Ions at the 4-position (Fig. 1a, compounds 17-21). While all of those analogues improved affinity and retained or improved selectivity, compound 17 appeared to become probably the most promising ligand generated as shown by the truth that it’s the only compound of this series detected at a printing concentration of 3 M in addition to a low hCD33 concentration (0.two g/ml, Fig. 1b bottom panel and Fig. S1, ESI). This was additional supported by experiments exactly where fluorescently labelled CHO cells expressing higher levels of hCD33 cells (CHO-hCD33) have been overlaid onto the array. In this case only 17 and 18 of this series can assistance binding of these cells, confirming that they exhibited highest avidity for CD33 (Fig. S3a, ESI). Having optimized substituents at the 3, 4, and five positions on the C9-benzamide ring we next asked when the additional addition in the previously identified C5 substituent, 4-cyclohexyl-1,2,3triazole (compound 2), would present further avidity.31 To accomplish the synthesis of a 9,5-disubstituted sialoside we employed a technique involving chemo-enzymatic synthesis of a sialoside orthogonally protected at the two positions (Scheme 1), as well as the aglycone. Within this tactic we employ a 3 enzyme one-pot reaction45, 46 that converts a 6azido-N-pentenoyl-mannosamine (E) into a 9-azido-5-N-pentenoyl sialic acid by condensation with pyruvate, which can be then activated for the corresponding CMP-sialic acid followed by sialyltransferase-mediated 2-6 sialylation on the von Hippel-Lindau (VHL) Degrader site lactoside (A) to yield the trisaccharide precursor (F). Subsequent deprotection with the pentenoyl group afforded (G) to which the 4-cyclohexyl-1,2,3-triazole was installed employing NHS chemistry. Reduction on the azide group at C9, followed by amine acylation, and hydrogenation with the Cbz group around the aglycone gave access to 22 in superior all round yield. As exemplified by the synthesis of 22, we believe this method represents a flexible strategy to synthesize 9,5-disubstitued sialosides. Microarray evaluation showed that 22 exhibited superior properties when compared with the monosubstituted compounds, for hCD33. In specific, 22 exhibited larger avidity than each parent compounds, 17 and 2 (Fig. 1b bottom panel and Fig. S1, ESI), and showed elevated selectivity for hCD33 more than hCD22 and mSn (Fig. 1c). This raise in avidity was additional supported by the fact that HL-60 cells, an AML cell line expressing intermediate levels ofChem Sci. Author manuscript; out there in PMC 2015 June 01.Rillahan et al.PagehCD33, bound only to compound 22, but not to any other analogue in our library (Fig. S3b, ESI).NIH-PA Author TBK1 Inhibitor Compound manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSince glycan microarrays supply only qualitative measures of avidity and selectivity, we analysed the relative affinities of those compounds employing solution-phase inhibition assays. Accordingly, IC50 values have been determined employing a flow cytometry assay, wherein compounds are evaluated for their capability to stop the binding of fluorescently labelled hCD33 to ligand-coated beads, and these values have been made use of to ascertain the relative inhibitory potency (rIP) for each compound compared to the native sialoside (rIP = 1). Encouragingly, the outcomes of those assays had been in remarkable agreement with the qualitative estimation of avidity gains obtained from our microarray studies (Fig. 2a). As anticipated the native sialoside (1) showed a relatively low affinity for hCD33 (IC50 = 3.78 mM).47 Relative to the native sialoside, the optimal 5-substituted a.
