For the d-glycero-d-gluco donor, on the other hand, modest axial selectivity isobserved. Selectivity patterns are discussed in terms of the side-chainconformation of the donors in combination with the electron-withdrawingeffect of the thioacetal group. After glycosylation, removal of thethiophenyl moiety and hydrogenolytic deprotection is achieved in asingle step with Raney nickel.The use of a phenylthiogroup (SPh) as a dummy ligandat the 6-positionto control the side-chain conformation of a series of hexopyranosyldonors is described. The SPh group limits side-chain conformationin a configuration-specific manner, which parallels that seen in theheptopyranosides, and so influences glycosylation selectivity. Withboth  This p mixture 1.5−713 systems in which the donor side chain predominantlyoccupies the tg conformation are less reactive andmore equatorially selective than those with a predominant gg conformation of the side chain, whereas those whose sidechain mainly populates the gt conformation have intermediatereactivity and selectivity. This is because (i) the tg conformation maximizes the electron-withdrawing effect of the C6–O6bond, thereby destabilizing nascent positive charge at the anomericcenter, so favoring bimolecular mechanisms, and (ii) the gg conformation is best placed to electrostatically stabilize the developingpositive charge at the anomeric center, while (iii) the gt conformation has intermediate behavior.15 Glycosylation reactivity and selectivity in the hepto- and highercarbon pyranosyl donors is therefore an inherent function of the relativeconfiguration of C4–C6 stereotriad and of the so-imposed side-chainconformations. In the hexopyranosides, donor reactivity can be correspondinglyinfluenced by locking the side-chain conformation with the aid of4,6-O-benzylidene acetals or related cyclic protectinggroups. However, as only one conformation is available in this mannerfor the gluco (tg) and galacto (gg) series, such options are limited.16In typical glycosylation reactions in which anelectrophilic glycosyldonor undergoes nucleophilic substitution of a leaving group by anacceptor alcohol,We now address the question of the control of side-chain conformationand glycosylation reactivity and selectivity in hexopyranosyl donorswithout the use of rigid bicyclic protecting groups at the 4,6-position.To this end, we envisaged that side-chain conformation in the hexopyranosidescould be achieved by installation of a temporary or dummy ligand atthe 6-position to mimic the C6–C7 bond in the hepto and highercarbon sugars. To be effective, such a ligand would need to be easilyinstalled and removed, of comparable electronegativity and stericbulk to a methyl group, and stable under typical glycosylation conditions,causing us to settle on the thioether moiety.5a–d began with the synthesisof alcohols 1 and 2,19 which wereoxidized to the corresponding aldehydesusing the Dess–Martin periodinane21 followed by the formation of dibenzylacetals 3 and 4 with tribenzyl orthoformate and a catalytic amount of p-TSA in 71 and 65% yields, respectively.22 