-azido acid chloride coupling companion, a comparison from the 13C NMR information of (S)-17 with these of (R/S)-17 [note that the (R/S) label denotes a 1:1 mixture of diastereoisomers epimeric at the stereogenic center positioned for the amide carbonyl group in 17] showed that no epimerization had occurred in the stereogenic center under the acylation reaction circumstances (see the Supporting Facts). Within the final step, Huisgen [3+2] dipolar cycloaddition52,53 in the azide in (S)-17 with alkyne 18 (see the Supporting Details) provided our target, biotinylated ThrCer (S)-10, in 77 isolated yield. Prior to progressing with biological evaluation of biotinylated ThrCer (S)-10 and to assess the importance of acquiring the appropriate stereochemistry in the tethering web-site, we utilised racemic 2azidohexacosanoic acid inside the synthetic sequence summarized in Scheme two to access the diastereoisomeric finish solution (R)10 [epimeric in the tethering site; the (R) label denotes theFigure five. Detection of biotinylated ThrCer ten (1:1 epimeric mixture) around the surface of APCs. C1R CD1d cells had been pulsed with the indicated concentrations of biotinylated ThrCer for 16 h then stained with (a) soluble iNKT cell TCR (APC), (b) fluorescent streptavidin (APC), or (c) the antibiotin antibody [labeled with fluorescein isothiocyanate (FITC)]. Panels a-c depict histogram overlays of the indicated fluorescence intensities. The dot plot shown in panel d depicts a double staining with the soluble iNKT cell TCR (APC) and antibiotin antibody (FITC).2-(Oxetan-3-yl)acetic acid Price The outcomes are representative of two separate experiments.1196155-05-1 supplier dx.doi.org/10.1021/bc300556e | Bioconjugate Chem. 2013, 24, 586-Bioconjugate Chemistry Scheme 3. Synthesis of Fluor 488-Labeled -GalCer C20:2ArticleFigure 6. In vitro activation of iNKT cells by Fluor 488-labeled GalCer C20:2 11. Splenocytes from C57 BL/6 mice have been cultured inside the presence of numerous concentrations of either unlabeled four or Fluor 488-labeled -GalCer C20:2 11 for 48 h. The supernatants have been then analyzed for the presence of IFN- by an ELISA. Data are means of duplicate wells and are representative of two independent experiments.PMID:23577779 absolute configuration with the stereogenic center located to the amide carbonyl group], which may very well be separated from its epimer (S)-10 by careful column chromatography. A 1:1 epimeric mixture (S/R)-10 and the single epimers of biotinylated ThrCer, (S)-10 and (R)-10, were subsequent tested separately for their capability to be loaded onto human CD1dmolecules as defined by staining with tetrameric human iNKT cell TCR29,54 (Figure 3a) and recognized by murine iNKT cells (Figure 3b).23 In the initially experiment, C1R-human CD1d cells have been loaded with ThrCer 5 as well as the corresponding biotinylated analogues and then incubated with fluorescent human iNKT cell TCR. Subsequent fluorescence-activated cell sorting (FACS) analysis revealed that (S)-10 and the epimeric mixture behaved like unlabeled ThrCer 5 in their ability to be recognized by the iNKT cell TCR, though the activity in the (R)-epimer, (R)-10, utilized on its own, was slightly attenuated (Figure 3a). Within a second assay, C1R-mouse CD1d cells have been very first loaded with ThrCer 5 and the corresponding biotinylated analogues and then cultured overnight within the presence of iNKT cell hybridoma (DN32). iNKT cell activation was assessed by measuring interleukin-2 (IL-2) production by an ELISA.55,56 When once more, and in line with our predictions, the two epimeric biotinylated ThrCer analogues behaved differently in their abili.
