and Heparin Analogs that Interact with Mycobacterial Heparin-binding Hemagglutinin SUPPORTING INFORMATION

Transcription

1 Glycosylation by DGlucosamineDerived Donors: Synthesis of Heparosan and Heparin Analogs that Interact with Mycobacterial Heparinbinding Hemagglutinin Medel Manuel L. Zulueta, a,b ShuYi Lin, a,c YaTing Lin, c ChingJui Huang, c ChunChih Wang, c ChiaoChu Ku, a Zhonghao Shi, a ChiaLin Chyan, d, * Deli Irene, d LiangHin Lim, d TsungI Tsai, a YuPeng Hu, a,c Susan D. Arco, b ChiHuey Wong, a and ShangCheng Hung a,e, * SUPPRTING INFRMATIN a Genomics Research Center, Academia Sinica, No. 128, Section 2, Academia Road, Taipei 115, Taiwan b Institute of Chemistry, University of the Philippines, Diliman, Quezon City 1101, Philippines c Department of Chemistry, National Tsing Hua University, No. 101, Section 2, KuangFu Road, Hsinchu 300, Taiwan d Department of Chemistry, National Dong Hwa University, No. 1, Sec. 2, TaHsueh Road, Shoufeng, Hualien 974, Taiwan e Department of Applied Chemistry, National Chiao Tung University, No. 1001, TaHsueh Road, Hsinchu 300, Taiwan S1

2 Table of Contents I. Synthetic procedures and characterization data... S3 A. General considerations... S3 B. Stereoselective glucosaminylation... S3 C. Synthesis of heparosan oligosaccharides... S21 D. Synthesis of heparin analogs... S36 II. Bioassay... S86 A. Preparation of the heparinbinding hemagglutinin (amino acid ) (HBHA )... S86 B. Isothermal titration calorimetry (ITC) measurements... S86 References... S88 NMR Spectra...S89 S2

3 I. Synthetic procedures and characterization data A. General considerations CH 2 Cl 2 was purified and dried from a safe purification system filled with anhydrous Al 2 3. All other reagents were obtained from commercial sources and used without further purification. Water was either distilled or MilliQpurified. Flash column chromatography was carried out on Silica Gel 60 ( mesh, E. Merck). TLC was performed on glass plates precoated with Silica Gel 60 F254 (0.25 mm, E. Merck); detection was executed by spraying with a solution of Ce(NH 4 ) 2 ( ) 6, (NH 4 ) 6 Mo 7 24, and H 2 S 4 in water and subsequent heating on a hot plate. Specific rotations were taken at ambient conditions and reported in 10 1 deg cm 2 g 1 ; the sample concentrations are in g dl 1. 1 H and 13 C NMR spectra were recorded on 400, 500, and 600 MHz spectrometers. Chemical shifts are in ppm calibrated using the resonances of the carbon and the residual proton of the deuterated solvent. Proton peaks were assigned with the aid of 2D NMR techniques ( 1 H 1 H CSY, HMQC, NESY, and HMBC). The hydrogen multiplicities of carbon peaks were determined using DEPT90 and DEPT135 experiments, the spectra of which were herein provided together with the powergateddecoupled 13 C NMR spectrum. B. Stereoselective glucosaminylation 2Naph H pbrbnbr, BH 3 THF, H Ag 2 2Naph Cu(Tf) 2 2NAP Bz pbrbn Bz pbrbn S1 S2 S3 Bz TBDPSCl, Et 3 N, DMAP TBDPS TBDPS CCl 3 CN, TBDPS 2NAP satd. NH 3 2NAP K 2 C 3 2NAP pbrbn Bz MeH/THF = 3/7 pbrbn pbrbn NH H CCCl 3 S4 S5 7 2Azido3(4bromobenzyl)6tertbutyldiphenylsilyl2deoxy4(2 naphthylmethyl)dglucopyranosyl trichloroacetimidate (7). (1) pbromobenzylation. A so S3

4 lution of compound S1 1 (24.6 g, 54.9 mmol) and pbromobenzyl bromide (20.6 g, 82.4 mol) in CH 2 Cl 2 (250 ml) containing freshly activated 4 Å molecular sieves (25.0 g) was stirred for 1 hour at room temperature under N 2 atmosphere. The mixture was protected from light and cooled to 0 C. Ag 2 was added to the mixture and the reaction was gradually warmed up to room temperature 10 min later. After 3 days of stirring, the mixture was filtered through Celite and the filtrate was concentrated under reduced pressure. Recrystallization of the residue in toluene provided the ether S2 (29.2 g, 86%) as a white solid. [ ] 19 D 12.5 (c 1.8, CHCl 3 ); mp C; IR (thin film): 2112, 1740, 1488, 1451, 1264, 1088, 1068, 1012, 821, 770, 712 cm 1 ; 1 H NMR (500 MHz, CDCl 3 ): (m, 2H; BzH), 7.95 (s, 1H; NaphH), (m, 3H; NaphH), 7.61 (t, J = 7.4 Hz, 1H; BzH), 7.56 (dd, J = 1.1, 8.5 Hz, 1H; NaphH), (m, 6H; ArH), 7.25 (d, J = 8.1 Hz, 2H; BrPhH), 5.81 (d, J = 8.3 Hz, 1H; 1H), 5.72 (s, 1H; NaphCH), 4.90 (d, J = 11.6 Hz, 1H; pbrphch 2 ), 4.79 (d, J = 11.6 Hz, 1H; pbrphch 2 ), 4.44 (dd, J = 5.0, 10.6 Hz, 1H; 6H eq ), (m, 4H; 6H ax, 4H, 2H, 3H), 3.67 (td, J = 5.0, 9.6 Hz, 1H; 5H); 13 C NMR (125 MHz, CDCl 3 ): (C), (C), (C), (CH), (C), (C), (CH), (CH), (CH), (CH), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (C), (CH), 93.6 (CH), 81.3 (CH), 79.3 (CH), 74.2 (CH 2 ), 68.4 (CH 2 ), 67.0 (CH), 65.1 (CH); HRMS (FAB): m/z Calcd for C 31 H 27 6 Br ([M + H] + ): , Found: (2) Regioselective 6 ring opening of 2naphthylmethylidene acetal. A 1 M solution of BH 3 THF in THF (236 ml, 236 mmol) was added to compound S2 (29.2 g, 47.3 mmol) at room temperature under N 2 atmosphere. The mixture was stirred until all solids dissolved and then cooled to 0 C. Copper(II) triflate [Cu(Tf) 2, 855 mg, 2.36 mmol] was added to the mixture which was, then, stirred for 6 h at icecold condition. The reaction was quenched by sequential S4

5 additions of Et 3 N (1 ml) and sufficient MeH (caution: H 2 was rapidly evolved; addition must be very slow) until all bubbling ceased. The resulting mixture was concentrated under reduced pressure and reconstituted with ethyl acetate and a dilute NaCl solution. The desired material was extracted with ethyl acetate and the combined organic layer was washed with brine, dried over anhydrous MgS 4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (ethyl acetate/hexanes = 1/3) to give the 6alcohol S3 (24.7 g, 84%) as a white solid. [ ] 19 D 9.8 (c 4.0, CHCl 3 ); mp C; IR (thin film): 3504, 2920, 2109, 1735, 1271, 1085, 1069, 1041 cm 1 ; 1 H NMR (500 MHz, CDCl 3 ): (m, 2H; BzH), (m, 3H; NaphH), 7.69 (s, 1H; NaphH), 7.60 (tt, J = 1.2, 7.4 Hz, 1H; Bz H), (m, 4H; BzH, NaphH), (m, 2H; BrPhH), 7.37 (dd, J = 1.6, 8.4 Hz, 1H; NaphH), (m, 2H; BrPhH), 5.73 (d, J = 8.4 Hz, 1H; 1H), 4.94 (d, J = 11.3 Hz, 1H; ArCH 2 ), 4.86 (d, J = 11.3 Hz, 1H; ArCH 2 ), 4.85, 4.80 (ABq, J = 11.2 Hz, 2H; ArCH 2 ), 3.92 (ddd, J = 2.3, 5.2, 12.3 Hz, 1H; 6H a ), (m, 2H; 4H, 6H b ), 3.69 (dd, J = 8.5, 9.8 Hz, 1H; 2 H), (m, 2H; 3H), 1.87 (dd, J = 5.7, 7.6 Hz, 1H; 6H); 13 C NMR (125 MHz, CDCl 3 ): (C), (C), (C), (CH), (C), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (C), 93.5 (CH), 83.0 (CH), 76.9 (CH), 76.3 (CH), 75.2 (CH 2 ), 74.8 (CH 2 ), 65.5 (CH), 61.2 (CH 2 ); HRMS (FAB): m/z Calcd for C 31 H 28 6 Br ([M] + ): , Found: (3) Silylation. Compound S3 (24.7 g, 39.9 mmol), Et 3 N (27.5 ml, 199 mmol), and DMAP (2.43 g, 19.9 mmol) were dissolved in CH 2 Cl 2 (247 ml) under N 2 gas and the reaction flask was immersed in an icewater bath. tertbutyldiphenylsilyl chloride (TBDPSCl, 20.4 ml, 79.7 mmol) was added to the solution, the icewater bath was removed, and the mixture was kept stirring for S5