These acetals were treated with 1.5 equiv of BF3·OEt2 and 1.5 equiv of thiophenol at −78 to−20 °C to obtain the S-phenyl-O-benzyl monothioacetals 5a and 5b in the galacto series and 5c and 5d in the gluco series 3 followedby treatment with catalytic OsO4 and N-methylmorpholine N-oxide to give the correspondinghemiacetals 6a–d as α:βmixtures.25 Finally, thetrichloroacetimidate donors 7a–d wereobtained by treatment with 5 equiv of Cl3CCN and catalyticamount of DBU at 0 °C in the form of α:β mixtures.29The allyl groups were removedfrom each of 2Cl2 solutions of donors 7a–d (1.0 equiv) with the acceptor (1.2 equiv)and 4 Å AWMSfor 1 h, followed by cooling to −78 °C and stirring foranother 0.5 h. TMSOTf (0.2 equiv) then was added to the mixture asa 10% solution in CH2Cl2.32 All glycosylations were maintained at −78°C and quenched with 0.2 mL of triethylamine at that temperaturebefore the mixtures were warmed to room temperature, filtered throughcelite, diluted with CH2Cl2, and washed withsaturated aqueous NaHCO3. The resulting glycosides werethen isolated by silica gel chromatography, leading to the resultspresented in Glycosylation reactions were carried out by stirring 0.15M CHd-glycero-d-galacto and l-glycero-d-galacto donors  plates. Detection of compounds was achieved by UV absorption(254 nm) and by staining with 10% sulfuric acid in ethanol. Purificationof crude residues was performed by silica gel chromatography usinga 230–400 mesh-grade 60 silica. Specific rotations [deg cm3 g–1 dm–1] were measuredin chloroform on an automatic polarimeter with a path length of 10cm. NMR spectra were recorded in C6D6, CD2Cl2, or CDCl3 at either 500, 600, or900 MHz as indicated. High-resolution mass spectra (HRMS) were recordedin the electrospray mode using an orbitrap mass analyzer (Thermo FisherESI-Orbitrap). Chemical shifts (δ) are recorded in ppm, andmultiplicities are abbreviated as follows: s (singlet), m (multiplet),br (broad), d (doublet), t (triplet), and q (quartet). Structuralassignments were made with additional information from gCOSY, gHSQC,and gHMBC experiments. All manipulations and reactions using osmiumtetroxide should be conducted in a well-ventilated fume hood.All reactions were carriedout under argon unless otherwise stated. 1-Adamantanol and 1,2:5,6-di-44This colorless oil was prepared accordingto the literature method and had spectral data consistent with theliterature .45Thiscolorless syrup was prepared accordingto the literature method and had spectral data consistent with theliterature .47This colorless oil was prepared accordingto the literature method and had spectral data consistent with theliterature .44Thiscolorless syrup was prepared accordingto the literature method and had spectral data consistent with theliterature .-d-galactose pentaacetate, starting with the synthesis of the allylglycoside and deacetylation,17 followedby trityl group protection of O6, benzylation, and the final tritylgroup removal .19 The spectral data isconsistent with the literature.481H NMR  δ 7.40–7.26 , 6.04–5.90 , 5.33 , 5.19 , 5.02–4.92 , 4.82 , 4.79 , 4.75 , 4.67 , 4.47–4.35 , 4.14 , 3.88 , 3.80–3.73 ,3.57–3.47 , 3.37 , 1.64 . 