쿠커비투릴 골격으로 이루어진 탄수화물 다량체와 염료-쿠커비투릴 복합체의 생물학적 응용

Abstract

Although the host-guest chemistry of cucurbit[n]uril (CB[n]) has been studied extensively over the last decade, their applications were little explored until recently due to the lack of functionalization methods and their poor solubility in common organic solvents. The direct functionalization method of CB[n] developed recently not only solved the solubility problem but also opened up the way to synthesize various tailor-made CB[n] derivatives. Furthermore, the functionalization of CB[n] allowed us to explore numerous applications of CB[n] derivatives. The aim of this thesis is to develop CB[n]-based functional materials useful for biological applications, such as targeted drug delivery, antiadhesion therapy, and biogenic amine sensor.Chapter 2 describes novel carbohydrate clusters in which a number of carbohydrate moieties are attached to the periphery of a rigid cucurbit[6]uril (CB[6]) scaffold like a wheel. Multiple copies of carbohydrates on a CB[6] scaffold enhance the affinity and selectivity in the binding with protein. CB[6]-based glucose, galactose, and mannose clusters (GlcCB[6], GalCB[6], and ManCB[6], respectively) were synthesized and fully characterized. Turbidimetric assay illustrated the specific and multivalent interaction between the CB[6]-based carbohydrate clusters and concanavalin A (ConA). Isothermal titration calorimetry established that ManCB[6] behaves as a trivalent ligand to ConA with a binding constant K = (1.9 ∂ 0.2) ⊥ 105 M？V1, which is almost 25 times higher than that of monomeric methyl-？？-mannopyranose (Me-？？Man) to ConA. The new type of carbohydrate clusters forms stable host-guest complexes with a wide range of guest molecules including FITC-spermine conjugate by taking advantage of the cavity provided by the CB[6] scaffold. Intracellular translocation experiments demonstrated the facile delivery of FITC-spermine@Gal-CB[6] into HepG2 cells which recognized the galactose moieties of the complex. The specificity and enhanced binding affinity due to the multivalency suggest the potential utility of the CB-based carbohydrate clusters in targeted drug delivery. Chapter 3 describes pseudopolyrotaxanes (ManCB[6]@PV) formed by ManCB[6]s threaded on a polyviologen (PV) linear polymer to increase the multivalent effects. This type of structures offer a flexible and dynamic platform for the multivalent display of ligands, which provides even stronger multivalent interactions with bacteria, and may thus show an effective inhibitory activity for bacterial binding onto epithelial cells. Mannose units on ManCB[6]@PV selectively interacted with the FimH proteins on ORN 178 E. coli strain and thus efficiently aggregated the bacteria. The hemagglutination inhibition assay showed that ManCB[6]@PV exhibit a 300-fold enhancement over Me-？？Man. The ability of ManCB[6]@PV to inhibit ORN 178 bacterial binding to the urinary epithelial cell (UROtsa cell) was investigated in vitro by flow cytometry. In the absence of the ManCB[6]@PV, the FITC-labeled ORN178 significantly interacts with UROtsa cell and thus gives strong fluorescence signal. However, after pre-treating of ManCB[6]@PV to ORN 178, the fluorescence signal was reduced as a result of inhibition of the binding between FITC-labeled bacteria and UROtsa cells. These results suggest ManCB[6]-PV pseudopolyrotaxanes are potentially useful in antiadhesion therapy by inhibiting infection of pathogens into the host cell. Chapter 4 describes the simple sensor elements based on the reversible interaction between dyes and CB[n] (n = 6, 7, or 8) for the discrimination of biogenic amines. NMR experiments and fluorescence enhancement study showed that 4-N,N-dimethylamino-4'-N'-methyl-stilbazolium iodide (DAMSI) forms a inclusion complex with CB[6] (D@CB[6], K = (3.9 ∂ 0.2) ⊥ 103 M？V1) and the complex emits 450 times enhanced fluorescence than the free dye. Another fluorescent dye, Hoechst 33258 showed an 100-fold enhancement in fluorescence when it forms a complex with CB[7] (H@CB[7], K = (3.91 ∂ 0.02) ⊥ 104 M？V1). Likewise, CB[8] also interact with Hoechst 33258 (H@CB[8]) and enhanced the fluorescence of the dye. In the presence of biogenic amines, which have higher affinities to CB[n] than those of the dyes, the dyes are released from the cavity of CB[n]. The exposure of biogenic amines to three dye@CB[n] complexes (D@CB[6], H@CB[7], and H@CB[8]) induced different levels of fluorescence changes. The patterns of fluorescence responses were analyzed by principle component analysis (PCA), which allowed us to discriminate eight biogenic amines with dye@CB[n] complexes.