)H], 7.99 [s, 1H, C(1)H], exchangeable hydrogens not observed; 13C NMR (125 MHz, CDCl3/CD3OD, 2:1) 14.3 [CH3, C(18), C(26) (resonance overlap)], 23.two (CH2), 26.1 [CH2, C(12)], 26.2 (CH2), 26.four (CH2), 28.eight [CH2, C(14)], [29.1, 29.5, 29.87, 29.90, 30.1, 30.19, 30.22, 32.5 (CH2, alkyl chain, some resonance overlap)], 36.two [CH2, C(11)], 39.8 [CH2, C(9)], 40.eight [CH2, C(18)], 51.3 [CH, C(2)], 56.three [CH, C(15)], 60.9 [CH, C(17)], 62.6 [CH, C(16)], 63.9 [CH2, C(four)], 64.7 [CH, C(two)], 64.8 [CH2, C(three)], 70.17 [CH2, C(4)], 70.24 (CH2), 70.5 (CH2), 70.6 (CH2), 70.eight (CH), 70.99 (CH2), 71.03 (CH2), 72.five (CH), 72.eight (CH), 73.four [CH2, C(1)], 75.0 [CH, C(3)], 123.3 [CH, C(1)], 145.three [C, C(two)], 165.1 (C, NHCONH), 169.1 [C, C(1)], 175.2 [C, C(10)]; MS (TOF ES+) m/z 1276.eight ([M + Na]+, 100 ), 649.9 (7), 398.1 (5); HRMS (TOF ES+) calcd for C67H127N7O12SNa [M + Na]+ 1276.9161, identified 1276.9166. Fluor 488-Labeled -GalCer (11) (1:1 mixture of regioisomers inside the label). A CuSO4 answer (6 L of a 0.five M solution, three mol) and also a sodium ascorbate solution (13 L of a 1.0 M remedy, 13 mol) had been added to a option of azide 22 (1.four mg, 1.7 mol) and alkyne 23 (1.0 mg, 1.7 mol) inside a t-BuOH/H2O mixture (0.5 mL, 1:1) at area temperature.dx.doi.org/10.1021/bc300556e | Bioconjugate Chem. 2013, 24, 586-Bioconjugate Chemistry The reaction mixture was heated for 10 h at 50 , diluted with CHCl3 (five mL), and washed with brine (1.five mL). The phases have been separated, as well as the aqueous layer was extracted with CHCl3 (two ?2.5 mL). The combined organic layers have been dried more than Na2SO4, along with the volatiles have been removed below decreased stress. Purification on the residue by flash column chromatography (CHCl3/MeOH/H2O, 65:25:4) afforded triazole 11 as a red strong (1.four mg, 59 , 1:1, mixture of regioisomers within the label): Rf = 0.32 (CHCl3/MeOH/H2O, 65:25:4); 1H NMR (400 MHz, CDCl3/CD3OD, two:1) 0.83 (t, J = six.7 Hz, 3H), 0.84 (t, J = six.7 Hz, 3H), 1.10-1.39 (stack, 44H), 1.40-1.65 (m, 1H), 1.65-1.89 (m, 1H), 1.92-2.03 (stack, 5H), two.12-2.30 (1H, m), 2.66-2.79 (stack, 2H), three.46- 3.79 (stack, 22H), 3.86 (dd, J = 10.eight, four.8 Hz, 1H), three.90 (br s, 1H), four.12-4.22 (m, 1H), 4.55-4.67 (m, 2H), 4.88 (d, J = 3.6 Hz, 1H), five.21-5.41 (stack, 5H), six.63-6.77 (stack, 4H), six.96- 7.09 (stack, 2H), 7.31 (d, J = 9.1217725-33-1 Chemical name 0 Hz, 0.83249-08-5 Formula 5H), 7.PMID:24982871 76 (br s, 0.5H), 7.92 (s, 1H), 8.13-8.24 (m, 1H), 8.31 (d, J = 9.0 Hz, 0.5H), eight.72 (br s, 0.5H); MS (TOF ES-) m/z 1396.eight ([M – H]-, 10 ), 1218.eight (5, [M – galactose]-), 1004.six (15), 915.6 (25), 850.7 (65), 815.7 (35), 451.five (95), 423.five (100), 179.1 (20, [galactose]-). Experimental procedures and characterization data for compounds 25, 26, 13a, 13b, 14, (2S)-methyl 2-azidohexacosanoate, (S)-15, (S)-17, 18, (S)-10, (rac)-15, 17, ten, 11, 27-30, and 20 may be discovered within the Supporting Details. Mice, Cell Lines, and Cultures. C57 BL/6 mice had been made use of in accordance using the Animals (Scientific Procedures) Act 1986. iNKT cells had been generated from wholesome blood donors as previously described.29,34 iNKT cell cultures have been maintained in Roswell Park Memorial Institute-1640 (RPMI-1640) supplemented with 2 mM L-glutamine, 1 nonessential amino acids, 1 pyruvate, 1 Pen/Strep, 5 human serum (Gibco), and 500 units of IL-2/mL. Functional assays and C1R CD1d lipid pulsing were performed in RPMI-1640 supplemented with two mM L-glutamine, 1 nonessential amino acids, 1 pyruvate, 1 Pen/Strep (Gibco), and ten fetal calf serum (FCS) (Sigma). iNKT Cell TCR Tetramer Staining. The synthesis of soluble iNKT TCR heterodimers ha.