13C{1H} NMR  δ 138.7 (C), 138.5 (C), 138.35 (C), 134.3 (CH), 128.7(Ar), 128.5 (Ar), 128.4 (Ar), 128.3 (Ar), 128.0 (Ar), 127.8 (Ar),127.7 (Ar), 127.6 (Ar), 117.2 (=CH2), 103.2 (CH1), 82.3(CH3), 79.7 (CH2), 75.3 (CH5), 74.6 (CH6), 74.2 (CH4), 73.5 (CH2), 73.0 (CH2), 70.4 (OCH2), 62.1 (CH2). HRMS (ESI) m/z: [M + Na]+ calcd for C30H34O6Na 513.2247; found 513.2242.This compound,a colorless oil, was prepared from β-d-glucose pentaacetate, starting with the synthesisof the allyl glycoside and deacetylation,17 followed by trityl group protection of O6, benzylation, and thefinal trityl group removal .19 The spectral data is consistent with the literature.491H NMR  δ 7.36–7.24 , 5.95, 5.34 , 5.21 , 4.98–4.89, 4.86 , 4.80 , 4.72 , 4.63 , 4.49 , 4.39 , 4.15 , 3.86 , 3.70 , 3.66 , 3.56, 3.44 , 3.35 , 1.87 . 13C{1H} NMR  δ 138.6 (Ar-H), 138.4 (Ar-H), 138.1 (Ar-H), 134.0(=CH), 128.6 (Ar-H), 128.5 (Ar-H), 128.3 (Ar-H), 128.2 (Ar-H), 128.0(Ar-H), 127.97 (Ar-H), 127.8 (Ar-H), 127.7 (Ar-H), 117.6 (CH2), 102.9 (CH1), 84.6 (CH4), 82.4 (CH3), 77.7 (CH2), 75.8 (CH5), 75.2(CH2-6), 75.12 (CH2), 75.07 (CH2),70.8 (CH2), 62.2 (CH2). HRMS (ESI) m/z: [M + Na]+ calcd for C30H34O6Na 513.2247; found 513.2240.Thiscompound, an amorphous white solid, was prepared from β1 or 2, 1 equiv, 0.5 M) in anhydrous CH2Cl2 was added Dess–Martin periodinane (1.5 equiv). Stirringwas continued for 1 h at room temperature. After the completion ofthe reaction as observed by thin-layer chromatography (TLC), saturatedaqueous Na2S2O3 (50 mL) and saturatedaqueous NaHCO3 (50 mL) were added to the reaction mixture,which was stirred for another 0.25 h. The organic layer was separated,and the aqueous layer was extracted with CH2Cl2 (3 × 50 mL). The combined organic layers were collected, driedover anhydrous Na2SO4, and concentrated underreduced pressure to afford a crude residue, which was used immediatelywithout further purification.To a stirred solution of alcohol (2SO4 (0.2 g/mmol) for 5 min before the addition of p-TSA (0.2 equiv) and subsequently the addition of CH(OBn)3 (2 equiv) in a dropwise manner. After complete addition,the reaction mixture was stirred at room temperature overnight; then,it was quenched with Et3N (2.0 mL), filtered, and concentratedunder reduced pressure to afford a crude residue. Purification ofthis crude product by silica gel chromatography using 100% hexaneto 5% ethyl acetate in hexane gave the dibenzylacetals.A 0.5 M solution of crude aldehyde(1 equiv) in anhydrous acetonitrile was stirred with Na1  following general procedure GP1, eluting from silica gel with 5% ethyl acetate in hexanes.White amorphous solid  [α]D21 = +27.7 . 