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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Org Chem. Author manuscript; available in PMC 2010 June 23.
Published in final edited form as:
PMCID: PMC2891570
NIHMSID: NIHMS91169

Stereoselective Synthesis of a C-linked Neuraminic Acid Disaccharide: Potential Building Block for the Synthesis of C-Analogs of Polysialic acids

Abstract

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C-Linked neuraminic acid disaccharide was synthesized in a diastereoselective manner from a sulfone donor and aldehyde acceptor, which was protected as propargyl ether, through a samarium mediated coupling reaction. The resulting disaccharide has acetal and phenyl sulfide functional groups that can be easily converted into aldehyde and phenyl sulfone groups by photolysis and oxidation reactions, to serve as disaccharide acceptor and donor, respectively.

Polysialic acid (PSA), a long linear α(2-8)-linked polymer of sialic acid, is widely distributed in nature, from bacteria to humans. PSA is expressed on the meningitis-causing bacteria, Neisseria meningitidis B, and the capsular polysaccharide of Escherichia coli K1.13 It has also been found on the cell surface in a number of important cancers and is believed to be associated with metastasis and correlates with tumor progression.4 PSA is most often found as an N-linked glycan on neural cell adhesion molecules (PSA-NCAM) and attenuates NCAM adhesion, facilitating neural cell migration and regeneration.5 PSA is also found in embryonic tissues and participates in developmental biology.6 In this respect, PSAs are important targets for biological studies, in the development of vaccines for protection against meningococcal infection, and for the treatment of various cancers including Wilms’ tumor and small cell lung carcinoma.7,8

PSAs are known to exhibit both chemical and enzymatic hydrolytic instability.9 The PSA glycosidic linkages are very labile and subject to self-cleavage under mildly acidic conditions. In contrast, C-glycosides, in which the interglycosidic oxygen atoms are replaced with carbon atoms, are resistant to chemical and enzymatic degradation.10 The unusual lability of PSAs, their participation in developmental biology and their reappearance in various tumors, makes their C-glycosidic analogs ideal targets for a wide array of experimental, biological and potential therapeutic applications.11

Over the past ten years, samarium mediated C-glycosylation reactions have been reported by our laboratory12 and Beau research group13 for the synthesis of C-linked α-sialosides. Recently, we reported the first synthesis of a C-linked neuraminic acid disaccharide, as a versatile precursor of C-analogs of oligosialic acids and gangliosides.12f However, this synthesis afforded a 1:1 ratio of R- and S-diastereoisomers at the bridge hydroxymethylene group, requiring additional steps to remove this hydroxyl group. The lack of stereoselectivity and the development of a more efficient strategy for C-oligosialic acids synthesis prompted us to investigate new protecting groups and a common donor-acceptor concept, widely used by others in oligosaccharide synthesis.14 In this effort, we found that propargyl protecting group chemistry afforded a single diastereoisomer with a photolabile protecting group, for sialic acid aldehyde protection. Furthermore, this protecting group was well suited for the development of a common donor-acceptor strategy owing to its easy introduction and removal under mild reaction conditions.

Herein, we report a diastereoselective synthesis of a C-linked neuraminic acid disaccharide that is useful as a donor-acceptor disaccharide for the further synthesis of C-linked α (2–8) oligosialic acids. The synthesis of C-glycosylation aldehyde acceptor 6 and sulfone donor 8 was straightforward from the known C-9 PMB protected compound 112f (Scheme 1). Propargylation of hydroxyl groups of 1 with propargyl bromide in the presence of barium hydroxide and barium oxide in DMF afforded the propargylated carboxylic acid derivative 2 in a modest 50% yield. Methylation of the carboxylic acid using TMSCHN2 gave methylester 3 in 95% yield. Acetylation of acetamido group in 3, required to prevent intramolecular cyclization reaction at later stage in the synthesis, was performed by reaction with isopropenyl acetate in the presence of a catalytic amount of p-toluenesulfonic acid, affording 4 in 96% yield. The PMB protecting group on C-9 of compound 4 was chemoselectively removed using trifluoroacetic acid in DCM at −20 °C, affording 5 in 75% yield. Oxidation of the resulting C-9 hydroxyl group using the mild Dess-Martin periodinane oxidizing agent, afforded the desired aldehyde acceptor 6 in 95% yield. The aldehyde group of 6 was protected with salicylic diol,15 a photolabile carbonyl protecting group, afforded the fully protected acetal derivative 7 as a single diastereomer in 83% yield. The aldehyde derivative 6 could be recovered by photochemical deprotection of 7 in 60% yield using a 450W medium pressure mercury lamp fitted with pyrex filter sleeve to minimize undesired side reactions, demonstrating the utility of this photolabile aldehyde protecting group. The target sulfone donor 8 was obtained in 85% yield through the oxidation of compound 7 with 3-chloroperbenzoic acid.