6 24 h. The reaction was quenched by adding MeH (10 ml) and the solvents were evaporated under reduced pressure. Ethyl acetate and water were added to the residue, and the desired material was extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhydrous MgS 4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (ethyl acetate/hexanes = 1/8) to furnish the silyl ether S4 (31.6 g, 93%) as a colorless syrup. [ ] 20 D 48.5 (c 5.0, CHCl 3 ); IR (thin film): 2930, 2857, 2111, 1743, 1488, 1471, 1427, 1266, 1113, 1086, 1065, 821, 741, 703, 505 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): (m, 2H; BzH), (m, 3H; ArH), (m, 6H; ArH), (m, 6H; ArH), (m, 2H; ArH), (m, 7H; ArH), 5.86 (d, J = 8.4 Hz, 1H; 1H), 5.12, 5.08 (ABq, J = 11.2 Hz, 2H; ArCH 2 ), 4.95, 4.92 (ABq, J = 11.1 Hz, 2H; ArCH 2 ), (m, 3H; 4H, 6H), 3.88 (dd, J = 8.6, 9.7 Hz, 1H; 2H), 3.71 (t, J = 9.4 Hz, 1H; 3H), 3.64 (d, J = 9.5 Hz, 1H; 5H), 1.16 (s, 9H; tbu); 13 C NMR (125 MHz, CDCl 3 ): (C), (C), (CH), (CH), (C), (CH), (CH), (C), (C), (C), (C), (CH), (CH), (CH), (CH), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (C), 93.4 (CH), 83.0 (CH), 77.2 (CH), 76.4 (CH), 75.2 (CH 2 ), 74.8 (CH 2 ), 65.5 (CH), 62.0 (CH 2 ), 26.8 (CH 3 ), 19.3 (C); HRMS (FAB): m/z Calcd for C 47 H 45 6 BrSi ([M + H] + ): , Found: (4) Anomeric debenzoylation. Ammonia gas was passed through a solution of compound S4 (31.6 g, 36.9 mmol) in a mixed solvent (MeH/THF = 3/7, 1.60 L) at icebath temperature for 30 min. After stirring at 0 C for 24 h, the solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (ethyl acetate/hexanes = 1/5) to furnish the hemiacetal S5 (24.0 g, 86%) as a colorless syrup. [ ] 20 D 17.3 (c 1.7, CHCl 3 ); IR (thin film): S6

7 3401, 2930, 2857, 2109, 1488, 1471, 1427, 1113, 1070, 1051, 506 cm 1 ; 1 H NMR (600 MHz, CDCl 3 ): (m, 1.6H; ArH), (m, 6.5H; ArH), (m, 3.2H; ArH), (m, 1.7H; ArH), (m, 3.3H; ArH), (m, 9.7H; ArH), (m, 4.6H; ArH), (m, 3.1H; ArH), 5.32 (t, J = 3.0 Hz, 1.0H; 1H), 4.96 (d, J = 11.2 Hz, 1.0H; ArCH 2 ), 4.93 (d, J = 11.2 Hz, 0.6H; ArCH 2 ), 4.89 (d, J = 11.2 Hz, 1.0H; ArCH 2 ), (m, 3.1H; ArCH 2 ), 4.75 (d, J = 11.0 Hz, 0.6H; ArCH 2 ), 4.50 (dd, J = 4.4, 7.9 Hz, 0.6H; 1H), (m, 2.0H; 3H, 6H a ), (m, 2.1H; 6H a, 5H), (m, 2.1H; 4H, 6H b ), 3.80 (t, J = 9.4 Hz, 0.6H; 4H), (m, 1.6H; 2H, 3H), (m, 1.1H; 5H, 2H), 2.94 (br s, 0.5H; 1H), 2.68 (br s, 1.0H; 1H), 1.06 (s, 14.4H; tbu); 13 C NMR (150 MHz, CDCl 3 ): (C), (CH), (CH), (CH), (CH), (C), (C), (C), (C), (C), (C), (C), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (C), 96.0 (CH), 92.1 (CH), 83.0 (CH), 80.2 (CH), 78.3 (CH), 77.5 (CH), 76.1 (CH), 75.1 (CH 2 ), 74.8 (CH 2 ), 72.0 (CH), 67.6 (CH), 64.1 (CH), 62.6 (CH 2 ), 62.5 (CH 2 ), 26.9 (CH 3 ), (C), (C); HRMS (FAB): m/z Calcd for C 40 H 43 5 BrSi ([M + H] + ): , Found: (5) Imidation. CCl 3 CN (7.7 ml, 77.2 mmol, 10 equiv) was added to a mixture of compound S5 (5.81 g, 7.72 mmol, 1 equiv) and anhydrous K 2 C 3 (5.34 g, 38.6 mmol, 5 equiv) in CH 2 Cl 2 (58 ml, 10 ml per gram of S5) at 0 C under N 2 atmosphere. Afterwards, the reaction was gradually warmed up to room temperature and kept stirring for 24 h. The resulting solution was filtered through Celite and sequentially washed with water and brine. The organic solution was dried over anhydrous MgS 4, filtered, and concentrated under reduced pressure to afford the S7

8 crude trichloroacetimidate 7 (6.93 g, 97%, / = 1/2.3), which was directly used in the ensuing reaction without further purification. 1 H NMR (600 MHz, CDCl 3 ): 8.75 (s, 2.2H; NH), 8.71 (s, 1.0H; NH), (m, 3.9H; ArH), (m, 7.3H; ArH), (m, 16.7H; ArH), 7.60 (s, 1.1H; ArH), (m, 7.4H; ArH), (m, 17.7H; ArH), (m, 16.0H; ArH), (m, 7.2H; ArH), 6.50 (d, J = 3.5 Hz, 1.0H; 1H), 6.68 (d, J = 8.5 Hz, 2.3H; 1H), 5.01 (d, J = 11.1 Hz, 2.4H; ArCH 2 ), (m, 3.7H; ArCH 2 ), (m, 5.9H; ArCH 2 ), 4.83 (d, J = 11.2 Hz, 2.5H; ArCH 2 ), 4.06 (dd, J = 8.4, 10.0 Hz, 1.1H; 3H), (m, 11.9H; 4H, 6H, 4H, 5H, 6H), 3.74 (dd, J = 8.5, 9.8 Hz, 2.4H; 2H), 3.70 (dd, J = 3.5, 10.2 Hz, 1.1H; 2H), 3.58 (t, J = 9.5 Hz, 2.4H; 3H), 3.51 (dt, J = 2.3, 9.7 Hz, 2.4H; 5H), (s 2, 32.9H; tbu); 13 C NMR (150 MHz, CDCl 3 ): (C), (C), (C), (C), (CH), (CH), (CH), (CH), (C), (C), (C), (C), (C), (C), (C), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (C), 96.6 (CH), 94.8 (CH), 91.0 (CH), 90.6 (CH), 82.9 (CH), 80.2 (CH), 77.8 (CH), 77.2 (CH), 76.4 (CH), 75.4 (CH 2 ), 75.3 (CH 2 ), 74.9 (CH 2 ), 74.8 (CH 2 ), 74.6 (CH), 65.8 (CH), 63.3 (CH), 62.1 (CH 2 ), 61.9 (CH 2 ), (CH 3 ), (CH 3 ), 19.4 (C), 19.3 (C). General procedure for the synthesis of compounds A solution of the trichloroacetimidate donor (1 7, 1.2 equiv) and the 4alcohol 8 2 (1.0 equiv) in CH 2 Cl 2 (10 ml per total gram of donor, acceptor, and molecular sieves) with freshly dried 4 Å molecular sieves (1.5 g per total gram of the donor and the acceptor) was stirred at room temperature for 1 h under N 2 atmosphere. The mixture was cooled to 78 C, trimethylsilyl triflate (TMSTf, 0.2 equiv) was added to the S8

