine with different pyridinium structures were designed and synthesized. Their reversible p H-dependent emissions in buffer solution containing 0.1% dimethyl sulfoxide(DMSO) locate in 625-850 nm with the fluorescent enhancement of 8.2-40.1 times, and their calculated p Ka values are 2.7, 5.8, and 7.1, respectively. A composite probe containing the three benzo[a]phenoxazines shows a linear p H-emission relationship in the range of p H 1.9-8.0. Real-time detection of intracellular p H using an in vitro assay with He La cells was also reported.2. Two benzo[a]phenoxazine-based p H probes(probes 6a-6b) bearing nitrile group or hydroxyl group in the benzo[a]phenoxazine skeleton were designed and synthesized. Probe 6a with p Ka = 5.0 exhibits OFF-ON emission of 625-850 nm upon excitation at 600 nm in aqueous buffers. The in cellulo imaging experiments with He La cells indicate that one of the probes(probe 6a) can serve as a lysosome-specific probe using red light excitation(633nm) with near infrared emission(650–790 nm).3. Two p H probes(probes 7a-7b) based on 3,11-diamino-7-(4-aminophenyl)-dibenzoxanthenium were reported. They both exhibited OFF-ON p H-dependent NIR emission in the range of p H 2.0-5.0, with p Ka values of 3.28 and 3.25, and increase time of 272 and 114 fold, respectively. The two probes were further applied in in vivo alimentary canal image. The work indicates that dibenzoxanthenium would be a new NIR fluorophore for PET type probes.4. Two dibenzoxanthenium derivatives(probes 10a-10b) bearing a 3,4-diaminophenyl group were prepared and evaluated as near-infrared probes for the detection of nitric oxide. One compound(probe 10a) gave a 3-fold emission enhancement towards nitric oxide at 681 nm, whereas another compound(probe 10b) exhibited OFF-ON emission behavior towards nitric oxide, resulting in a 35-fold increase in the fluorescence intensity with an emission maximum of 750 nm. Probes showed independent emission in the presence of common cations, amino acids, reactive oxygen and nitrogen species; furthermore, it can work under acidic and neutral conditions.