1H NMR  δ 7.41–7.19 , 5.94, 5.29 , 5.17 , 5.02 , 5.00–4.92 , 4.82–4.73 , 4.74–4.63 , 4.48 , 4.44–4.36 , 4.26, 4.11 , 4.03 , 3.88 , 3.52 ,3.49 ; 13C{1H} NMR  δ 138.9 (C), 138.8 (C), 138.6(C), 138.3 (C), 137.6 (C), 134.2 (=CH), 128.5 (Ar), 128.44 (Ar), 128.4(Ar), 128.2 (Ar), 128.1 (Ar), 128.0 (Ar), 127.9 (Ar), 127.83 (Ar),127.8 (Ar), 127.65 (Ar), 127.6 (Ar), 127.5 (Ar), 117.3 (=CH2), 103.2 (C1), 100.5 (C6), 82.4 (C3), 79.5 (C2), 75.7 (CH2), 75.3 (C5), 74.7 (CH2), 74.5 (C4), 73.3 (CH2), 70.3 (CH2), 69.7 (OCH2), 69.4 (CH2). HRMS (ESI) m/z:[M + Na]+ calcd for C44H46O7Na 709.3136; found, 709.3118.Prepared fromcompound 2 following general procedure GP1, eluting from silicagel with 5% ethyl acetate in hexanes. White amorphous solid  [α]D21 = +3.9 . 1H NMR  δ7.43–7.16 , 7.09 , 5.96 , 5.34 , 5.20 , 5.04 , 5.00 , 4.94 , 4.87–4.71 , 4.59 , 4.47 , 4.43 , 4.34 , 4.12 , 3.73 , 3.68 , 3.63 , 3.57 . 13C{1H} NMR  δ 138.46 (C), 138.4 (C), 138.22(C), 138.2 (C), 137.5 (C), 134.0 (=CH), 128.34 (Ar), 128.30 (Ar),128.24 (Ar), 128.2 (Ar), 128.10 (Ar), 128.07 (Ar), 127.77 (Ar), 127.7(Ar), 127.59 (Ar), 127.56 (Ar), 127.54 (Ar), 127.5 (Ar), 127.4 (Ar),117.2 (CH2), 102.7 (C1), 98.3 (C6), 84.6 (C3), 81.9 (C2),78.1 (C4), 76.0 (C5), 75.6 (CH2), 74.6 (CH2),74.4 (CH2), 70.1 (CH2), 68.8 (CH2), 68.4 (CH2). HRMS (ESI) m/z: [M + Na]+ calcd for C44H46O7Na 709.3136; found 709.3137.Prepared from compound 3 or 4, 1 equiv) was coevaporated withtoluene and dried overnight under high vacuum. The residue was dissolvedin anhydrous toluene to give a 0.2 M solution and cooled down to −78°C. Thiophenol (1.5 equiv) was added at the same temperaturebefore. BF3·OEt2 (1.5 equiv) was addedin a dropwise manner. After complete addition, the reaction mixturewas stirred for 10 min at the same temperature and then graduallybrought to −20 °C over a period of 3 h. The reaction mixturewas stirred at −20 °C for 0.5 h and then quenched withsaturated aqueous NaHCO3 (20 mL) and extracted with EtOAc(3 × 20 mL). The combined organic layers were collected, driedover anhydrous sodium sulfate, and concentrated under reduced pressureto afford a crude residue, which was purified by silica gel chromatographyusing 5–10% ethyl acetate and hexane as eluents to afford thecorresponding monothioacetals.Dibenzylacetalfollowing general procedure GP2, eluting from silicagel with 5% ethyl acetate in hexanes. White amorphous solid  [α]D23 = −32.1 . 1H NMR  δ 7.57–7.48 , 7.39–7.19 , 6.02 , 5.37 ), 5.21 ), 5.14, 5.00 , 4.95 , 4.88 , 4.71 , 4.68 , 4.49 , 4.40 , 4.32 , 4.26 , 4.18 , 4.06 , 3.73 ,3.41 , 3.19 . 13C {1H} NMR  δ 139.1 (C), 139.0 (C), 138.7 (C), 137.2 (C),134.7 (CHo-SPh), 134.5 (=CH), 131.8(C), 128.9 (Ar), 128.5 (Ar), 128.4 (Ar), 128.28 (Ar), 128.26 (Ar),128.19 (Ar), 128.18 (Ar), 128.08 (Ar), 128.01 (Ar), 127.6 (Ar), 127.54(Ar), 127.50 (Ar), 127.4 (Ar), 116.8 (=CH2), 102.8 (C1),87.0 (C6), 82.3 (C3), 80.0 (C2), 75.0 (CH2), 74.8 (CH2), 74.5 (C4), 73.4 (CH2), 73.0 (C5), 70.3 (CH2), 70.2 (OCH2). HRMS (ESI) m/z: [M + Na]+ calcd for C43H44O6SNa 711.2750; found 711.2734.Prepared from compound 3 following general procedure GP2, eluting from silicagel with 5% ethyl acetate in hexanes. White amorphous solid . [α]D23 = +22.8  1H NMR  δ 7.47–7.41 ,7.37–7.31 , 7.32–7.21 , 7.15–6.97, 6.96–6.85 ,5.87–5.70 , 5.37–5.27 ,5.19 ,4.98–4.89 , 4.77 , 4.73 , 4.65 , 4.54 , 4.43 , 4.38 , 4.36–4.30, 4.17 , 4.07 , 3.95, 3.41 , 3.27 . 