9 solution, and the mixture was gradually warmed up to 0 C. After stirring for h, Et 3 N (0.3 equiv) was added to quench the reaction and the mixture was filtered through Celite. The filtrate was concentrated under reduced pressure to furnish a residue, which was purified by flash column chromatography to give the expected disaccharide. The yields of compounds 9 15 are listed in Table 1. 1,6Anhydro4[2azido3,6dibenzyl2deoxy4(2naphthylmethyl) Dglucopyranosyl]2benzoyl3benzyl Lidopyranose (9). [ ] 23 D (c 1.31, CHCl 3 ); IR (thin film): 3063, 2917, 2108, 1724, 1270, 1095 cm 1 ; 1 H NMR (500 MHz, CDCl 3 ): 8.04 (dd, J = 8.4, 1.2 Hz, 2H; BzH), (m, 1H; ArH), (m, 2H; ArH), (m, 2H; ArH), (m, 20H; ArH), 5.53 (d, J = 1.6 Hz, 1H; 1H), 5.31 (d, J = 3.8 Hz, 1H; 1'H), 5.06 (dd, J = 7.7, 1.6 Hz, 1H; 2H), 4.94, 4.66 (ABq, J = 11.1 Hz, 2H; ArCH 2 ), 4.93 (s, 2H; ArCH 2 ), 4.88, 4.75 (ABq, J = 1.9 Hz, 2H; ArCH 2 ), 4.66 (dd, J = 5.2, 4.0 Hz, 1H; 5H), 4.57, 4.48 (ABq, J = 12.1 Hz, 2H; ArCH 2 ), 4.16 (d, J = 7.7 Hz, 1H; 6H a ), 4.06 (dd, J = 8.1, 7.7 Hz, 1H; 3H), 4.03 (dd, J = 8.1, 4.0 Hz, 1H; 4H), 3.98 (dd, J = 10.3, 8.1 Hz, 1H; 3'H), 3.76 (dd, J = 7.7, 5.2 Hz, 1H; 6H b ), (m, 4H; 4'H, 5'H, 6'H a, 6'H b ), 3.44 (dd, J = 10.3, 3.8 Hz, 1H; 2'H); 13 C NMR (125 MHz, CDCl 3 ): (C), (C), (C), (C), (C), (C), (C), (C), (CH), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), 99.7 (CH), 99.3 (CH), 80.0 (CH), 79.3 (CH), 79.0 (CH), 78.2 (CH), 77.0 (CH), 75.5 (CH 2 ), 75.4 (CH 2 ), 75.0 (CH 2 ), 74.3 (CH), 73.7 (CH 2 ), 71.6 (CH), 68.2 (CH 2 ), 65.8 (CH 2 ), 63.5 (CH); HRMS (FAB): m/z Calcd for C 51 H Na ([M + Na] + ): , Found: S9

10 Dglucopyranosyl]2benzoyl3benzyl Lidopyranose (10). [ ] 28 D (c 2.0, CHCl 3 ); mp C; IR (thin film): 2902, 2108, 1724, 1602, 1452, 1315, 1269, 1094, 1072, 1042, 1026, 971, 746, 711 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): 8.07 (dd, J = 8.5, 1.3 Hz, 2H; BzH), 7.98 (dd, J = 8.4, 1.2 Hz, 2H; BzH), (m, 1H; ArH), (m, 1H; ArH), (m, 3H; ArH), (m, 12H; ArH), (m, 6H; ArH), 5.79 (dd, J = 10.8, 8.8 Hz, 1H; 3'H), 5.53 (d, J = 1.7 Hz, 1H; 1H), 5.47 (d, J = 3.8 Hz, 1H; 1'H), 5.08 (dd, J = 7.9, 1.7 Hz, 1H; 2H), 4.80, 4.75 (ABq, J = 10.8 Hz, 2H; ArCH 2 ), (m, 5H; 5H, ArCH 2 ), 4.22 (d, J = 7.9 Hz, 1H; 6'H a ), (m, 2H; 3H, 4H), (m, 1H; 4'H), (m, 1H; 5'H), (m, 3H; 6H a, 6H b, 6'H b ), 3.37 (dd, J = 10.8, 3.8 Hz, 1H; 2'H); 13 C NMR (100 MHz, CDCl 3 ): (C), (C), (C), (C), (C), (CH), (CH), (C), (C), (CH), (CH), (C), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), 99.7 (CH), 99.3 (CH), 79.8 (CH), 77.8 (CH), 77.3 (CH), 76.0 (CH), 75.1 (CH 2 ), 74.9 (CH 2 ), 74.3 (CH), 73.8 (CH 2 ), 72.4 (CH), 71.3 (CH), 68.0 (CH 2 ), 65.9 (CH 2 ), 61.5 (CH); HRMS (FAB): m/z Calcd for C 51 H ([M + H] + ): , Found: ; Anal. Calcd for C 51 H : C, 69.77; H, 5.40; N, Found: C, 70.05; H, 5.0; N, ,6Anhydro4[2azido3benzoyl6benzyl2deoxy4(2naphthylmethyl) 1,6Anhydro4[2azido6benzoyl3benzyl2deoxy4(2naphthylmethyl) Dglucopyranosyl]2benzoyl3benzyl Lidopyranose (11). [ ] 27 D (c 0.52, CHCl 3 ); IR (thin film): ν 2928, 2108, 1729, 1260, 1068 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): S10

11 8.04 (d, J = 8.4 Hz, 2H; BzH), 7.95 (d, J = 8.4 Hz, 2H; BzH), (m, 3H; ArH), 7.69 (s, 1H; ArH), (m, 2H; ArH), (m, 13H; ArH), (m, 4H; ArH), 5.53 (d, J = 1.6 Hz, 1H; 1H), 5.31 (d, J = 3.7 Hz, 1H, 1'H), 5.06 (dd, J = 7.7, 1.6 Hz, 1H; 2H), 5.04 (d, J = 10.9 Hz, 1H; ArCH 2 ), 4.99 (d, J =10.6 Hz, 1H; ArCH 2 ), 4.95 (d, J =10.6 Hz, 1H; ArCH 2 ), 4.88 (d, J = 10.9 Hz, 1H; ArCH 2 ), 4.82 (d, J = 10.9 Hz, 1H; ArCH 2 ), 4.76 (d, J = 10.9 Hz, 1H; ArCH 2 ), (m, 2H; 5H, 6'H a ), 4.47 (dd, J = 12.0, 5.1 Hz, 1H; 6'H b ), 4.19 (d, J = 7.8 Hz, 1H; 6H a ), 4.11 (t, J = 7.7 Hz, 1H; 3H), (m, 2H; 4H, 3'H), 3.96 (ddd, J = 9.6, 5.1, 1.7 Hz, 1H; 5'H), 3.78 (dd, J = 7.8, 5.2 Hz, 1H; 6H b ), 3.68 (t, J = 9.6 Hz, 1H; 4'H), 3.45 (dd, J = 10.4, 3.7 Hz, 1H; 2'H); 13 C NMR (100 MHz, CDCl 3 ): (C), (C), (C), (C), (C), (CH), (CH), (C), (C), (CH), (CH), (C), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), 99.5 (CH), 99.3 (CH), 80.1 (CH), 79.4 (CH), 78.6 (CH), 78.2 (CH), 77.0 (CH), 75.7 (CH 2 ), 75.6 (CH 2 ), 74.9 (CH 2 ), 74.3 (CH), 70.1 (CH), 65.7 (CH 2 ), 63.5 (CH), 63.2 (CH 2 ); HRMS (MALDI): m/z Calcd for C 51 H Na ([M + Na] + ): , Found: ; Anal. Calcd for C 51 H : C 69.77, H 5.40, N 4.79; Found: C 69.65; H 5.39; N ,6Anhydro4[2azido3,6dibenzoyl2deoxy4(2naphthylmethyl) Dglucopyranosyl]2benzoyl3benzyl Lidopyranose (12). [ ] 23 D (c 0.96, CHCl 3 ); IR (thin film): 3068, 2910, 2108, 1721, 1267, 1177 cm 1 ; 1 H NMR (500 MHz, CDCl 3 ): 8.05 (d, J = 7.3 Hz, 4H; BzH), 7.98 (d, J = 7.3 Hz, 2H; BzH), (m, 6H; ArH), 7.51 (s, 1H; ArH), (m, 8H; ArH), (m, 6H; ArH), 5.88 (dd, J = 10.7, 9.0 Hz, 1H; 3'H), 5.53 (d, J = 1.6 Hz, 1H; 1H), 5.46 (d, J = 3.7 Hz, 1H; 1'H), 5.06 (dd, J = 8.1, 1.6 Hz, 1H; 2H), S11