Having the proper sulfone donor 8 and aldehyde acceptor 6 in hand, C-glycosylation was performed under the standard Barbier conditions.16 As shown in Scheme 2, only one diastereoisomer 9R was obtained in 70% yield, based on the consumed donor. Structural analysis of 9R was performed using 1D and 2D NMR experiments including 1H-NMR, 13C-NMR, COSY, HSQC, TOCSY, and ROESY (see supporting information). Initially, all protons and carbons were assigned. Next, their through-bond and through-spatial correlations were determined. The α-configuration of the newly formed C-glycoside was confirmed by the presence of NOEs, in the 2D-ROESY spectrum, between H-4, H-6 and the methyl ester protons, such NOEs would only be observed in an α-sialoside adopting 5C2 conformation. The absolute configuration of the newly formed hydroxymethylene group was also identified in the same 2D-ROESY spectrum, which showed close spatial proximity between H-3ax and H-9′. The Felkin-Ahn model17 can be used to predict the stereochemical outcome of a kinetically controlled addition of a nucleophile to a prochiral aldehyde. The bulkiest ligand a to the carbonyl group in 9R, the oxygen atom bearing propargyl group at C-8′, has a perpendicular relationship to the plane of the carbonyl group. The incoming nucleophile approaches to the carbonyl group through the Bürgi-Dunitz trajectory18 anticlinal to the bulkiest oxygen atom at C-8′, resulting in R-configuration. Based on this analysis, shown in Scheme 2, and the presence of an NOE between H-3ax and H-9′, R-configuration was confirmed.

Experimental Section

Methyl [phenyl 5-acetamido-9-O-(p-methoxy)benzyl-3,5-dideoxy-4,7,8-tri-O-propargyl-2-thio-D-glycero-α-D-galacto-non-2-ulopyranosid]onate (3)

Propargyl bromide (18.7 mL, 168 mmol), Ba(OH)2·8H2O (13.2 g, 42 mmol), and BaO (12.9 g, 84 mmol) were added successively to a solution of compound 1 (4.5 g, 8.4 mmol) in anhydrous DMF (100 mL) at room temperature. After stirring overnight, the reaction mixture was quenched by adding of TsOH·H2O (15.9 g, 84 mmol), and then diluted with ethyl acetate (300 mL). After washing with saturated aqueous NH4Cl (300 mL), the organic phase was dried (MgSO4), filtered, and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (DCM/MeOH, 20/1) to afford phenyl 5-acetamido-9-O-(p-methoxy)benzyl-3,5-dideoxy-4,7,8-tri-O-propargyl-2-thio-D-glycero-α-D-galacto-non-2-ulopyranosidic acid (2) (2.67 g, 50%): Rf = 0.15 (DCM/MeOH, 6/1). TMSCHN2 (2 M in diethyl ether, 7.5 mL, 15 mmol) was added to a solution of 2 in methanol/diethyl ether (1/1, 100 mL) at room temperature. After stirring for 3 h at the same temperature, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (33% ethyl acetate in hexane) to afford 3 (2.60 g, 95%): Rf = 0.25 (ethyl acetate/hexane, 2/1); 1H NMR (500 MHz, CDCl3) δ 7.55-7.53 (m, 2H, aromatic), 7.37-7.28 (m, 5H, aromatic), 6.89-6.87 (m, 2H, aromatic), 5.48 (d, 1H, J = 8.5 Hz), 4.53 (s, 2H), 4.47 (dd, 1H, J = 2.5, 15.5 Hz), 4.26-4.19 (m, 2H), 4.15 (dd, 1H, J = 2.5, 15.5 Hz), 4.05 (dd, 1H, J = 2.5, 15.5 Hz), 3.97-3.93 (m, 2H), 3.89-3.86 (m, 2H), 3.80 (s, 3H), 3.74-3.68 (m, 3H), 3.62 (s, 3H), 3.61-3.59 (m, 1H), 2.94 (dd, 1H, J = 4.5, 12.5 Hz), 2.48 (t, 1H, J = 2.5Hz), 2.44 (t, 1H, J = 2.5Hz), 2.38 (t, 1H, J = 2.5Hz), 1.95 (s, 3H), 1.79 (dd, 1H, J = 11.5, 12.5 Hz); 13C NMR (125 MHz, CDCl3) δ170.8, 169.1, 159.4, 137.1, 137.0, 130.5, 129.7, 129.6, 129.4, 128.9, 128.8, 113.9, 87.1, 81.1, 81.0, 80.2, 77.8, 76.5, 75.1, 74.9, 74.7, 74.5, 74.4, 74.2, 73.9, 73.2, 60.5, 58.2, 56.6, 55.6, 52.9, 50.7, 37.5, 24.0; HR MALDI-TOF MS: m/z: calcd for C35H39NO9S [M+Na]+: 672.2238; found: 672.2240.

