Rease affinity and selectivity for hCD22 more than other siglecs. To compare these analogues straight, a custom array containing 1, four, 12, 22, and 23, printed at one hundred M and 3 M printing concentration, was constructed. Using a sensitive 2-step detection mTOR Modulator Molecular Weight method (see Techniques section) and evaluating binding at different concentrations on the hCD22-Fc, compound 4 showed a larger avidity than compound 12 (Fig. 3a and Fig. S4, ESI). However, the related analogue, 23, had comparable avidity to compound 4, and also exhibited exceptional selectivity for hCD22 over other siglecs (Fig. 3b and Fig. S4, ESI). To confirm these final results, a solution-phase, competitive inhibition assay was utilized to establish IC50 values of compounds 1, 4, and 23 for hCD22. With this assay, the all-natural sialoside (1) yielded an IC50 worth inside the array of prior observations (IC50 = 99 M).47?9 The 4-biphenyl derivative (4) had an IC50 of 0.35 M, whilst compound 23 gave a roughly 2-fold higher value (IC50 = 0.65 M). So that you can enhance the affinity of compound 23 but retain selectivity for hCD22, we hypothesized that a N-fluoroacetamide group could possibly be installed at the C5 position determined by previous reports which documented that this modification yields a selective increase in affinity for hCD22 over Sn.36, 50 As such, both the mono- and disubstituted 5-N-fluoroacetamide containing compounds, 24 and 25, respectively, had been synthesized (see ESI). As hoped, the 5-N-fluoroacetamide group gave an additive affinity increase (roughly 3-fold), with all the most potent compound 25 yielding an IC50 of 0.2 M. According to our previous outcomes with compound (4)-displaying liposomes,28 we were confident that liposomes bearing 25 would bind avidly to CD22-expressing cells. It was uncertain, nonetheless, when the minor decrease in affinity of 23 would yield comparable results. In testing these liposomes together with the hCD22-expressing, non-Hodgkin’s lymphoma B-cell line, Ramos, both 23- and 25-displaying liposomes, at four molar ligand concentration, show fantastic binding and, not surprisingly, the 25-bearing liposomes are superior (Fig. S5, ESI). Both of those ligand-bearing liposomes were then assessed for selectivity applying our panel of siglec expressing cell lines (Fig. 3d). Notably, no binding was detected with mSn-expressing CHO cells or any other siglec in 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 furthermore, the binding correlates with CD22 intensity (Fig. 3e). As expected due to 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 higher affinity hCD22-specific sialic analogues without the need of PDE7 Inhibitor web cross-reactivity to other siglecs, opening the door for future research 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 possess utility as novel chemical probes for elucidating the organic 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.