12 4.79, 4.75 (ABq, J = 10.8 Hz, 2H; ArCH 2 ), 4.74 (s, 2H; ArCH 2 ), (m, 2H; 6'H a, 5H), 4.57 (dd, J = 12.0, 5.4 Hz, 1H; 6'H b ), 4.25 (d, J = 7.9 Hz, 1H; 6H a ), 4.17 (t, J = 8.1 Hz, 1H; 3 H), (m, 2H; 4H, 5'H), (m, 1H; 6H b ), 3.82 (t, J = 9.0 Hz, 1H; 4'H), 3.37 (dd, J = 10.7, 3.7 Hz, 1H; 2'H); 13 C NMR (125 MHz, CDCl 3 ): (C), (C), (C), (C), (CH), (CH), (CH), (C), (C), (CH), (CH), (CH), (C), (C), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), 99.4 (CH), 99.3 (CH), 79.8 (CH), 77.5 (CH), 77.2 (CH), 76.3 (CH), 75.3 (CH 2 ), 74.9 (CH 2 ), 74.3 (CH), 72.4 (CH), 69.8 (CH), 65.8 (CH 2 ), 63.2 (CH 2 ), 61.4 (CH); HRMS (FAB): m/z Calcd for C 51 H ([M + H] + ): , Found: ; Anal. Calcd for C 51 H : C 68.68, H 5.09, N 4.71; Found: C 68.82; H 5.03; N ,6Anhydro4(2azido3benzoyl6tertbutyldiphenylsilyl2deoxy4(2 naphthylmethyl) Dglucopyranosyl)2benzoyl3benzyl Lidopyranose (13). [ ] 27 D (c 1.0, CHCl 3 ); mp C; IR (thin film): 3009, 2921, 2097, 1720, 1263, 1214, 758, 665 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): (m, 4H; BzH), (m, 5H; ArH), (m, 4H; ArH), (m, 13H; ArH), (m, 5H; ArH ), 7.10 (dd, J = 8.4, 1.5 Hz, 1H; ArH), 5.84 (dd, J = 10.8, 8.8 Hz, 1H; 3'H), 5.52 (d, J = 1.6 Hz, 1H; 1H), 5.43 (d, J = 3.8 Hz, 1H; 1'H), 5.10 (dd, J = 8.1, 1.6 Hz, 1H; 2H), 4.84, 4.78 (ABq, J = 10.8 Hz, 2H; ArCH 2 ), 4.70 (t, J = 11.4 Hz, 2H; ArCH 2 ), 4.57 (t, J = 4.6 Hz, 1H; 5H), 4.21 (d, J = 7.8 Hz, 1H; 6H a ), 4.16 (t, J = 8.1 Hz, 1H; 3H), 4.07 (dd, J = 8.1, 4.6 Hz, 1H; 4H), 3.95 (d, J = 2.6 Hz, 2H; 6'H a, 6'H b ), 3.89 (t, J = 8.8 Hz, 1H; 4'H), 3.83 (dt, J = 8.8, 2.7 Hz, 1H; 5'H), 3.74 (dd, J = 7.8, 4.6 Hz, 1H; 6H b ), 3.34 (dd, J = 10.8, 3.8 Hz, 1H; 2'H), 1.11 (s, 9H; tbu); 13 C NMR (100 MHz, S12

13 CDCl 3 ): (C), (C), (C), (CH), (CH), (C), (CH), (C), (C), (C), (C), (CH), (CH), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), 99.6 (CH), 99.3 (CH), 79.7 (CH), 77.9 (CH), 77.2 (CH), 76.3 (CH), 75.3 (CH 2 ), 74.9 (CH 2 ), 74.2 (CH), 72.5 (CH), 65.8 (CH 2 ), 62.6 (CH 2 ), 61.7 (CH), 26.9 (CH 3 ), 19.3 (C); HRMS (FAB): m/z Calcd for C 60 H SiNa ([M + Na] + ): , Found: ; Anal. Calcd for C 60 H Si: C 70.22, H 5.79, N 4.09; Found: C 70.54, H 5.78, N ,6Anhydro4[2azido3benzyl6tertbutyldiphenylsilyl2deoxy4(2 naphthylmethyl) Dglucopyranosyl]2benzoyl3benzyl Lidopyranose (14). [ ] 27 D (c 0.64, CHCl 3 ); IR (thin film): 3058, 2928, 2109, 1724, 1269, 1109 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): 8.03 (dd, J = 8.5, 1.4 Hz, 2H; BzH), (m, 1H; ArH), (m, 2H; ArH), (m, 4H; ArH), (m, 2H; ArH), (m, 21H; ArH), 5.50 (d, J = 1.6 Hz, 1H; 1H), 5.27 (d, J = 3.8 Hz, 1H; 1'H), 5.06 (dd, J = 8.0, 1.6 Hz, 1H; 2H), 4.99, 4.77 (ABq, J = 10.8 Hz, 2H; ArCH 2 ), 4.95 (s, 2H; ArCH 2 ), 4.90, 4.76 (ABq, J = 10.8 Hz, 2H; ArCH 2 ), 4.57 (t, J = 4.5 Hz, 1H; 5H), 4.12 (d, J = 7.7 Hz, 1H; 6H a ), 4.06 (t, J = 8.0 Hz, 1H; 3 H), 4.01 (dd, J = 8.0, 4.5 Hz, 1H; 4H), 4.00 (t, J = 10.4 Hz, 1H; 3'H), (m, 2H, 6'H a, 6'H b ), 3.73 (dd, J = 10.4, 8.7 Hz, 1H; 4'H), (m, 2H; 4H, 5'H), 3.41 (dd, J = 10.4, 3.8 Hz, 1H; 2'H), 1.05 (s, 9H; tbu); 13 C NMR (125 MHz, CDCl 3 ): (C), (C), (C), (CH), (CH), (C), (CH), (C), (C), (C), (C), (CH), (CH), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), S13

14 126.1 (CH), (CH), 99.6 (CH), 99.2 (CH), 80.0 (CH), 79.3 (CH), 78.9 (CH), 78.2 (CH), 77.0 (CH), 75.6 (CH 2 ), 75.5 (CH 2 ), 75.0 (CH 2 ), 74.2 (CH), 72.8 (CH), 65.7 (CH 2 ), 63.6 (CH), 62.6 (CH 2 ), 26.8 (CH 3 ), 19.2 (C); HRMS (FAB): m/z Calcd for C 60 H SiNa ([M + Na] + ): , Found ; Anal. Calcd for C 60 H Si: C 71.19, H 6.07, N 4.15; Found: C 70.75; H 5.82; N ,6Anhydro4[2azido3(4bromobenzyl)6tertbutyldiphenylsilyl2deoxy4 (2naphthylmethyl) Dglucopyranosyl]2benzoyl3benzyl Lidopyranose (15). [ ] 23 D (c 1.0, CHCl 3 ); IR (thin film): 3048, 2936, 2107, 1723, 1269, 1111, 705 cm 1 ; 1 H NMR (600 MHz, CDCl 3 ): (m, 2H; BzH), 7.81 (dd, J = 5.9, 3.4 Hz, 1H; ArH), (m, 2H; ArH), (m, 4H; ArH), (m, 2H; ArH), (m, 2H; ArH), (m, 6H; ArH), 7.35 (t, J = 7.4 Hz, 2H; ArH), 7.31 (t, J = 7.4 Hz, 2H; ArH), (m, 8H; ArH), 5.50 (d, J = 1.4 Hz, 1H; 1H), 5.27 (d, J = 3.7 Hz, 1H; 1'H), 5.06 (dd, J = 8.0, 1.6 Hz, 1H; 2H), 4.91 (d, J = 10.9 Hz, 1H; ArCH 2 ), (m, 3H; ArCH 2 ), 4.77 (d, J = 11.8 Hz, 1H; ArCH 2 ), 4.75 (d, J = 11.2 Hz, 1H; ArCH 2 ), 4.56 (t, J = 4.5 Hz, 1H; 5H), 4.11 (d, J = 7.7 Hz, 1H; 6H a ), 4.04 (t, J = 8.1 Hz, 1H; 3H), 4.00 (dd, J = 8.0, 4.2 Hz, 1H; 4H), 3.96 (dd, J = 10.0, 9.1 Hz, 1H; 3'H), (m, 2H; 6'H), 3.72 (t, J = 9.4 Hz, 1H; 4'H), (m, 2H; 6H b, 5'H), 3.39 (dd, J = 10.4, 3.8, 1H; 2'H), 1.05 (s, 9H; tbu); 13 C NMR (150 MHz, CDCl 3 ): (C), (C), (C), (CH), (CH), (C), (CH), (C), (C), (C), (C), (CH), (CH), (CH), (CH), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (C), 99.6 (CH), 99.2 (CH), 80.1 (CH), 79.3 (CH), 78.8 (CH), 78.3 (CH), 77.0 (CH), 75.5 (CH 2 ), 74.9 S14

15 (CH 2 ), 74.7 (CH 2 ), 74.2 (CH), 72.8 (CH), 65.7 (CH 2 ), 63.5 (CH), 62.5 (CH 2 ), 26.8 (CH 3 ), 19.2 (C); HRMS (ESI): m/z Calcd for C 60 H BrSiNa ([M + Na] + ): , Found: NAP pbrbn TBDPS NH CCCl Ac Ph Ac H Ph 21 5 TMSTf TBDPS 2NAP pbrbn Ph Ph Ac Ac 22 6[2azido3(4bromobenzyl)6tertbutyldiphenylsilyl2deoxy4(2 naphthylmethyl) Dglucopyranosyl]2,4di[(S)2acetyloxy2phenylacetyl]1,3,5methylideneDmyoinositol (22). A solution of the trichloroacetimidate 7 (80.8 mg, 0.09 mmol) and the 6alcohol 21 (32 mg, 0.06 mmol) in CH 2 Cl 2 (3 ml) with freshly dried 4 Å molecular sieves (0.15 mg) was stirred at room temperature for 1 h under N 2 atmosphere. The mixture was cooled to 40 C, TMSTf (2.2 L, 0.01 mmol) was added to the solution, and the mixture was gradually warmed up to 20 C. Trichloroacetimidate 21 3 (27 mg, 0.03 mmol) and TMSTf (1.2 L, mmol) was added, then the mixture was gradually warmed up to 0 C. After stirring for 2 h, Et 3 N was added to quench the reaction and the mixture was filtered through Celite. The filtrate was concentrated under reduced pressure to furnish a residue, which was purified by flash column chromatography (ethyl acetate/hexanes = 1/4) to give the adduct 22 (54 mg, 71%). [ ] 21 D (c 2.8, CHCl 3 ); IR (thin film): 2930, 2109, 1751, 1230, 1163, 875, 771, 700 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): (m, 6H; ArH), (m, 5H; ArH), (m, 13H; ArH), (m, 7H; ArH), 5.97 (s, 2H; PhCH(Ac)C(=)), (m, 1H; 4H), 5.48 (d, J = 1.12 Hz, 1H; methylideneh), (m, 1H; 2H), 5.04 (d, J = 3.7 Hz, 1H; S15