Methyl [phenyl 5-(N-acetylacetamido)-9-O-(p-methoxy)benzyl-3,5-dideoxy-4,7,8-tri-O-propargyl-2-thio-D-glycero-α-D-galacto-non-2-ulopyranosid]onate (4)

A catalytic amount of TsOH·H2O (190 mg, 1.0 mmol) was added to a solution of 3 (2.55 g, 3.92 mmol) in isopropenyl acetate (50 mL) at room temperature. The reaction mixture was heated to 60 °C, and stirred for 5 h at the same temperature. After quenching by adding of NEt3 (140 μL, 1.0 mmol), the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (25% ethyl acetate in hexane) to afford 4 (2.60 g, 96%): Rf = 0.65 (ethyl acetate/hexane, 2/1); 1H NMR (500 MHz, CDCl3) δ7.55-7.53 (m, 2H, aromatic), 7.35-7.24 (m, 5H, aromatic), 6.89-6.87 (m, 2H, aromatic), 4.67 (dd, 1H, J = 2.5, 15.5 Hz), 4.54 (d, 1H, J = 11.5 Hz), 4.47 (dd, 1H, J = 2.5, 15.5 Hz), 4.45 (d, 1H, J = 11.5 Hz), 4.67 (dt, 1H, J = 5.0, 10.5 Hz), 4.27-4.17 (m, 3H), 4.15 (dd, 1H, J = 2.5, 15.5 Hz), 4.10-4.06 (m, 2H), 4.03-4.00 (m, 1H), 3.92 (t, 1H, J = 10.0 Hz), 3.80 (s, 3H), 3.75 (t, 1H, J = 3.0 Hz), 3.69 (dd, 1H, J = 7.0, 11.0 Hz), 3.53 (s, 3H), 3.12 (dd, 1H, J = 5.0, 13.0 Hz), 2.45 (s, 3H), 2.44 (t, 1H, J = 2.5 Hz), 2.39-2.37 (m, 2H), 2.38 (s, 3H), 1.78 (dd, 1H, J = 10.5, 13.0 Hz); 13C NMR (125 MHz, CDCl3) δ175.9, 175.4, 168.5, 159.3, 136.9, 136.8, 130.9, 129.6, 129.4, 129.3, 129.0, 113.9, 88.5, 80.6, 79.5, 79.4, 79.3, 76.3, 75.7, 75.5, 75.4, 75.0, 74.7, 74.5, 73.2, 72.4, 59.9, 58.7, 58.0, 57.3, 55.6, 52.8, 40.0, 28.5, 26.0; HR MALDI-TOF MS: m/z: calcd for C37H41NO10S [M+Na]+: 714.2343; found: 714.2352.

