Rotaxanes are a class of interlocked compounds that have been extensively investigated for their potential utility as switches or sensors. We recently demonstrated that Rotaxanes have further application as agents that transport material into cells. This novel finding prompted our investigation into the mechanism by which Rotaxanes are involved in transmembrane transport. Presented in the following dissertation are four major research focus areas in the development of host-Rotaxanes and investigation of their binding abilities and intracellular transport mechanism of the host-Rotaxanes.
The first chapter is about the mechanism study of a cleft-[2]Rotaxane as a intracellular transport agent. Two-dimensional NMR analysis showed that a cleft-containing rotaxane exists in two dominant conformations (‘closed’ and ‘open’). The rotaxane was chemically modified to lock it in the closed conformation in order to determine the importance of conformational flexibility on the ability of the rotaxanes to bind guests and transport material into cells. Charged guests interact less favorably with the locked rotaxane, as compared to the unmodified rotaxane, in both an aqueous solution and in DMSO. The locked rotaxane exhibited a reduced capacity to transport a fluoresceinated peptide into cells, whereas, the unmodified rotaxane efficiently delivers the peptide. Flow cytometry experiments demonstrated that a high percentage of the cells contained the delivered peptide, the level of delivery is concentration dependent, and the rotaxanes and peptide have low toxicity. Cellular uptake of the peptide was largely temperature and ATP independent, suggesting that the rotaxane-peptide complex passes through the cellular membrane without requiring active cell-mediated processes. The results show that the sliding motion of the wheel is necessary for the delivery of materials into cells and can enhance the association of guests.
The second chapter is about the synthesize, binding study, and intracellular transport mechanism study of a novel host-[3]rotaxane. In order to improve the affinity, selectivity, and delivery of guests, a host-[3]rotaxane (Cy3R) was constructed. The properties of Cy3R were compared to a new host-[2]rotaxane (Cy2R), which has the same cyclophane pocket but only a single wheel. The second wheel of Cy3R can form additional salt-bridges, cation-π interactions with appropriately functionalized guests and is required for the delivery of Fl-AVWAL and Fl-QEAVD into COS 7 cells. Flow cytometry experiments demonstrated that a high percentage of the cells, in the presence of Cy3R (10 µM) and a Fl-peptide (10 µM), contained Fl-AVWAL (76%) and a moderate level of cells contained Fl-QEAVD (26%). The level of delivery is concentration dependent and largely temperature and ATP independent, suggesting that the Cy3R•peptide complexes pass through the cellular membrane without requiring active cell-mediated processes. Cy2R, on the other hand, does not deliver materials into cells at the same or higher concentrations of components that were used for Cy3R and other host-[2]rotaxanes. Cy2R forms weak complexes with the Fl-peptides, which most likely is responsible for its inability to deliver materials into cells. Host-rotaxanes that bind fluorescein tightly in DMSO (KA > 1 x 105 M-1) are efficient transporters. Although Cy3R binds fluorescein in buffered water, DMSO and CHCl3 with KA’s of 4 x 104 M-1, it does not transport fluorescein. This result suggests that strong association in DMSO, similar in polarity to the cell surface, is required for intracellular delivery.
The third chapter is about the total synthesize of novel Rhodamine-[B] host-[2]Rotaxanes. In order to investigate the Rotaxanes intracellular location when it acts as a intracellular transporter, we designed two novel Rhodamine-B host-[2]Rotaxanes that linked to the rotaxane architecture, and we assume that Rhodamine-B rotaxane will delivery material as usual, so its position in the cell can be determined by simply observing the red fluorescence in the cells and quantified via flow cytometry experiment.
The fourth chapter is about design and synthesize of a series of reversible covalent bond forming Rotaxanes. We assume that aldehydic rotaxane should form reversible imine bonds with guests, and these Rotaxanes should associate with amino guests at very low concentrations.