16 1'H), 4.86, 4.77 (d, J = 10.8 Hz, 2H; ArCH 2 ), 4.62 (s, 2H; ArCH 2 ), (m, 1H; 6H), (m, 1H; 1H), (m, 1H; 5H), (m, 4H; 3'H, 5'H, 6'H a, 6'H b ), (m, 2H; 3H, 4'H), 3.49 (dd, J = 10.8, 3.7 Hz, 1H; 2'H), 2.19 (s, 3H; Ac), 1.87 (s, 3H; Ac), 1.07 (s, 9H; tbu); 13 C NMR (100 MHz, CDCl 3 ): C), (C), (C), (C), (C), (CH), (CH), (C), (C), (C), (C), (C), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), 97.5 (CH), 80.5 (CH), 78.0 (CH), 75.1 (CH 2 ), 74.5 (CH), 74.4 (CH 2 ), 73.8 (CH), 72.7 (CH), 71.9 (CH), 68.8 (CH), 68.5 (CH), 68.3 (CH), 67.9 (CH), 64.0 (CH), 63.5 (CH), 62.3 (CH 2 ), 27.0 (CH 3 ), 26.8 (CH 3 ), 20.6 (CH 3 ), 20.2 (CH 3 ), 19.3 (C); HRMS (ESI): m/z Calcd for C 67 H BrSi ([M + H] + ): , Found: TBDPS 2NAP pbrbn TBDPS NH CCCl H Bn Bn 23 TMSTf 2NAP pbrbn Bn Bn 24 1,6Anhydro4[2azido3(4bromobenzyl)6tertbutyldiphenylsilyl2deoxy4 (2naphthylmethyl) Dglucopyranosyl]2,3dibenzyl Lidopyranose (24). A solution of the trichloroacetimidate 7 (86 mg, 0.10 mmol) and the 4alcohol 23 4 (32 mg, 0.08 mmol) in CH 2 Cl 2 (2 ml) with freshly dried 4 Å molecular sieves (100 mg) was stirred at room temperature for 1 h under N 2 atmosphere. The mixture was cooled to 78 C, TMSTf (2.7 L, mmol) was added to the solution, and the mixture was gradually warmed up to 0 C. After stirring for 2 h, Et 3 N was added to quench the reaction and the mixture was filtered through Celite. S16

17 The filtrate was concentrated under reduced pressure to furnish a residue, which was purified by flash column chromatography (ethyl acetate/hexanes = 1/5) to give the disaccharide 24 (66 mg, 77%). [ ] 26 D (c 1.2, CHCl 3 ); IR (thin film): 2927, 2856, 2107, 1488, 1264, 1036, 805, 741, 701 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): (m, 1H; ArH), (m, 6H; Ar H), 7.56 (s, 1H; ArH), (m, 2H; ArH), (m, 18H; ArH), (m, 3H; ArH), 5.30 (s, 1H; 1H), 5.25 (d, J = 3.9 Hz, 1H; 1'H), 4.95 (d, J = 10.7 Hz, 2H; ArCH 2 ), (m, 4H; ArCH 2 ), 4.77 (d, J = 10.7 Hz, 2H; ArCH 2 ), 4.71, 4.69 (ABq, J = 11.9 Hz, 2H; ArCH 2 ), 4.49 (t, J = 4.4 Hz, 1H; 5H), 4.06 (d, J = 7.6 Hz, 1H; 6H a ), 3.95 (t, J = 10.2 Hz, 1H; 3'H), (m, 4H; 3H, 4H, 6'H a, 6'H b ), (m, 3H; 4'H, 5'H, 6H b ), 3.50 (d, J = 1.6 Hz, 1H; 2H), 3.37 (dd, J = 10.2, 3.9 Hz, 1H; 2'H), 1.05 (s, 9H; tbu); 13 C NMR (100 MHz, CDCl 3 ): (C), (C), (C), (CH), (CH), (C), (C), (C), (C), (C), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (C), 99.5 (CH), 99.4 (CH), 82.8 (CH), 81.3 (CH), 80.0 (CH), 79.1 (CH), 78.3 (CH), 75.5 (CH 2 ), 75.4 (CH 2 ), 74.6 (CH 2 ), 74.1 (CH), 72.9 (CH 2 ), 72.7 (CH), 65.6 (CH 2 ), 63.6 (CH), 62.6 (CH 2 ), 26.8 (CH 3 ), 19.2 (C); HRMS (ESI): m/z Calcd for C 60 H 62 9 BrSiNa ([M + Na] + ): , Found: TMS TMS TMS STol TMS S6 1. PhCH, TMSTf 2. PhCH, Et 3 SiH, TMSTf 3. Bz 2, TMSTf 4. TFA H H Bn STol Bz S7 Ac 2, Et 3 N Ac H Bn STol Bz 25 4Methylphenyl 6acetyl2benzoyl3benzyl1thio Dglucopyranoside (25). (1) Regioselective onepot protection. A mixture of the compound S6 5 (106 mg, 0.19 mmol), benzaldehyde (19 L, 0.19 mmol), and freshly dried 3 Å molecular sieves (160 mg) in CH 2 Cl 2 S17

18 (1.6 ml) was stirred at room temperature for 30 min under N 2 atmosphere. The reaction flask was cooled to 0 C, TMSTf (6.2 L, 0.03 mmol) was added to the solution, and the mixture was kept stirring at the same temperature for 2 h. The mixture was next cooled to 78 C, Et 3 SiH (33 L, 0.20 mmol), benzaldehyde (19 L, 0.19 mmol), and TMSTf (3.4 L, 0.02 mmol) were sequentially added, and the resulting mixture was stirred for another 3 h. The temperature was gradually warmed up to 0 C, and benzoic anhydride (130 mg, 0.56 mmol) and TMSTf (3.4 L, 0.02 mmol) were then added. The reaction flask was gradually warmed up to room temperature and the mixture was stirred at the same temperature for 18 h. A 70% aqueous solution of trifluoroacetic acid (TFA, 3 ml) was added to the resulting solution and the mixture was continuously stirred at room temperature for another 3 h. The reaction was quenched by saturated NaHC 3(aq), and the desired material was extracted with ethyl acetate. The combined organic layer was washed with brine, dried over MgS 4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (ethyl acetate/hexanes = 1/1) to afford the 4,6diol S7 (67 mg, 75%). [ ] 22 D (c 1.0, CHCl 3 ); mp C; IR (thin film): 3630, 3100, 1724, 1267, 1254, 1094, 1070 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): (m, 2H; ArH), (m, 1H; ArH), (m, 2H; ArH), 7.30 (d, J = 8.1 Hz, 2H; ArH), (m, 5H; ArH), 7.06 (d, J = 7.9 Hz, 2H; ArH), 5.20 (dd, J = 10.0, 9.0 Hz, 1H; 2H), 4.75 (d, J = 10.0 Hz, 1H; 1H), 4.70 (d, J = 11.4 Hz, 1H; ArCH 2 ), 4.55 (d, J = 11.4 Hz, 1H; ArCH 2 ), 3.90 (ddd, J = 12.0, 6.6, 3.4 Hz, 1H; 6H a ), (m, 1H; 6H b ), (m, 2H; 3H, 4H), (m, 1H; 5H), 2.37 (d, J = 2.2 Hz, 1H; 4H), 2.30 (s, 3H; ArCH 3 ), 2.11 (t, J = 6.6 Hz, 1H, 6H); 13 C NMR (100 MHz, CDCl 3 ): (C), (C), (C), (CH), (CH), (CH), (CH), (C), (CH), (CH), (CH), 86.6 (CH), 83.9 (CH), 79.4 (CH), 74.8 (CH 2 ), 72.4 (CH), 70.3 (CH), S18

