After deacylation of 5, subsequent values found in their mass spectra, and their purity was verified by TLC and NMR spectroscopy. O-specific polysaccharides to determine the mode of antigenCantibody binding in the O:1, serotype Inaba and Ogawa systems.11,12 The disaccharide described herein is equipped with a linker (spacer) allowing conjugation to proteins. The hydroxyl group at C-4II in the intermediate alcohol 9 is positioned to allow further build-up of the molecule and, eventually, construction of the complete hexasaccharide. 2. Results and discussion The synthesis of 10 was planned with the future construction of the hexasaccharide A (Fig. 1) in mind, and conjugation of the Rabbit polyclonal to ACBD6 latter to protein carriers using, for example, squaric acid chemistry.13,14 Accordingly, one of its intermediates should allow extension of the oligosaccharide chain at HO-4II, and protecting groups present should be stable during a variety of chemical transformations leading to the hexasaccharide. We anticipate that the last step before conversion of the antigenic carbohydrate to the squaric acid monoester14 would be reduction of azide in the spacer into amine. Therefore, benzyl-protecting groups seemed to be the natural choice, as those would be removed simultaneously with the reductive azideamine conversion. The synthesis of 10 (Scheme 1) started with the known thioglycoside 3, prepared Amotosalen hydrochloride as described,15 except that the glycosylation reaction of halide 1 and acceptor 2 was carried out at almost neutral conditions. This minimized formation of byproducts15 due to hydrolysis of the benzylidene ring in 3. Treatment of 3 with 8-azido-3,6-dioxaoctan-1-ol6 gave the spacer-equipped disaccharide 4 whose benzylidene acetal underwent HanessianCHullar ring opening16,17 to regioselectively form 5, a bromide at the 6 position, in 77% yield. After deacylation of 5, subsequent values found in their mass spectra, and their purity was verified by TLC and NMR spectroscopy. Palladium-on-charcoal catalyst (5%) (Escat? 103) was purchased from Engelhard Industries. Rubber septa used to close reaction flasks containing organic solvents were protected with a thin Teflon? sheet, to avoid leaching. Solutions in Amotosalen hydrochloride organic solvents were dried with anhydrous Na2SO4, and concentrated at 40 C/2 kPa. 3.2. Ethyl 4,6-1.8, CHCl3); 1H NMR (600 MHz, CDCl3) = 7.4 Hz, 1H, NH), 5.55 (s, 1H, PhCH), 5.31 (dd, = 9.6 Hz, 1H, H-3I), 4.34 (dd, = 5.0 Hz, 2H, H-6), 2.11 (s, 3H, COCH3), 2.00 (s, 3H, COCH3), 1.95 (s, 3H, COCH3), 1.91 (s, 3H, COCH3); NMR (150 MHz, CDCl3) 2.1, CHCl3); 1H NMR (600 MHz, CDCl3) = 7.6 Hz, 1H, NH), 5.20 (d, = 9.5 Hz, 1H, Amotosalen hydrochloride H-4I), 5.08 (d, = 9.0 Hz, 1H, H-3I), 4.01C3.99 (m, 1H, = 2.6, 11.4 Hz, 1H, = 5.1, 9.0 Hz, 1H, 1.7, CHCl3); 1H NMR (600 MHz, CD3OD) = 8.2, 10.5 Hz, 1H, H-3I), 3.96C3.93 (m, 1H, = 2.1, 11.2 Hz, 1H, = 5. 0 Hz, H-6); NMR (150 MHz, CD3OD) 0.9, CHCl3); 1H NMR (600 MHz, CDCl3) = 7.1 Hz, 1H, NH), 6.84C6.82 (m, 2H, aromatic protons), 5.45 (s, 1H, 4-MeOPhC= 10.5 Hz, 1H, PhC= 10.5 Hz, 1H, PhC= 10.5 Hz, 1H, PhC= 1.5, 12.8 Hz, 1H, H-6IIa)), 4.09 (d, 1.7, MeOH); 1H NMR (600 MHz, CDCl3) = 10.3 Hz, 1H, PhC= 11.6 Hz, 1H, PhC= 11.6 Hz, 1H, PhC= 11.6 Hz, 1H, PhC= 11.6 Hz, 1H, PhC= 10.3 Hz, 1H, PhC= 7.4, 11.5 Hz, 1H, = 1.8, 11.3 Hz, 1H, 1.8, CHCl3); 1H NMR (600 MHz, CDCl3) = 11.3 Hz, 2H, PhC= 4.7, 5.9 Hz, 2H, H-6), 2.56 (d, = 1.4 Hz, 1H, 4II-OH); NMR (150 MHz, CDCl3) = 9.2 Hz, 1H, H-4I), 3.21 (t, = 5.4 Hz, 2H, H-6), 2.04 (s, 3H, COCH3), 1.36 (d, em J /em 5,6 = 6.2 Hz, 3H, H-6I); NMR (150 MHz, D2O) em /em : 174.5 (CO), 174.4 (CO), 103.0 (C-1II), 100.8 (C-1I), 82.5 (C-3I), 75.3 (C-5II), 74.0 (C-4I), 72.6 (C-3II), 71.5 (C-5I), 70.2 (C-2II), 70.0 (C-4II), 69.7C65.3 (5C, C-1, C-2, C-3, C-4, C-5), 54.6 (C-2I), 39.1 (C-6), 22.2 (CO em C /em H3), 16.7 (C-6I); TOF-HRMS, em m /em / em z /em : calcd for C20H35N2O13 [MCH]+: Amotosalen hydrochloride 511.2139, found: 511.2132. Acknowledgment This research was supported by the Intramural Research Program of the NIH, NIDDK. Dedicated to Professor Dr. Andrs Liptk on the occasion of his 75th.