Synthesis Of A 2,2’-bipyridyl-linked Berberine Dimer As A Fluorescent Probe For Selective Detection Of Metal Ions And DNA

During the past decade, the development of fluorescent sensors for biomacromolecules has received considerable attention due to their high sensitivity, selectivity and easy manipulation, low cost and high potential in clinical diagnostic and therapeutic advances. Till now, some known fluorescent sensors, such as coumarin and rhodamine, are available for the detection of various cations, anions and small molecules. However, few of them have been explored for the detection of biomacromolecules because of their poor water solubility. As a consequence, it is still of urgent need to develop novel sensing platforms based on easily obtained materials with good water solubility.In this thesis, we designed and synthesized a 2,2’-bipyridyl-linked berberine dimer with relatively good water solubility and investigated its sensing properities as a fluorescent sensor for metal ions, biomacromolecules G-quadruplex and HIV DNA. The obtained results are summarized below.1. Synthesis, characterization and properties of compound 1A berberine dimer 1 tethered with 2,2’-bipyridine was synthesized and characterized by 1H NMR,13C NMR, ESI-MS and HR-MS. Compound 1 is soluble in good water containing 1‰ DMSO. It exhibits aggregation-induced emission in DMSO/H2O with the increasing ratio of water. Compound 1 exhibits an increase in fluorescence intensity in acidic pH from 2.0 to 7.0, and a decrease in basic pH from 7.0 to 10.5. These results convincingly suggest that the pH-dependent fluorescence intensity of compound 1 is a likely consequence of the varying protonation of compound 1 at different pH.2. Fluorescent detection of metal ions by compound 1UV absorption and fluorescence spectra showed that compound 1 forms a 2:1 ratio complex with Cu2+, thus can detect Cu2+selectively in 5 mM Tris-HCl buffer at pH 7.0 and not interfered with other metal ions with the limit detection of 1.08 μM. Compound 1 also showed a corresponding difference in reaction time with Cu2+, Ni2+ and Co2+ of instant,2 h and 10 h. The reaction rate constant of compound 1 with Co2+ and Ni2+ was estimated to be 9.83 ×10-3 min-1 and 9.60×20-2 min-1, respectively.3. Fluorescent detection of G-quadruplex by compound 1Compound 1 has strong affinity with G-quadruplex, single-stranded DNA and double-stranded DNA with the binding constants in the range from 105 to 106 M-1. G-quadruplex could enhance the fluorescent intensity of compound 1, while single-stranded DNA and double-stranded DNA with decreasement, which can be detected with naked eyes. Compound 1 prefers binding to parallel G-quadruplex over anti-parallel G-quadruplex. It also had strong binding ability with human telomere sequence 22AG to form anti-parallel structure. CD melting results indicated that the ATm for compound 1 with parallel G-quadruplex of c-kit2, anti-parallel of 22AG (Na+) and disordered of 22AG are> 24 ℃,1.5 ℃ and 14 ℃, respectively. It was further confirmed that compound 1 could stabilize parallel G4s. Inhibition of the peroxidase activity of G4/hemin indicated that compound 1 may interact with parallel G-quadruplex by means of π-πstacking. Compound 1 could be used to image nucleic acids within the Hela cell.4. Fluorescent detection of HIV DNA by compound 1Compound 1 contains aromatic rings and positively charged pyridinium cation centers. These functional groups may offer π-π stacking, hydrogen bonding and electrostatic interactions with DNA. To test this hypothesis, we investigated the sensing properties of compound 1 toward HIV DNA. The results indicated that compound 1 could quench the fluorensence of probe single FAM-labeled DNA with quenching efficiency of 92%. Upon addition of target complementary duplex HIV DNA (ds-DNA), the fluorescence intensity could be recovered with recovery efficiency of 7.42. Compound 1 is capable of distinguishing complementary ds-DNA from mismatched target sequences with the detection limit of 0.11 nM.