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Rease affinity and selectivity for hCD22 more than other siglecs. To compare these analogues straight, a custom array containing 1, 4, 12, 22, and 23, printed at 100 M and 3 M printing concentration, was constructed. Utilizing a sensitive 2-step detection strategy (see Procedures section) and evaluating SIK2 Inhibitor Formulation binding at many concentrations in the hCD22-Fc, compound four showed a larger avidity than compound 12 (Fig. 3a and Fig. S4, ESI). However, the related analogue, 23, had comparable avidity to compound 4, as well as exhibited outstanding selectivity for hCD22 more than other siglecs (Fig. 3b and Fig. S4, ESI). To confirm these final results, a solution-phase, competitive inhibition assay was utilized to identify IC50 values of compounds 1, four, and 23 for hCD22. With this assay, the all-natural sialoside (1) yielded an IC50 worth in the range of prior observations (IC50 = 99 M).47?9 The 4-biphenyl derivative (four) had an IC50 of 0.35 M, while compound 23 gave a roughly 2-fold larger worth (IC50 = 0.65 M). To be able to increase the affinity of compound 23 however retain selectivity for hCD22, we hypothesized that a N-fluoroacetamide group could be installed in the C5 position based on preceding reports which documented that this modification yields a selective raise in affinity for hCD22 over Sn.36, 50 As such, each the mono- and disubstituted 5-N-fluoroacetamide containing compounds, 24 and 25, respectively, were synthesized (see ESI). As hoped, the 5-N-fluoroacetamide group gave an additive affinity increase (roughly 3-fold), using the most potent compound 25 yielding an IC50 of 0.2 M. Depending on our previous final PKCĪ³ Activator Source results with compound (4)-displaying liposomes,28 we had been confident that liposomes bearing 25 would bind avidly to CD22-expressing cells. It was uncertain, even so, in the event the minor decrease in affinity of 23 would yield equivalent outcomes. In testing these liposomes with all the hCD22-expressing, non-Hodgkin’s lymphoma B-cell line, Ramos, both 23- and 25-displaying liposomes, at four molar ligand concentration, show exceptional binding and, not surprisingly, the 25-bearing liposomes are superior (Fig. S5, ESI). Each of these ligand-bearing liposomes had been then assessed for selectivity working with our panel of siglec expressing cell lines (Fig. 3d). Notably, no binding was detected with mSn-expressing CHO cells or any other siglec inside the series (Fig. 3d). Experiments with white blood cells isolated from peripheral human blood showed that only cells expressing CD22 are targeted,NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptChem Sci. Author manuscript; offered in PMC 2015 June 01.Rillahan et al.Pageand moreover, the binding correlates with CD22 intensity (Fig. 3e). As expected as a result of the restricted expression of CD22 on B cells, this CD22+-liposome+ cell population consists completely of CD19+ B cells (data not shown). In summary, we’ve got developed high affinity hCD22-specific sialic analogues devoid of cross-reactivity to other siglecs, opening the door for future studies aimed at targeting hCD22 for therapeutic gain.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptConclusionsSelective, high affinity ligands of siglecs have confirmed to have utility as novel chemical probes for elucidating the natural function of those receptors,30, 51, 52 and for targeting nanoparticles to siglec-expressing cells in vivo.28, 29 By loading these nanoparticles with various therapeutic payloads, siglec-targeted nanoparticles represent a versatile platform for cell-targ.