Application Of CBT-Cys Click Condensation Reaction In Molecule Imaging, Cancer Therapy, And Cell Bridging

Click chemistry is a highly selective and efficient chemical approach which uses a wide scope of reagents and does not need purification. In the early years of this century, Sharpless and coworkers defined a click reaction as the one that is wide in scope, easy to perform, uses only readily available reagents, and is insensitive to oxygen and water. Work-up and product isolation of a click reaction must be simple, without requiring chromatographic purification. Applications of click reactions are now widely found in drug discovery, clinical medicine, combinatorial chemistry, material sciences, proteomics, and DNA research.The condensation reaction between2-cyanobenzothiazole (CBT) and D-cysteine (D-Cys) is a classical click chemistry reaction occurring in the body of firefly. This reaction owns the advantages of mild conditions, high reaction rate and good biocompatibility. The properties of the condensation products or the assembled nanostructures of the click reaction such as molecular weight, size, and morphology can be controlled by the structures of the monomers or the reaction conditions used (i.e., pH, reduction or enzymatic cleavage). Thus this click reaction can be applied in material sciences for the preparation of oligomeric nanoparticles and crosslinked polymers. Through the change of functional group on monomer, this condensation reaction can also be used in molecule imaging, including optical imaging and magnetic resonance (MR) imaging, biomolecule modification, drug design, functional proteomics, pathology, etc.On the basis of this condensation reaction, we synthesized a panel of functional substrates, and applied them in different biological areas and systems, which contain the following seven parts:(1) Based on the CBT-Cys condensation reaction, upon reduction by GSH in vitro or in cells under physiological conditions (pH7.4in buffer), the small molecule CBT-Cys(SEt) condenses and self-assembles into nanorings, increasing the optical absorbance at380nm. This method is selective to GSH rather than Cys in biological samples. To better get insight into the mechanism of the nanoring self-assembly, the condensation products of CBT-Cys(SEt) formed at different concentrations of GSH and different reaction times were characterized by transmission electron microscopy (TEM). (2) We developed a AB2type (Cys(SEt)-Lys[Cys(SEt)]-CBT) small molecule with a CBT motif and two disulfide bond-protected Cys motifs, which self-assembled to nanoparticles with a large number of Cys residues on surface after the reduction-controlled condensation. By the conjugation of-NH2on Cys residues with-COOH on the surfaces of near-infrared (NIR) QDs, and the linkage between-SH groups on Cys residues and maleimide-conjugated folic acid (Mal-FA), we obtained a kind of biocompatible nanoparticles with NIR emitting for targeted imaging of FA receptor carring tumors in vivo.(3) There have been no reports on enzyme-controlled disassembly of self-quenched NIR fluorescent nanoparticles with fluorescence turning on for specific detection/imaging of an enzyme’s activity in vitro and in vivo. We reported the rational design of a new NIR probe Cys(StBu)-Arg-Val-Arg-Arg-Lys(FPR-675)-CBT whose fluorescence signal is self-quenched upon reduction-controlled condensation and subsequent assembly to nanoparticles. Disassembly of the nanoparticles by Furin turns the fluorescence on. With this " off-on " strategy, we successfully applied this probe for the NIR detection of Furin in vitro and in vivo.(4) We reported a new type of dual-functional probe Cys(StBu)-Asp-Glu-VaI-Asp-Lys(FMBA)-CBT (1), which can sequentially detect GSH and Casp3/7activity. A3,5-Bis-trifluoromethyl-benzoic acid motif conjugating to the side chain of a lysine (Lys) motif was used to generate the19F NMR/MRI signal. When the probe was uptaken into cells, the intracellular GSH would reduce the disulfide bond of Cys, leading to the condensation between Cys and CBT, and the condensation product then self-assembled to nanoparticles. Because the formation of nanoparticles induced fast transverse relaxation of19F magnetic spin for its slower molecular tumbling, the19F NMR signal became broad and intensity decreased. Under the effect of Casp3/7, nanoparticles were disassemblied to free monomers, and the19F NMR signal recovered. Through this on-off-on process, we could sequentially detect GSH and Casp3/7activity.(5) Based on the study (4), we reported a probe Cys(StBu)-Ala-Ala-Asn-Lys(FMBA)-CBT (2) and its control compound Ac-Ala-Ala-Asn-Cys(StBu)-Lys(FMBA)-CBT (2-A), both of which could detect the Lgmn activity specifically. When the probe2was uptaken into cells, the intracellular GSH reduced the disulfide bond of Cys, leading to the condensation between Cys and CBT. The condensation product then self-assembled to nanoparticles, and therefore, the19F NMR signal became broad and intensity decreased. Under the effect of Lgmn, nanoparticles were disassemblied to free monomers, and the19F NMR signal recovered. Through this on-off-on process, we could sequentially detect GSH and Lgmn activity. Following a different way, the control compound2-A, whose NH2group of disulfided Cys motif is caged by AAN, was firstly subjected to GSH-reduction of the disulfide bond to yield2-A-Red inside cells, keeping the19F NMR signal "on". Subsequent cleavage of the AAN substrate of2-A-Red by Lgmn initiated the condensation reaction and self-assembly of the nanoparticles (i.e.,2-A-NPs) occurred, turning the19F NMR signal "off. It offers an on-on-off process to monitor Lgmn activity.(6) Multidrug resistance (MDR) has remained the biggest challenge facing the treatment of cancers. Current general strategies for overcoming MDR include inhibiting MDR efflux pumps or using nanocarries to encapsulate large amount of drugs. We proposed a new strategy of intracellular self-assembly of nanodrugs for overcoming MDR. Employing the CBT-Cys condensation reaction, we rationally designed a taxol derivative CBT-Taxol which could be subjected to Furin-controlled condensation and self-assembly of taxol nanoparticles (Taxol-NPs). In vitro and in vivo studies indicated that, compared with taxol, CBT-Taxol showed4.5folds and1.5folds increase of anti-MDR effects on taxol-resistant HCT116cancer cells without imposing toxicity on the mice. Our results demonstrate that structuring protease-susceptible agents and intracellular assembling them into nanodrugs could be a new optimal strategy for overcoming MDR.(7) Cell-cell interactions play a crucial role in the development and function of multicellular organisms. To study cell-cell interactions in vitro, it is a big challenge for researchers to artificially build up cell junctions to bridge different types of cells. Herein, by employing two orthogonal click reactions, we rationally designed four click reagents Mal-CBT, Mal-Cys, Mal-AIkyne, and Mal-N3and successfully applied them for bridging cells in three colors. Orthogonality between the two click reactions was initially validated in solution through characterization with HPLC and ESI-MS analyses. After modifications of fluorescent protein-expressing prokaryotic E. Coli cells (or eukaryotic HEK293T cells) in three colors with respective Mal-Cys, Mal-CBT and Mal-AIkyne, or Mal-N3, the cells were sequentially bridged. The HEK293T cells showed higher efficiency of cell bridging than that of E. Coli cells due to the higher content of thiol groups on the surface of HEK293T cells. Our results demonstrate that it is possible to use two (or n) orthogonal click reactions to bridge three (or n+1) types of cells. Taken the biological importance of cell junctions into consideration, we anticipate our method of bridging three types of cells with two bio-orthogonal click reactions to be a useful tool for biologists to study cell-cell interactions with more convenience and efficiency.This dissertation aims at using the CBT-Cys condensation click reaction to develop novel applications in molecule imaging, cancer therapy, and cell assembly. This efficient and biocompatible click reaction offers new directions for biomolecule sensing, imaging, and modification.