The synthesis and study of TF antigen functionalized dendrimers and dendrimer end group characterization and indium(III) promoted glycosylation

Abstract

Polyamidoamine (PAMAM) dendrimers were functionalized with the Thomsen-Friedenreich (TF) antigen to study the multivalent effects on the galectin-3 mediated homotypic aggregation of A549 cells. TF antigen functionalized dendrimers of generations 2, 3, 4, and 6 were found to induce cellular aggregation. This is in contrast with previously observed results using lactose functionalized dendrimers, in which lactose functionalized generation 2 dendrimers were able to inhibit cellular aggregation. Additionally, TF antigen functionalized generation 6 dendrimers did not induce cellular aggregation as effectively as lactose functionalized generation 6 dendrimers. These preliminary results suggest that when compared to lactose functionalized dendrimers, the stronger galectin-3 binding affinity for TF antigen dendrimers may allow for more galectin-3 recruitment, creating aggregates with less freedom to rearrange into an optimized conformation. This suggests the reversibility of the binding event is important for effective protein interactions. The synthesis of TF antigen was achieved using indium triflate catalyzed glycosylation reactions. The development of indium(III) as a glycosylation promoter involved the analysis of indium bromide, indium chloride, and indium triflate for use in glycosylation reactions with a variety of alcohol acceptors. In(OTf) 3 mediated glycosylations of acetonide protected mannosides afforded exclusively alpha products in high yields. Acetylated mannosides gave moderate yields of exclusively alpha products using InBr 3 and InCl 3. Benzylated galactosides gave moderate yields of alpha, beta product mixtures using In(OTf) 3, with beta products formation being favored. Indium(III) was also used to synthesize alpha-1-2-dimannoside and alpha-galactose-1-2-mannoside in high yield. Additionally, spin labelled PAMAM dendrimers were preliminarily studied via electron paramagnetic resonance (EPR) to analyze the spatial arrangement of end groups. Dendrimers were functionalized with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) covalently tethered as a dimer. At low percent loading, a strong effect arising from the dimeric spin-labeled end groups was observed. EPR spectra of dendrimers bearing a higher loading of the dimeric spin-labeled end groups indicated that the end group arrangement approached a random distribution at approximately 40 to 50 percent loading. This suggests that covalently clustered pairs of end groups are significantly different from randomly distributed end groups on PAMAM dendrimers at low loading and become equivalent to randomly functionalized dendrimers around 50% functionalization.