Abstract
Carbohydrate-lectin interactions involve in various biological recognitions and mediate many processes such as cellular recognition, adhesion, signal transduction, glycoprotein clearance, immunomodulation, inflammation, and host-pathogen recognition. To fully understand the biological implications of carbohydrate-lectin interactions in living organisms, it is imperative to investigate and profile their functions under pathological conditions. However, it is quite difficult to probe the carbohydrate interacting protein(s) due to the intrinsic characters of carbohydrate-lectin interaction: non-covalent, reversible, and weak binding affinity. Thus, for low affinity interaction between carbohydrate ligand and lectin, the monovalent carbohydrate ligand conjugated with photoaffinity probe may not be suitable for target lectin labeling. The multivalent carbohydrate structures which greatly enhance the weak affinity of individual mono-ligands to their binding lectins is a solution to overcome the weak affinity problem. We designed a novel and multifuntional photoaffinity scaffold that derived from a glutamic acid core, carried a powerful photo-cross-linker, 3-trifluoromethyl-3-phenyl-diazirine, and two well-defined orthogonal synthetic handles that empower subsequent conjugations. Multivalent carbohydrate and tag of the probe were conveniently conjugated via orthogonal reactions, Cu(I) catalyzed click reaction and amide bond formation. The tri-valent (compound 43) galactose photoaffinity probe demonstrate that the designed probe is able to selectively label the target lectin, RCA120 (Ricinus communis Agglutinin) in E. Coli. lysates and ASGP-R (Asialoglycoprotein receptor) on intact HepG2 cell membrane. Moreover, we had synthesized a second generation carbohydrate photoaffinity probe for enrichment of lectin by combination of multivalent carbohydrate, bio-orthogonal chemistry, photochemical cleavage group, and functional magnetic nanoparticle.