13C{1H} NMR  δ 139.5 (C), 139.3 (C), 139.0 (C), 138.0(C), 134.6 (=CH), 133.5 (Ar), 132.7 (Ar), 128.9 (Ar), 128.4 (Ar),128.29 (Ar), 128.2 (Ar), 128.1 (Ar), 128.0 (Ar), 127.97 (Ar), 127.91(Ar), 127.86 (Ar), 127.78 (Ar), 127.73 (Ar), 127.68 (Ar), 127.6 (Ar),127.5 (Ar), 127.4 (Ar), 127.2 (Ar), 116.3 (=CH2), 103.3(C1), 87.2 (C6), 82.2 (C3), 79.7 (C2), 76.1 (C4), 76.0 (C5), 75.3(CH2), 74.7 (CH2), 73.3 (CH2), 70.7(CH2), 69.7 (OCH2). HRMS (ESI) m/z: [M + Na]+ calcd for C43H44O6SNa 711.2750; found 711.2750.Prepared from compound 4 following general procedure GP2, eluting from silicagel with 5% ethyl acetate. Colorless syrup . [α]D20 = +6.3 . 1H NMR  δ 7.48–7.44, 7.37–7.27 ,7.26–7.20 , 7.09–6.84 , 5.81, 5.59, 5.27 , 5.03–4.98 , 4.93 , 4.83, 4.72 , 4.66 , 4.53 , 4.47 , 4.42 , 4.36, 4.04 , 3.94 ,3.75 , 3.62 . 13C{1H} NMR  δ139.17 (C), 139.10 (C), 139.0 (C), 137.6 (C), 136.9 (C), 134.3 (=CH),131.3 (SPh-CHo), 129.0 (Ar), 128.26 (Ar), 128.21 (Ar), 128.18 (Ar),128.0 (Ar), 127.9 (Ar), 127.7 (Ar), 127.5 (Ar), 127.4 (Ar), 127.3(Ar), 126.7 (Ar), 116.8 (=CH2), 103.3 (C1), 88.4 (C6),84.8 (C3), 82.4 (C2), 79.6 (C5), 78.5 (C4), 75.3 (CH2),74.6 (CH2), 74.5 (CH2), 69.9 (CH2), 69.8 (-OCH2-). HRMS (ESI) m/z: [M + Na]+ calcd for C43H44O6NaS 711.2751; found 711.2741.Prepared from compound 4 following general procedure GP2, eluting from silicagel with 5% ethyl acetate in hexanes. White amorphous solid . [α]D20 = +6.6 . 1H NMR  δ 7.43–7.37 ,7.31–7.28 , 7.26–7.21 , 7.18–7.12, 7.12–6.91 , 6.90–6.81 , 5.74 , 5.62 , 5.17 , 5.00–4.93, 4.89 , 4.79 , 4.71–4.68 ,4.64 , 4.45 , 4.38 , 4.25 , 4.01 , 3.94–3.85 , 3.67, 3.62 . 13C{1H} NMR  δ 139.09 (C), 139.03 (C), 138.7 (C), 138.0 (C),136.5 (Ci-SPh), 134.3 (=CH-), 131.4 (CoH-SPh), 128.9 (Ar-H), 128.3 (Ar), 128.24(Ar), 128.21 (Ar), 128.0 (Ar), 127.8 (Ar), 127.6 (Ar), 127.5 (Ar),127.45 (Ar), 127.42(Ar), 127.37 (Ar), 127.31 (Ar), 126.6 (Ar), 116.6(=CH2), 102.8 (C1), 88.9 (C6), 84.6 (C3), 82.2 (C2), 79.0(C4), 78.7 (C5), 75.1 (CH2), 74.6 (CH2), 74.5(CH2), 69.8 (CH2), 69.5 (CH2). HRMS (ESI) m/z: [M + Na]+ calcd for C43H44O6NaS 711.2751;found 711.2741.Prepared from compound 2(PPh3)3 (0.1 equiv)in ethyl alcohol(0.1 M) was added DBU (1.0 equiv). The resulting mixture was heatedto reflux for 5 h before the solvent was evaporated under reducedpressure to afford a crude residue that was taken up in CH2Cl2 (50 mL) and washed with water (50 mL). The organiclayer was separated, and the aqueous layer was extracted with CH2Cl2 (2 × 50 mL). The organic layers were combined,dried over Na2SO4 addition, filtered, and concentratedunder reduced pressure to give a crude mixture, which was purifiedwith silica gel column chromatography using 10% ethyl acetate in hexaneas an eluent to afford a colorless syrup.To a stirred solution of allyl glycoside (1.0equiv) and RuCl4  and stirred until completion (monitoredby TLC) at room temperature. The reaction was quenched with saturatedaqueous Na2S2O3, diluted with ethylacetate, washed with brine, and dried over Na2SO4. The organic layer was filtered and concentrated under reduced pressureto afford a crude residue that was purified by silica gel column chromatographyusing 15–25% ethyl acetate in hexane