Design and synthesis of high affinity ligands for the asialoglycoprotein receptor (ASGP-R)

摘要

The asialoglycoprotein receptor (ASGP-R) is a carbohydrate-binding protein fromudthe C-type lectin family that is expressed exclusively and in high numbers onudmammalian hepatocytes. The human ASGP-R is a transmembrane protein,udconsisting of two homologous subunits (H1 and H2), that recognizes and bindsuddesialylated glycoproteins with terminal galactose or N-acetylgalactosamineudresidues. The binding process is followed by receptor-mediated endocytosis ofudthe receptor-ligand complex by the parent hepatocyte. The ASGP-R is thenudrecycled back to the surface, whereas the ligand is ferried to the lysosomes forudenzymatic degradation. Due to its location and efficient ligand uptake, the ASGPRudhas for a long time been a validated target for liver-specific drug delivery.udFurthermore, there is substantial evidence that the ASGP-R is involved inudhepatitis B and C virus entry into the liver cells.udThe focus of this thesis was to design and synthesize various high affinity ligandsudfor the ASGP-R that could be used as (1) drug carriers for liver-specific druguddelivery, (2) small molecular weight inhibitors of hepatitis B/C entry, (3) a spinlabeledudGalNAc-based molecular probe for second binding site screening byudNMR, and (4) a set of trivalent compounds for investigating the localudconcentration effect on ligand affinity towards the ASGP-R by surface plasmonudresonance (BIACORE).udThe trivalent drug carrier for liver-specific drug delivery was shown to bind withudhigh affinity and selectivity to the ASGP-R, and is now awaiting the next step,udnamely, its conjugation to a therapeutic agent and in vivo testing.udThe TEMPO spin-labeled GalNAc derivative was successfully used as a first-siteudligand for second-site screening by NMR, in which imidazole was identified as audpotential second-site ligand. Therefore, after the removal of the TEMPO spinudlabel the first-site ligand will be used in further studies, involving “in situ click chemistry”, in order to find the appropriate linker for joining the first- and secondsiteudligands.udThe four trivalent compounds synthesized for investigating the localudconcentration effect had an identical molecular mass and scaffold, but differed inudthe ratio of D-galactose to D-glucose moieties per molecule. Since the affinity ofudglucose towards the ASGP-R is > 20 mM, and that of galactose is 2.2 mM, theudaffinity was expected to increase with increasing number of galactose moieties.udHowever, the compound bearing two galactose and one glucose residueudunexpectedly showed an affinity greater than that for a compound with threeudgalactose residues. The phenomenon is yet to be explained and verified byudfurther experiments. Nevertheless, the results presented in this work did confirmudthat the statistical local concentration effect has a weaker influence onudmultivalency than the chelate effect.