19 62.6 (CH 2 ), 21.1 (CH 3 ); HRMS (FAB): m/z Calcd for C 27 H 29 6 S ([M + H] + ): , Found: ; Anal. Calcd for C 27 H 28 6 S: C 67.48, H 5.87; Found: C 67.42; H (2) Regioselective acetylation. Compound S7 (1.21 g, 2.51 mmol) was dissolved in CH 2 Cl 2 (12 ml), and the reaction flask was immersed in an ice bath. Acetic anhydride (0.26 ml, 2.77 mmol) and Et 3 N (3.10 ml, mmol) were added to the solution, and the mixture was kept stirring at 0 C for 1 h. Methanol was added to quench the reaction, the mixture was stirred for another 30 min, and the resulting solution was evaporated under reduced pressure. Water was added to the residue, the crude target material was extracted with ethyl acetate, and the combined organic layer was sequentially washed with saturated NaHC 3(aq) and brine. The organic portion was dried over anhydrous MgS 4, filtered, and concentrated under reduced pressure to give a residue, which was purified by flash column chromatography (ethyl acetate/hexanes = 1/3) to furnish the 4alcohol 25 (1.13 g, 86%); [ ] 20 D 3.1 (c 0.99, CHCl 3 ); mp C; IR (thin film): 3486, 2872, 1720, 1493, 1451, 1251, 1115, 1071, 1039 cm 1 ; 1 H NMR (500 MHz, CDCl 3 ): 8.05 (dd, J =8.0, 1.2 Hz, 2H; BzH), (m, 1H; BzH), 7.46 (t, J = 8.0 Hz, 2H; BzH), (m, 2H; ArH), (m, 5H; ArH), 7.05 (d, J = 8.0 Hz, 2H; ArH), 5.18 (dd, J =10.0, 9.0 Hz, 1H; 2H), 4.71 (d, J =10.0 Hz, 1H; 1H), 4.69, 4.62 (ABq, J =11.4 Hz, 2H; ArCH 2 ), 4.41 (dd, J =12.0, 4.6 Hz, 1H; 6H a ), 4.36 (dd, J =12.0, 2.2 Hz, 1H; 6H b ), 3.67 (t, J = 9.0 Hz, 1H; 3H), 3.58 (td, J = 9.0, 3.3 Hz, 1H; 4H), 3.53 (ddd, J = 9.0, 4.6, 2.2 Hz, 1H; 5H), 2.76 (d, J = 3.3 Hz, 1H; 4H), 2.30 (s, 3H; ArCH 3 ), 2.10 (s, 3H, 6Ac); 13 C NMR (125 MHz, CDCl 3 ): (C), (C), (C), (C), (CH), (CH), (CH), (C), (CH), (CH), (CH), (CH), 86.6 (CH), 83.5 (CH), 77.7 (CH), 74.9 (CH 2 ), 72.1 (CH), 69.9 (CH), 63.2 (CH 2 ), 21.1 (CH 3 ), 20.8 (CH 3 ); HRMS (FAB): m/z Calcd S19

20 for C 29 H 31 7 S ([M + H] + ): , Found: ; Anal. Calcd for C 29 H 30 7 S: C 66.65, H 5.79; Found: C 66.70; H NAP pbrbn TBDPS NH CCCl Ac H Bn STol Bz 25 AgTf 2NAP pbrbn TBDPS Bn 26 Ac STol Bz 4Methylphenyl 6acetyl4[2azido3(4bromobenzyl)6tertbutyl diphenylsilyl2deoxy4(2naphthylmethyl) Dglucopyranosyl]2benzoyl3 benzyl1thio Dglucopyranoside (26). A solution of the trichloroacetimidate 7 (16.1 g, 17.9 mmol) and the thioglycoside 25 (7.8 g, 14.9 mmol) in CH 2 Cl 2 (350 ml) with freshly dried 4 Å molecular sieves (10 g) was stirred at room temperature for 1 h under N 2 atmosphere. The mixture was cooled to 40 C, and silver(i) triflate (AgTf, 20 g, 74.9 mmol) was added. The resulting solution was allowed to gradually warm up to 0 C. After 4 h, the reaction was quenched with Et 3 N and the whole mixture was filtered through Celite. The filtrate was concentrated under reduced pressure and the residue was purified by flash column chromatography (ethyl acetate/hexanes = 1/7.5) to give the disaccharide 26 (13.3 g, 71%). [ ] 20 D 1.46 (c 0.8, CHCl 3 ); IR (thin film): 2929, 2857, 2106, 1736, 1263, 1221, 1110, 1070, 1037, 773, 709 cm 1 ; 1 H NMR (600 MHz, CDCl 3 ): 8.12 (dd, J = 1.2, 8.4 Hz, 2H; ArH), (m, 1H; ArH), (m, 2H; ArH), (m, 6H; ArH), (m, 22H; ArH), 7.08 (d, J = 7.9 Hz, 2H; ArH), 5.57 (d, J = 3.9 Hz,1H; 1'H), 5.32 (t, J = 9.0 Hz, 1H; 2H), 4.96, 4.94 (ABq, J = 11.2 Hz, 2H; ArCH 2 ), 4.87, 4.81 (ABq, J = 11.0 Hz, 2H; ArCH 2 ), 4.83, 4.72 (ABq, J = 10.2, 2H; ArCH 2 ), 4.75 (d, J = 9.0 Hz, 1H; 1H), 4.49 (dd, J = 11.9, 2.2 Hz, 1H; 6H a ), 4.09 (dd, J = 11.9, 4.8 Hz, 1H; 6H b ), 4.03 (dd, J = 12.0, 2.5 Hz, 1H; 6'H a ), 4.02 (t, J = 9.0 Hz, 1H; 3H), (m, 2H; S20

21 3'H, 4'H), 3.88 (dd, J =9.6, 9.0 Hz, 1H; 4H), (m, 1H, 6'H b ), (m, 2H; 5H, 5'H), 3.30 (dd, J = 9.6, 3.9 Hz, 1H; 2'H), 2.33 (s, 3H; ArCH 3 ), 1.80 (s, 3H; Ac), 1.07 (s, 9H; tbu); 13 C NMR (150 MHz, CDCl 3 ): (C), (C), (C), (C), (C), (CH), (CH), (C), (CH), (CH), (C), (C), (C), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (C), 97.8 (CH), 86.1 (CH), 84.8 (CH), 79.9 (CH), 78.0 (CH), 76.4 (CH), 75.2 (CH 2 ), 74.6 (CH 2 2), 73.4 (CH), 72.9 (CH), 72.7 (CH), 63.3 (CH), 63.0 (CH 2 ), 62.0 (CH 2 ), 26.9 (CH 3 ), 21.2 (CH 3 ), 20.6 (CH 3 ), 19.3 (C); HRMS (FAB): m/z Calcd for C 69 H BrSSiNa ([M + Na] + ): , Found: C. Synthesis of heparosan oligosaccharides 2NAP pbrbn TBDPS Bn 26 Ac STol Bz Bn H Bn Me 27 NIS, TMSTf 2NAP pbrbn TBDPS Bn Ac Bz Bn 28 Bn Me Methyl 4[2azido3(4bromobenzyl)6tertbutyldiphenylsilyl2deoxy4(2 naphthylmethyl) Dglucopyranosyl](1 4)[6acetyl2benzoyl3benzyl Dglucopyranosyl](1 4)2azido2deoxy3,6dibenzyl Dglucopyranoside (28). A solution of the thioglycoside 26 (298 mg, 0.24 mmol) and 4alcohol 27 6 (72.5 mg, 0.18 mmol) in CH 2 Cl 2 (7 ml) with freshly dried 3 Å molecular sieves (370 mg) was stirred at room temperature for 1 h under N 2 atmosphere. The mixture was cooled to 78 C, Niodosuccinimide (NIS, 21.6 S21