as an eluent to afforda colorless syrup as a mixture of anomers.A stirred 0.1 M solutionof the compound prepared in the previous step in 1,4-dioxane/water(4:1) was treated with NMO (3.0 equiv) and OsOGP3 from 5a  as a mixture of 2.4:1, ax:eq anomers after eluting from silica gel with 15–25%gradientof ethyl acetate in hexanes .Prepared using general protocol 1H NMR  δ 7.67–7.58 , 7.40–7.26, 5.39 , 5.19–5.12, 5.09–4.99 , 4.93 , 4.85–4.76 , 4.75–4.68 , 4.56 , 4.48–4.40, 4.32–4.26 , 4.17 , 4.10–4.05 , 3.91 , 3.88 ,3.74 , 3.45 , 3.20 , 2.77. 13C{1H} NMR  δ 139.10, 138.9, 138.8, 138.7, 138.5,138.4, 137.3, 137.1, 135.4, 135.0, 131.9, 131.2, 129.0, 128.8, 128.68,128.65, 128.5, 128.48, 128.47, 128.40, 128.38, 128.37, 128.35, 128.30,128.16, 128.13, 128.10, 127.88, 127.8, 127.68, 127.66, 127.64, 127.61,127.57, 127.55, 127.53, 127.51, 127.49, 97.9, 92.0, 87.1, 86.8, 82.6,81.0, 79.2, 76.6, 75.4, 75.2, 74.8, 74.7, 74.3, 73.8, 73.5, 73.0,70.4, 70.3, 70.1. HRMS (ESI) m/z: [M + Na]+ calcd for C40H40O6SNa 671.2438; found 671.2427.Mixture of anomers:colorless syrup. GP3 from 5b  as a mixture of 2.4:1, ax:eq anomers after eluting from silica gel with 15–25%gradientof ethyl acetate in hexanes .Prepared using general protocol 1H NMR  δ 7.73–6.65 , 5.33 , 5.18–5.09 , 4.99 , 4.96–4.91 , 4.87 , 4.85–4.59 , 4.48–4.42, 4.35 , 4.21 , 4.14–4.03 , 3.88 , 3.80 , 3.65 , 3.38 , 3.33 . 13C NMR  δ 138.99,138.91, 138.90, 138.8, 138.6, 138.5, 137.2, 137.1, 133.6, 133.0, 132.7,131.8, 129.17, 129.16, 128.58, 128.57, 128.56, 128.50, 128.49, 128.47,128.46, 128.38, 128.33, 128.31, 128.2, 128.1, 128.04, 128.00, 127.9,127.88, 127.86, 127.81, 127.79, 127.75, 127.74, 127.6, 127.53, 127.51,127.0, 98.0, 91.9, 86.7, 86.1, 82.2, 80.42, 79.2, 76.3, 76.1, 75.6,75.0, 74.97, 74.95, 74.8, 73.35, 73.33, 73.2, 71.5, 70.4, 69.9. HRMS (ESI) m/z: [M + Na]+ calcd for C40H40O6SNa 671.2438;found 671.2430.Mixture of anomers:colorless syrup. GP3 from 5c  as a mixture of 1.5:1, ax:eq anomers after eluting from silica gel with 15–25%gradientof ethyl acetate in hexanes .Prepared using general protocol 1H NMR  δ 7.55–7.49 , 7.39–7.33, 7.32–7.08 , 7.06–7.01 , 5.35–5.31, 5.30 ,5.00–4.87 , 4.86–4.67 , 4.54 , 4.43 , 4.39 , 4.21 , 4.17 , 4.13 ,4.02 , 3.83 , 3.75 , 3.70 , 3.66, 3.61 , 3.45 , 3.37 , 3.12 . 13C{1H} NMR  δ 138.6 (C), 138.5 (C), 138.4 (C), 138.3 (C), 138.0 (C),137.0 (C), 136.9 (C), 136.1 (C), 135.7 (C), 132.5 (Ar), 132.4 (Ar),129.22 (Ar), 129.18 (Ar), 128.7 (Ar), 128.65 (Ar), 128.61 (Ar), 128.55(Ar), 128.50 (Ar), 128.46 (Ar), 128.4 (Ar), 128.3 (Ar), 128.2 (Ar),128.1 (Ar), 128.0 (Ar), 127.8 (Ar), 127.68 (Ar), 127.6 (Ar), 127.5(Ar), 98.1 (C1 β anomer), 91.5 (C1 α anomer), 88.2 (CH),87.2 (CH), 84.7 (CH), 83.2 (CH), 81.9 (CH), 80.0 (CH), 79.5 (CH),78.4 (CH), 78.2 (CH), 76.0 (CH2), 75.8 (CH2),75.3 (CH2), 74.8 (CH2), 74.7 (CH2), 73.4 (CH), 70.6 (CH2), 70.2 (CH2). HRMS (ESI) m/z: [M + Na]+ calcd for C40H40O6NaS 671.2438;found 671.2432.Mixture of anomers:colorless syrup. GP3 from 5d  as a mixture of 9.8:1, ax:eq anomers after eluting from silica gel with 15–25%gradientof ethyl acetate in hexanes .Prepared using general protocol 1H NMR  δ 7.41–7.13 , 7.01–6.98, 5.38 , 5.24, 4.97 , 4.86 , 4.83–4.74 ,4.71 , 4.63 , 4.43–4.36 , 4.03 ,3.79 , 3.60 , 2.91 . 