Methyl [phenyl 5-(N-acetylacetamido)-3,5-dideoxy-4,7,8-tri-O-propargyl-2-thio-D-glycero-α-D-galacto-non-2-ulopyranosid]onate (5)

Trifluoroacetic acid (7.0 mL) was added to a solution of 4 (2.60 g, 3.75 mmol) in DCM (50 mL) at −20 °C. After stirring for 1 h at the same temperature, the reaction mixture was quenched with saturated aqueous NaHCO3 (100 mL). After separation, the organic phase was dried (MgSO4), filtered, and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (25% ethyl acetate in hexane) to afford 5 (1.59 g, 75%): Rf = 0.27 (ethyl acetate/hexane, 1/1); 1H NMR (500 MHz, CDCl3) δ7.56-7.54 (m, 2H, aromatic), 7.44-7.35 (m, 3H, aromatic), 4.57 (dd, 1H, J = 2.0, 9.5 Hz), 4.49 (dd, 1H, J = 2.5, 15.5 Hz), 4.32 (dt, 1H, J = 5.0, 10.5 Hz), 4.30 (dd, 1H, J = 2.5, 15.5 Hz), 4.19-4.06 (m, 5H), 3.92 (t, 1H, J = 10.0 Hz), 3.76-3.72 (m, 3H), 3.60 (s, 3H), 3.15 (dd, 1H, J = 5.0, 13.0 Hz), 2.47 (t, 1H, J = 2.5 Hz), 2.45 (s, 3H), 2.42 (t, 1H, J = 2.5 Hz), 2.40 (s, 3H), 2.39 (t, 1H, J = 2.5 Hz), 1.81 (dd, 1H, J = 10.5, 13.0 Hz); 13C NMR (125 MHz, CDCl3) δ175.8, 175.5, 168.6, 137.2, 130.5, 129.2, 129.0, 88.4, 82.5, 80.2, 79.4, 79.3, 75.9, 75.8, 75.7, 75.1, 75.0, 74.9, 72.1, 62.1, 59.9, 58.4, 58.3, 57.3, 52.9, 39.9, 28.6, 26.0; HR MALDI-TOF MS: m/z: calcd for C29H33NO9S [M+Na]+: 594.1768; found: 594.1765.

Methyl [phenyl 5-(N-acetylacetamido)-3,5-dideoxy-4,7,8-tri-O-propargyl-2-thio-D-glycero-α-D-galacto-non-2-(9-oxa-ulopyranosid)]onate (6)

Dess-Martin periodinane solution (0.3 M in DCM, 36 mL, 10.7 mmol) was added slowly to a solution of 5 (1.53 g, 2.67 mmol) in DCM (100 mL) at 0 °C. After stirring for 1 h at 0 °C, the reaction mixture was warmed to room temperature, and then stirred for another 2 h. After quenching by adding of aqueous 0.1 M Na2S2O3 solution (100 mL), the reaction mixture was separated. The organic phase was washed with saturated aqueous NaHCO3 (100 mL) and dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (20% ethyl acetate in hexane) to afford 6 (1.44 g, 95%): Rf = 0.50 (ethyl acetate/hexane, 1/1); 1H NMR (500 MHz, CDCl3) δ9.76 (s, 1H), 7.50-7.35 (m, 5H, aromatic), 4.75 (dd, 1H, J = 2.5, 9.5 Hz), 4.48 (d, 1H, J = 1.0 Hz), 4.37 (dt, 1H, J = 5.0, 10.5 Hz), 4.33-4.29 (m, 3H), 3.94 (t, 1H, J = 10.0 Hz), 3.90 (dd, 1H, J = 1.5, 2.0 Hz), 3.66 (s, 3H), 3.16 (dd, 1H, J = 5.0, 13.0 Hz), 2.42 (s, 3H), 2.41 (s, 3H), 2.42-2.40 (m, 2H), 2.39 (t, 1H, J = 2.5 Hz), 1.84 (dd, 1H, J = 11.0, 13.0 Hz); 13C NMR (125 MHz, CDCl3) δ200.2, 175.7, 175.5, 168.7, 137.1, 130.6, 129.2, 129.0, 88.6, 84.9, 79.4, 78.8, 78.5, 77.1, 76.2, 76.0, 75.8, 75.1, 74.9, 72.2, 59.8, 58.2, 57.5, 57.3, 53.1, 40.0, 28.7, 26.0; HR MALDI-TOF MS: m/z: calcd for C29H31NO9S [M+Na]+: 592.1612; found: 592.1615.