22 mg, 0.10 mmol) and TMSTf (8.5 L, 0.05 mmol) were added to the reaction flask, and the mixture was gradually warmed up to room temperature for 3 h. Afterwards, Et 3 N (2.2 L, 0.02 mmol) was added to quench the reaction. The whole mixture was filtered through Celite, and the filtrate was sequentially washed with Na 2 S 2 3(aq) and brine. The organic layer was dried over anhydrous MgS 4, filtered, and concentrated under reduced pressure to get the crude product, which was purified by flash column chromatography (ethyl acetate/hexanes = 1/3) to get the trisaccharide 28 (240 mg, 86%). [ ] 25 D +1.1 (c 8.2, CHCl 3 ); IR (thin film): 3030, 2931, 2846, 2108, 1739, 1264, 1113, 1041, 701 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): (d, J = 7.5 Hz, 2H; ArH), (m, 3H; ArH), (m, 6H; ArH), (m, 11H; ArH), (m, 19H; ArH), 5.55 (d, J = 3.9 Hz, 1H; 1''H), 5.32 (t, J = 8.6 Hz, 1H; 2'H), 5.09 (d, J = 11.4 Hz, 1H, ArCH 2 ), 4.96 (s, 2H; ArCH 2 ), 4.92 (d, J = 11.0 Hz, 1H; ArCH 2 ), (m, 3H; ArCH 2 ), 4.71 (d, J = 3.5 Hz, 1H; 1H), (m, 3H; 1'H, ArCH 2 ), 4.38 (d, J = 12.2 Hz, 1H; ArCH 2 ), 4.19 (dd, J = 11.8, 2.0 Hz, 1H; 6'H a ), (m, 5H; 4H, 6'H b, 3''H, 4'' H, 6''H a ), (m,. 5H; 3H, 6H a, 3'H, 4'H, 6''H b ), (m, 1H; 5H), (m, 2H; 5'H, 5''H), (m, 3H; 2H, 6H b, 2''H), 3.28 (s, 3H; CH 3 ), 1.56 (s, 3H; 6 Ac), 1.06 (s, 9H; tbu); 13 C NMR (100 MHz, CDCl 3 ): (C), (C), (C), (C), (C), (C), (CH), (CH), (C), (CH), (C), (C), (C), (C), (CH), (CH), (CH), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (C), 99.9 (CH), 98.7 (CH), 97.6 (CH), 83.2 (CH), 79.8 (CH), 78.3 (CH), 78.0 (CH), 77.2 (CH), 75.1 (CH 2 ), 74.8 (CH 2 ), 74.6 (CH 2 ), 74.3 (CH 2 ), 74.2 (CH), 73.8 (CH 2 ), 73.5 (CH), 72.8 (CH), 72.2 (CH), 69.9 (CH), 67.2 (CH 2 ), 63.4 (CH), 63.1 (CH 2 ), 62.8 (CH), 62.0 (CH 2 ), 55.3 (CH 3 ), 26.9 (CH 3 ), 20.2 (CH 3 ), 19.3 S22

23 (C); HRMS (ESI): m/z Calcd for C 83 H N 6 BrSiNa ([M + Na] + ): , Found: NAP pbrbn TBDPS Bn Ac Bz Bn 28 Bn Me DDQ H pbrbn TBDPS Bn Ac Bz Bn S8 Bn Me 26, NIS, TfH 2NAP pbrbn TBDPS Bn 29 Ac Bz Bn 2 Bn Me Methyl [2azido3(4bromobenzyl)6tertbutyldiphenylsilyl2deoxy4(2 naphthylmethyl) Dglucopyranosyl](1 4)[6acetyl2benzoyl3benzyl D glucopyranosyl](1 4)[2azido3(4bromobenzyl)6tertbutyldiphenylsilyl2deoxy Dglucopyranosyl](1 4)[6acetyl2benzoyl3benzyl Dglucopyranosyl] (1 4)2azido2deoxy3,6dibenzyl Dglucopyranoside (29). (1) Cleavage of the 2 NAP group. 2,3Dichloro5,6dicyano1,4benzoquinone (DDQ, 44.6 mg, mmol) was added in three equal portions at halfhour intervals to a solution of compound 28 (151 mg, mmol) in a mixed solvent (CH 2 Cl 2 /H 2 = 18/1, 7.5 ml) at room temperature. After stirring for 2 h, the reaction was quenched by the addition of saturated NaHC 3(aq), and the crude target material was extracted with CH 2 Cl 2. The combined organic layer was consecutively washed with saturated NaHC 3(aq) and brine, dried over anhydrous MgS 4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (ethyl acetate/hexanes = 1/3) to give the 4''alcohol S8 (108 mg, 78%). [ ] 25 D +3.3 (c 2.3, CHCl 3 ); IR (thin film): 3468, 2929, 2108, 1735, 1263, 1113, 1044, 701 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): S23

24 7.90 (d, J = 7.6 Hz, 2H; ArH), (m, 6H, ArH), (m, 18H; ArH), (m, 8H; ArH), 5.48 (d, J = 3.8 Hz, 1H; 1''H), 5.24 (t, J = 8.6 Hz, 1H; 2'H), 5.05 (d, J = 11.4 Hz, 1H; ArCH 2 ), 4.92 (d, J = 11.6 Hz, 1H; ArCH 2 ), 4.83 (d, J = 12.0 Hz, 1H; ArCH 2 ), 4.81 (d, J = 11.6 Hz, 1H; ArCH 2 ), 4.74 (d, J = 10.3 Hz, 1H; ArCH 2 ), 4.69 (d, J = 3.6 Hz, 1H; 1H), 4.73 (d, J = 11.4 Hz, 1H; ArCH 2 ), 4.57 (d, J = 8.0 Hz, 1H; 1'H), 4.56 (d, J = 10.3 Hz, 1H; ArCH 2 ), 4.36 (d, J = 12.0 Hz, 1H; ArCH 2 ), 4.24 (dd, J = 11.9, 2.2 Hz, 1H; 6'H a ), (m, 2H; 6'H a, 4'' H), 3.88 (dd, J = 11.0, 3.4 Hz, 1H; 6''H a ), (m, 7H; 3H, 4H, 6H a, 3'H, 4'H, 3''H, 6''H b ), (m, 1H; 5H), (m, 2H; 5'H, 5''H), 3.35 (dd, J = 10.7, 1.2 Hz, 1H; 6''H b ), 3.31 (dd, J = 10.2, 3.6 Hz, 1H; H2), 3.26 (s, 3H; CH 3 ), 3.21 (dd, J = 9.9, 4.1 Hz, 1H; 2''H), 1.71 (s, 3H; 6'Ac), 1.03 (s, 9H; tbu); 13 C NMR (100 MHz, CDCl 3 ): (C), (C), (C), (C), (C), (C), (CH), (CH), (CH), (C), (C), (CH), (CH), (CH), (CH), (CH), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (C), 99.9 (CH), 98.7 (CH), 97.5 (CH), 83.2 (CH), 79.2 (CH), 77.7 (CH), 77.0 (CH), 74.8 (CH 2 ), 74.3 (CH 2 ), (CH 2 ), (CH), 73.9 (CH 2 ), 73.6 (CH), 72.6 (CH), 72.5 (CH), 71.9 (CH), 69.9(CH), 67.3 (CH 2 ), 63.8 (CH 2 ), 63.2 (CH 2 ), 62.9 (CH), 62.6(CH), 55.4 (CH 3 ), 26.9 (CH 3 ), 20.5 (CH 3 ), 19.2 (C); HRMS (ESI): m/z Calcd for C 72 H N 6 BrSiNa ([M + Na] + ): , Found: (2) Glycosylation. A solution of the thioglycoside 26 (424 mg, 0.34 mmol) and acceptor S8 (313 mg, 0.23 mmol) in CH 2 Cl 2 (15 ml) with freshly dried 3 Å molecular sieves (750 mg) was stirred at room temperature for 1 h under N 2 atmosphere. After cooling to 78 C, NIS (101 mg, 0.46 mmol) and triflic acid (TfH, 3.9 L, mmol) were added to the reaction flask. The mixture was gradually warmed up to room temperature for a period of 2 h. Afterwards, Et 3 N S24

25 was added to quench the reaction. The whole mixture was filtered through Celite, and the filtrate was sequentially washed with Na 2 S 2 3(aq) and brine. The organic layer was dried over anhydrous MgS 4, filtered, and concentrated under reduced pressure to get the crude product, which was purified by flash column chromatography (ethyl acetate/hexanes = 1/3) to get the pentasaccharide 29 (375 mg, 64%). [ ] 25 D (c 2.5, CHCl 3 ); IR (thin film): 3401, 3063, 3030, 2931, 2894, 2860, 2108, 1736, 1601, 1453, 1428, 1364, 1263, 1113, 1071, 1037, 823, 802, 753, 702 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): (m, 9H; ArH), (m, 8H; ArH), (m, 48H; ArH), 5.56 (d, J = 3.8 Hz, 1H; 1''''H), (m, 2H; 2'H, 1''H), 5.15 (t, J = 8.5 Hz, 1H; 2'''H), 5.11 (d, J = 11.3 Hz, 1H; ArCH 2 ), (m, 5H; ArCH 2 ), (m, 3H; 1'H, ArCH 2 ), 4.71 (d, J = 10.1 Hz, 1H; ArCH 2 ), 4.67 (d, J = 3.5 Hz, 1H; 1H), (m, 3H; ArCH 2 ), 4.45 (d, J = 10.1 Hz, 1H; ArCH 2 ), 4.40 (d, J = 8.1 Hz, 1H; 1'''H), 4.29 (d, J = 12.1 Hz, 1H; ArCH 2 ), 4.23 (dd, J = 12.4, 2.5 Hz, 1H; 6'''H a ), 4.16 (t, J = 9.3 Hz, 1H; 4'H), (m, 2H; 6'H a, 6'''H a ), (m, 4H; 4H, 4''H, 4'''H, 4''''H), (m, 6H; 3H, 3'H, 6'H b, 6''H a, 3''''H, 6''''H a ), (m, 6H; 6H a, 3''H, 6''H b, 3'''H, 5''''H, 6'''' H b ), (m, 1H; 5'''H), (m, 1H; 5H), (m, 7H; 2H, 6H b, 5'H, 2'''' H, CH 3 ), (m, 2H; 2''H, 5''H), 1.50 (s, 3H; Ac), 1.36 (s, 3H; Ac), 1.07 (s, 9H; t Bu), 1.02 (s, 9H; tbu); 13 C NMR (100 MHz, CDCl 3 ): (C), (C), (C), (C), (C), (C), (C 2), (C), (CH), (CH), (CH), (C), (C), (CH), (C), (C), (C), (C), (CH), (CH), (CH), (CH), (CH 2), (CH 2), (C), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH 2), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (C), (C), 99.6 (CH), 99.5 (CH), 98.7 (CH), 98.1 (CH), 97.9 (CH), S25

