Synthesis And Bioactivity Of Selective Fluorescent Probes For Biologically Important Molecules

It is well known that the organism has many kinds of biomolecules, such as amino acids, ions, nucleic acids and proteins. These biological molecules play very important roles in the functioning of biological systems.On one hand, amino acids are one kinds of important biomolecules. Thiol-containing molecules, such as cysteine (Cys), homocysteine (Hey) and glutathione (GSH), play an essential role in human physiological activities. The deficiency of Cys may lead to many disorders, such as slowed growth, hair depigmentation, edema, lethargy, liver damage and skin lesions. Hey is an intermediate product of methionine in vivo. Elevated Hey in the blood is a well-known risky factor for cardiovascular diseases, Alzheimer’s diseases and osteoporosis. Reduced GSH is critical for maintaining cellular redox equilibrium. Therefore, detection of biothiols has attracted wide public concerns. In this regard, naphthalimide derivatives have served as an attractive fluorophore for the detection of thiols because of their optical and chemical stability, large stokes’ shifts, intense fluorescence and high fluorescence quantum yields. However, naphthalimide-based probes have been largely confined to 4-substituted 1,8-naphthalimide derivatives, for example 4-amino-1,8-naphthalimide. As a matter of facts, all the amino-substituted 1, 8-naphthalimides, including 4-amino,3-amino and 3,6-diamino-substituted derivatives emit strong fluorescence. However, few molecular sensors based on 3-amino or 3,6-bis-substituted 1,8-naphthalimides have been reported to date.On the other hand, nucleic acids are another kinds of important biomolecules and play an essential role in living processes. G-quadruplexes are special secondary structures of DNA, which are important in cell growth, proliferation, apoptosis, the formation and development of tumor. To date, major interest in G-quadruplex sensing has been focused on the development of rapid and simple approaches for the seclective and sensitive detection of single-stranded G-quadruplexes. It is known that telomeric terminal overhang is composed of ca.200 nucleotides. Although most of them are prone to form monomeric G-quadruplex, a small number of G-rich sequences tend to form more-complex G-quadruplex structures consisting of consecutive G-quadruplex units connected by TTA subunits. In addition, there are some diseases, such as ALS and Fragile X syndromes that are associated with multimeric G-quadruplexes. Thus highly selective probes for dimeric and/or multimeric G-quadruplexes may help to better understand the structures and functions of quadruplexes and provide useful guidance for the rational design of G-quadruplex DNA-targeting anticancer drugs.In this regard, new probes for biothiols and dimeric G-quadruplexes were constructed and the obtained results are summarized below.Firstly, a new 1,8-naphthalimide derivative bearing 3-amino and 6-(2,4-dinitro-benzenesulfonamido) groups was designed and synthesized as a fluorescent "turn-on" probe for biothiols, that is compound 1. Compound 1 was fully characterized on the basis of ESI MS (LR and HR) and NMR (1H and 13C) data. The selectivity of compound 1 for thiol-containing molecules was studied by fluorescence spectra. The data indicate that compound 1 possesses high selectivity. Among Cys, GSH and Hcy, compound 1 exhibited the highest response to Cys with the detection limit being 2.0 ×10-7 M. The time course study indicates compound 1 exhibited rapid response to Cys with the rate constant of 3.0×10-2 min-1. In addition, the sensing mechanism of compound 1 to Cys was investigated by 1H-NMR and ESI MS and verified that the mechanism involves the nucleophilic attack of thiols on the DNs group of compound 1 to give highly fluorescent N-butyl-3,6-diamino-1,8-naphthalimide, leading to imm ediate fluorescence regeneration. Moreover, probe 1 was successfully applied to detect biological thiols in human liver cancer (HepG2).Secondly, a 9,9’-diglycol-tethered dimeric berberine (2) was designed and synth esized as a fluorescent probe for dimeric G-quadruplex. Compound 2 was character ized on the basis of ESI MS and NMR (1H and 13C) data. Photo fluorimetric titrations with G-quadruplexes suggest that compound 2 exhibits faster response to dimeric G-quadruplexes than monomeric G-quadruplex, and to antiparallel dimeric G-quadruplex than hybrid-type dimeric G-quadruplex. The detection limit of compound 2 was measured by means of spectrofluorimetric titrations and was 7.8×10-10 M toward A(GGGTTA)7G3 (antiparallel G2T1),9.6×10-9 M toward A(G3TTA)3G3 (antiparallel G1),2.16×10-9 M toward hybrid-type G2T1 and 1.23×10-8 M toward hybrid-type G1, respectively. Thus, compound 2 exhibits the highest sensitivity for anti-parallel dimeric G-quadruplexes. The apparent binding constants of compound 2 are 1.48×108 M-1 toward antiparallel G2T1,3.58×105 M-1 toward antiparallel G1,4.96×107M-1 toward hybrid-type G2T1 and 1.54×107 M-1 toward hybrid-type G1, respectively, indicating that compound 2 showed higher seclectivity toward dimeric G-quadruplex than monomeric G-quadruplex, and toward antiparallel dimeric G-quadruplex than hybrid-type dimeric G-quadruplex. Native polyacrylamide gel electrophoresis further proved that compound 2 showed higher seclectivity toward dimeric G-quadruplex than monomeric G-quadruplex. The detection limit and the binding ability of 2 to G2T1 is equal to 9,9’-triethylene glycol-tethered dimeric berberine (118), indicating that compound 2 serves as a good DNA-binder and fluorescent probe for dimeric G-quadruplex.