Methyl [phenyl 5-(N-acetylacetamido)-3,5-dideoxy-4,7,8-tri-O-propargyl-2-thio-D-glycero-α-D-galacto-non-2-(9-oxa-ulopyranosid)]onate (1′,1′)-diphenyl-5-methoxysalicylic alcohol acetal (7)

A catalytic amount of TsOH·H2O (57 mg, 0.3 mmol) was added to a solution of 6 (570 mg, 1.0 mmol) and (1′,1′)-diphenyl-5-methoxysalicylic alcohol (920 mg, 3.0 mmol) in anhydrous benzene (20 mL) at room temperature. After stirring for 1 day at the same temperature, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (15% ethyl acetate in hexane) to afford 7 (712 mg, 83%): Rf = 0.45 (ethyl acetate/hexane, 2/3); 1H NMR (500 MHz, CDCl3) δ7.41-7.22 (m, 13H, aromatic), 7.14-7.11 (m, 2H, aromatic), 6.97 (d, 1H, J = 9.0 Hz, aromatic), 6.77 (dd, 1H, J = 3.0, 9.0 Hz, aromatic), 6.40 (d, 1H, J = 3.0 Hz, aromatic), 5.51 (d, 1H, J = 3.0 Hz), 4.78 (dd, 1H, J = 2.5, 9.5 Hz), 4.49 (dd, 1H, J = 2.5, 15.5 Hz), 4.37 (dt, 1H, J = 5.0, 10.5 Hz), 4.33 (dd, 1H, J = 2.5, 15.5 Hz), 4.23 (d, 2H, J = 2.0 Hz), 4.15-4.07 (m, 3H), 3.99 (t, 1H, J = 10.0 Hz), 3.87 (dd, 1H, J = 2.5, 15.5 Hz), 3.62 (s, 3H), 3.31 (s, 3H), 3.10 (dd, 1H, J = 5.0, 12.5 Hz), 2.48 (s, 3H), 2.42 (t, 1H, J = 2.5 Hz), 2.40 (s, 3H), 2.38 (t, 1H, J = 2.5 Hz), 2.21 (t, 1H, J = 2.5 Hz), 1.73 (dd, 1H, J = 11.0, 12.5 Hz); 13C NMR (125 MHz, CDCl3) δ176.0, 175.2, 168.5, 153.3, 146.1, 146.0, 143.8, 136.8, 129.9, 129.6, 128.7, 128.4, 128.2, 128.1, 127.7, 126.0, 118.4, 114.9, 114.4, 95.3, 88.0, 84.6, 80.3, 79.8, 79.6, 77.7, 75.6, 75.0, 74.8, 74.6, 72.5, 59.6, 59.5, 57.3, 56.8, 55.8, 52.5, 39.8, 28.6, 25.9; HR MALDI-TOF MS: m/z: calcd for C49H47NO11S [M+Na]+: 880.2762; found: 880.2774.

Photochemical reaction to remove the photolabile aldehyde protecting group

A solution of acetal 7 (20 mg, 0.023 mmol) in acetonitrile (20 mL) was photolyzed at room temperature for 80 min. After photolysis, the reaction mixture was concentrated in vacuo, and then purified by silica gel column chromatography (20% ethyl acetate in hexane) to afford aldehyde 6 (8.6 mg, 60%)

Methyl 5-(N-Acetylacetamido)-3,5-dideoxy-4,7,8-tri-O-propargyl-2-phenylsulfonyl-D-glycero-α-D-galacto-non-2-(9-oxa-ulopyranosid)]onate (1′,1′)-diphenyl-5-methoxysalicylic alcohol acetal (8)