26 83.4 (CH), 82.9 (CH), 79.6 (CH), 78.0 (CH), 77.5 (CH), 77.2 (CH), 77.1(CH), 76.2 (CH), 75.9 (CH), 75.2 (CH 2 ), 74.7 (CH), 74.6 (CH 2 4), 74.3 (CH), 74.1 (CH 2 ), 73.9 (CH 2 ), 73.4 (CH), 72.9 (CH), 72.5 (CH), 72.2 (CH), 71.9 (CH), 69.8 (CH), 67.1 (CH 2 ), 63.2 (CH), 62.9 (CH 2 ), 62.8 (CH), 62.6 (CH), 62.3 (CH 2 ), 62.1 (CH 2 ), 60.9 (CH 2 ), 55.3 (CH 3 ), 27.0 (CH 3 ), 26.9 (CH 3 ), 20.2 (CH 3 ), 20.0 (CH 3 ), 19.4 (C), 19.3 (C); HRMS (ESI): m/z Calcd for C 134 H N 9 Br 2 Si 2 Na ([M + Na] + ): , Found: NAP pbrbn TBDPS Bn Ac Bz Bn 28 Bn Me AcSH, Pyr 2NAP pbrbn TBDPS AcHN Bn Ac Bn Bz Bn AcHN S9 Me NaMe, MeH 2NAP pbrbn TBDPS AcHN Bn 30 H H Bn Bn AcHN Me Methyl [2acetamido3(4bromobenzyl)6tertbutyldiphenylsilyl2deoxy4(2 naphthylmethyl) Dglucopyranosyl](1 4)(3benzyl Dglucopyranosyl)(1 4)2 acetamido2deoxy3,6dibenzyl Dglucopyranoside (30). (1) Azidotoacetamido conversion. Pyridine (4.6 ml, 10 ml per gram of compound 28) and thioacetic acid (4.6 ml, 10 ml per gram of compound 28) were consecutively added to a solution of compound 28 (462 mg, mmol) in CHCl 3 (4.6 ml, 10 ml per gram of compound 28) at room temperature. After stirring for 36 h, the mixture was diluted with ethyl acetate and sequentially washed with 1 N HCl (aq), saturated NaHC 3, water, and brine. The organic layer was dried over anhydrous MgS 4, filtered, and concentrated under reduced pressure to get a residue, which was purified by flash column chromatography (ethyl acetate/hexanes = 3/1) to afford the Nacetylated compound S9 (406 mg, 88%). [ ] 26 D +1.7 (c 1.9, CHCl 3 ); IR (thin film): 3440, 3063, 2930, 2859, 1733, S26

27 1662, 1264, 1242, 1112, 1070, 1043, 753, 702 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): (d, J = 7.9 Hz, 2H; ArH), (m, 3H; ArH), (m, 6H; ArH), (m, 23H; ArH), (m, 7H; ArH), 6.32 (d, J = 9.8 Hz, 1H; NH), 5.33 (t, J = 8.7 Hz, 1H; 2'H), 5.03 (d, J = 8.5 Hz, 1H; NH), (m, 6H; 1''H, ArCH 2 ), (m, 7H; 1H, 1'H, 6' H a, ArCH 2 ), (m, 2H; 2''H, ArCH 2 ), 4.45 (dd, J = 11.8, 4.2 Hz, 1H; 6'H b ), (m, 6H; 2H, 4H, 4''H, 5''H, 6''H a, 6''H b ), (m, 4H; 6H a, 3'H, 4'H, 3''H), 3.49 (t, J = 10.6 Hz, 1H; 3H), (m, 2H; 5H, 6H b ), 3.27 (m, 1H; 5'H), 3.16 (s, 3H; CH 3 ), 1.88 (s, 3H; Ac), 1.70 (s, 3H; Ac), 1.35 (s, 3H; Ac), 1.05 (s, 9H; tbu); 13 C NMR (100 MHz, CDCl 3 ): (C), (C), (C), (C), (C), (C), (C), (C), (CH), (CH), (C), (CH), (C), (C), (C), (C), (CH), (CH), (CH), (CH), (CH), (C), (CH), (CH), (CH), (CH 2), (CH), (CH), (CH), (CH 2), (CH), (CH), (CH), (CH), (CH), (CH), (C), (CH), 99.9 (CH), 98.3 (CH), 82.1 (CH), 81.4 (CH), 77.5 (CH), 77.3 (CH), 77.2 (CH), 77.0 (CH), 75.5 (CH 2 ), 75.1 (CH 2 ), 74.9 (CH 2 ), 74.0 (CH), 73.9 (CH 2 ), 73.8 (CH), 73.6 (CH 2 ), 73.2 (CH), 70.1 (CH), 67.6 (CH 2 ), 62.4 (CH 2 ), 62.2 (CH 2 ), 55.1 (CH 3 ), 53.0 (CH), 52.2 (CH), 26.8 (CH 3 ), 23.2 (CH 3 ), 22.5 (CH 3 ), 20.7 (CH 3 ), (C); HRMS (ESI): m/z Calcd for C 83 H N 2 BrSiNa ([M + Na] + ): , Found: (2) Deacylation. Sodium methoxide (34 mg, 0.63 mmol, 3 equiv) was added to a solution of compound S9 (328 mg, 0.21 mmol, 1 equiv) in MeH (3.3 ml, 10 ml per gram of S9) at room temperature. The mixture was kept stirring for 16 h, and the reaction solution was neutralized by Dowex 50WX4200 acidic resin. The resulting mixture was filtered through paper, and the filtrate was concentrated under reduced pressure. Purification of the residue by flash column S27

28 chromatography (ethyl acetate/hexanes = 4/1) provided the diol 30 (254 mg, 84%). [ ] 26 D +1.2 (c 4.5, CHCl 3 ); IR (thin film): 3415, 3309, 3066, 2930, 2929, 2860, 1660, 1536, 1366, 1112, 1044, 745, 701 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ): (m, 9H; ArH), (m, 25H; Ar H), 7.08 (m, 2H; ArH), 6.70 (d, J = 9.8 Hz, 1H; NH), 5.38 (d, J = 9.2 Hz, 1H; NH), (m, 3H; 1''H, ArCH 2 ), (m, 4H; ArCH 2 ), 4.66 (d, J = 3.5 Hz, 1H; 1H), (m, 4H; ArCH 2 ), 4.43 (d, J = 7.4 Hz, 1H; 1'H), (m, 1H; 2''H), 4.25 (td, J = 9.9, 3.5 Hz, 1H; 2H), (m, 4H; 4H, 6H a, 4''H, 6''H a ), (m, 10H; 3H, 5H, 6H b, 4'H, 5'H, 6'H a 6'H b, 3''H, 5''H, 6''H b ), 3.50 (m, 1H, 2'H), 3.39 (t, J = 9.9 Hz, 1H; 3'H), 3.32 (s, 3H; CH 3 ), 3.29 (m, 1H; 2'H), 2.89 (m, 1H; 6'H), 1.82 (s, 3H; Ac), 1.40 (s, 3H; Ac), 1.03 (s, 9H; tbu); 13 C NMR (100 MHz, CDCl 3 ): (C), (C), (C), (C), (C), (CH), (C), (CH), (C), (C), (C), (C), (C), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH), (CH) (C), 102.5(CH), 99.9 (CH), 98.6 (CH), 83.0 (CH), 81.8 (CH), 78.8 (CH), 77.6 (CH), 77.1 (CH), 76.6 (CH), 75.8 (CH), 75.7 (CH), 75.2 (CH 2 ), 75.1 (CH 2 ), 74.2 (CH 2 ), 74.1 (CH 2 ), 73.8 (CH 2 ), 73.3 (CH), 70.4 (CH), 68.6 (CH 2 ), 62.3 (CH 2 ), 61.1 (CH 2 ), 55.1 (CH 3 ), 53.3 (CH), 52.3 (CH), 26.7 (CH 3 ), 23.3 (CH 3 ), 22.5 (CH 3 ), 19.2 (C); HRMS (ESI): m/z Calcd for C 78 H N 2 BrSiNa ([M + Na] + ): , Found: NAP pbrbn TBDPS AcHN Bn 30 H H Bn Bn AcHN Me TEMP, BAIB 2NAP pbrbn TBDPS AcHN Bn C 2 H 32 H Bn Bn AcHN Me S28