3-Chloroperbenzoic acid (233 mg, 60%, 0.94 mmol) was added to a solution of 7 (270 mg, 0.31 mmol) in DCM (15 mL) at 0 °C. After stirring for 1 h at the same temperature, the reaction mixture was warmed to room temperature, and then stirred another 1 h to complete the reaction. Then, the reaction mixture was quenched by aqueous 0.1 M Na2S2O3 solution (30 mL), separated, and washed with saturated aqueous NaHCO3 (30 mL). The organic phase was dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (15% ethyl acetate in hexane) to afford 8 (233 mg, 85%): Rf = 0.35 (ethyl acetate/hexane, 2/3); 1H NMR (500 MHz, CDCl3) δ7.84-7.82 (m, 2H, aromatic), 7.64-7.61 (m, 1H, aromatic), 7.50-7.47 (m, 2H, aromatic), 7.37-7.25 (m, 10H, aromatic), 6.92 (d, 1H, J = 9.0 Hz, aromatic), 6.77 (dd, 1H, J = 3.0, 9.0 Hz, aromatic), 6.40 (d, 1H, J = 3.0 Hz, aromatic), 5.32 (d, 1H, J = 2.5 Hz, H9), 4.83 (dd, 1H, J = 2.0, 10.0 Hz, H6), 4.53 (dt, 1H, J = 4.5, 10.5 Hz, H4), 4.27 (dd, 1H, J = 2.5, 15.5 Hz, OCH2CCH), 4.21 (dd, 1H, J = 2.5, 15.5 Hz, OCH2CCH), 4.16 (dd, 1H, J = 2.5, 15.5 Hz, OCH2CCH), 4.11 (dd, 1H, J = 2.5, 15.5 Hz, OCH2CCH), 3.99 (t, 1H, J = 10.0 Hz, H5), 3.95 (dd, 1H, J = 2.5, 15.5 Hz, OCH2CCH), 3.88 (dd, 1H, J = 2.5, 15.5 Hz, OCH2CCH), 3.76 (s, 3H, COOCH3), 3.74 (dd, 1H, J = 2.0, 8.5 Hz, H7), 3.65 (dd, 1H, J = 2.0, 8.5 Hz, H8), 3.63 (s, 3H, OCH3), 3.25 (dd, 1H, J = 4.5, 12.5 Hz, H3eq), 2.47 (s, 3H, CH3CO), 2.42 (t, 1H, J = 2.5 Hz, OCH2CCH), 2.40 (t, 1H, J = 2.5 Hz, OCH2CCH), 2.36 (s, 3H, CH3CO), 2.09 (dd, 1H, J = 11.0, 12.5 Hz, H3ax), 2.01 (t, 1H, J = 2.5 Hz, OCH2CCH); 13C NMR (125 MHz, CDCl3) δ 175.9, 175.0, 165.7, 153.5, 145.8, 145.7, 143.9, 134.8, 134.4, 131.2, 129.8, 129.0, 128.4, 128.3, 128.2, 127.8, 125.9, 118.4, 115.0, 114.4, 95.6, 94.7, 84.9, 80.2, 79.9, 79.2, 76.7, 75.6, 75.1, 75.0, 74.8, 73.8, 71.7, 59.6, 58.8, 57.1, 56.8, 55.8, 53.9, 32.6, 28.4, 25.; HR MALDI-TOF MS: m/z: calcd for C49H47NO13S [M+Na]+: 912.2660; found: 912.2676.

Methyl 5-(N-acetylacetamido)-3,5-dideoxy-2-C-{(9′R)-[methyl (phenyl 5′-(N-acetylacetamido)-3′,5′-dideoxy-4′,7′,8′-tri-O-propargyl-2′-thio-D-glycero-α-D-galacto-non-2′-ulopyranosid)]onate}-4,7,8-tri-O-propargyl-D-glycero-α-D-galacto-non-2-(9-oxa-ulopyranosid)]onate (1′,1′)-diphenyl-5-methoxysalicylic alcohol acetal (9R)

SmI2 solution (0.63 mL, 0.1M in THF, 0.063 mmol) was added to a mixture of sulfone donor 8 (14 mg, 0.0157 mmol) and aldehyde acceptor 6 (12 mg, 0.021 mmol) and stirred for 5 min at room temperature. After quenching by aqueous 0.1 M Na2S2O3 solution (2 mL), the reaction mixture was diluted with DCM (3 mL), separated, and washed with saturated aqueous NaHCO3 (5 mL). The organic phase was dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (20% ethyl acetate in hexane) to afford 9R {8 mg, 70%, calculated based on consumed donor 8 (8 mg)}: Rf = 0.45 (ethyl acetate/hexane, 1/1); 1H NMR (500 MHz, CDCl3) δ7.57-7.23 (m, 15H, aromatic), 6.92 (d, 1H, J = 9.0 Hz, aromatic), 6.77 (dd, 1H, J = 3.0, 9.0 Hz, aromatic), 6.42 (d, 1H, J = 3.0 Hz, aromatic), 5.53 (d, 1H, J = 4.5 Hz, H9), 5.23 (dd, 1H, J = 2.0, 10.0 Hz, H6), 4.75 (dd, 1H, J = 2.5, 10.0 Hz, H6′;), 4.54-4.40 (m, 4H, 4 × OCH2CCH), 4.35-4.29 (m, 3H, 2 × OCH2CCH, H4′;), 4.27-4.18 (m, 4H, OCH2CCH, H9;, H4, H8), 4.10-4.02 (m, 4H, 3 × OCH2CCH, H8′;), 4.00 (dd, 1H, J = 2.5, 15.5 Hz, OCH2CCH), 3.91 (d, 1H, J = 2.5 Hz, H7′;), 3.87 (t, 1H, J = 10.0 Hz, H5), 3.85 (s, 3H, COOCH3′;), 3.82-3.75 (m, 3H, OCH2CCH, H5;, H7), 3.64 (s, 3H, OCH3), 3.54 (s, 3H, COOCH3), 3.12 (dd, 1H, J = 4.5, 13.5 Hz, H3eq), 3.04 (d, 1H, J = 10.5 Hz, OH), 2.77 (dd, 1H, J = 5.0, 13.0 Hz, H3;eq), 2.50 (s, 3H, CH3CO), 2.46 (t, 1H, J = 2.5 Hz, OCH2CCH), 2.45 (s, 3H, CH3CO), 2.41 (t, 1H, J = 2.5 Hz, OCH2CCH), 2.40 (t, 1H, J = 2.5 Hz, OCH2CCH), 2.38 (s, 6H, 2 × CH3CO), 2.32 (t, 1H, J = 2.5 Hz, OCH2CCH), 2.30 (t, 1H, J = 2.5 Hz, OCH2CCH), 2.28 (t, 1H, J = 2.5 Hz, OCH2CCH), 1.76 (dd, 1H, J = 11.5, 13.5 Hz, H3ax), 1.53 (dd, 1H, J = 10.5, 13.0 Hz, H3;ax); 13C NMR (125 MHz, CDCl3) δ176.0, 175.9, 175.4, 174.8, 171.7,3 169.5, 153.4, 146.1, 145.5, 143.7, 137.3, 130.4, 129.6, 129.3, 128.9, 128.5, 128.4, 128.2, 128.1, 127.7, 125.9, 118.4, 115.1, 114.5, 95.0, 87.8, 84.6, 84.3, 81.4, 80.4, 80.1, 79.7, 79.5, 79.4, 78.9, 78.1, 77.8, 76.2, 75.7, 75.3, 75.2, 75.1, 74.9, 74.7, 74.4 (2), 72.6 (2), 72.0, 60.5, 59.8, 59.6, 59.2, 58.7, 57.6, 57.3, 56.4, 55.8, 53.1, 52.4, 40.0, 37.3, 29.9, 28.6, 28.4, 25.9 (2); HR MALDI-TOF MS: m/z: calcd for C72H74N2O20S [M+Na]+: 1341.4448; found: 1341.4452.

Supplementary Material

1_si_001

Supporting Information Available:

General procedures and 1H, 13C NMR spectra for all compounds 3-9R and COSY, HSQC, TOCSY, ROESY spectra and HR ESI-MS spectrum for disaccharide 9R. This material is available free of charge via the internet at http://pubs.acs.org.

Acknowledgments

We thank Dr. Scott McCallum and Dr. Herb Schwartz for their help with 2D NMR and support from the NIH in the form of grant AI065787.

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