13C{1H} NMR  δ 138.6 (C),138.2 (C), 137.9 (C), 137.5 (C), 135.6 (C), 131.9 (Ar), 129.1 (Ar),128.6 (Ar), 128.5 (Ar), 128.4 (Ar), 128.4 (Ar), 128.11 (Ar), 128.10(Ar), 128.08 (Ar), 128.0 (Ar), 127.8 (Ar), 127.6 (Ar), 127.5 (Ar),127.2 (Ar), 91.3 (C1), 89.0 (C6), 81.6 (C4), 80.2 (C2), 79.1 (C3),75.7 (CH2), 74.9 (CH2), 74.1 (CH2), 73.3 (C5), 70.0 (CH2). HRMS (ESI) m/z: [M + Na]+ calcd for C40H40O6NaS 671.2438 found 671.2424.Mixture of anomers:colorless syrup. 2Cl2 (0.2 M) and cooled down to 0 °C beforetrichloroacetonitrile (5.0 equiv) and then DBU (1 drop) were added,and the reaction mixture was stirred for 0.5 h at the same temperature.After completion, the reaction mixture was diluted with dichloromethaneand washed with water. The aqueous layer was extracted with CH2Cl2 (2 × 20 mL). The organic layers combinedwere dried over Na2SO4 and concentrated underreduced pressure. The crude product was passed through a silica gelcolumn, eluting with 10–20% ethyl acetate in hexane to givethe trichloroacetimidate, which was used in the next step withoutfurther purification.The hemiacetal (1.0 equiv) was dissolved in CHGP4 from hemiacetal 6a  eluting with 10–20% ethyl acetate in hexane togive the trichloroacetimidate. Colorless syrup. HRMS (ESI) m/z: [M + Na]+ calcdfor C42H40O6NCl3NaS 814.1534;found 814.1517.Prepared  by following thegeneral protocol GP4 from hemiacetal 6b  eluting with 10–20% ethyl acetate in hexane togive the trichloroacetimidate. Colorless syrup. HRMS (ESI) m/z: [M + Na]+ calcdfor C42H40O6NCl3NaS 814.1534;found 814.1506.Prepared  by following thegeneral protocol GP4 from hemiacetal 6c  eluting with 10–20% ethyl acetate in hexane togive the trichloroacetimidate. Colorless syrup. HRMS (ESI) m/z: [M + Na]+ calcdfor C42H40O6NCl3NaS 814.1534;found 814.1529.Prepared  by following thegeneral protocol GP4 from hemiacetal 6d  eluting with 10–20% ethyl acetate in hexane togive the trichloroacetimidate. Colorless syrup. HRMS (ESI) m/z: [M + Na]+ calcdfor C42H40O6NCl3NaS 814.1534;found 814.1511.Prepared  by following thegeneral protocol 2Cl2 (0.15 M) and stirred for 1 h with activated4 Å AWMS (2 g/mmol of the donor) at room temperature under argonbefore cooling to −78 °C. After 0.5 h of continuous stirring,the reaction mixture was treated with TMSOTf (0.2 equiv) and stirredfor 4–5 h at −78 °C before it was quenched withtriethylamine (0.2 mL). The reaction mixture was diluted with dichloromethane(10 mL), filtered through a pad of celite, and washed with saturatedaqueous NaHCO3. The organic layer was separated, driedover Na2SO4, filtered, and concentrated underreduced pressure. Purification by flash column chromatography on silicagel (hexane/ethyl acetate) afforded the corresponding α/β-glycopyranosides.The anomeric ratio of the products was determined by integration ofthe anomeric signals in the1H NMR spectra of the crudeproduct mixtures unless otherwise stated.A mixture of the donor (1.0 equiv) and acceptor(1.2 equiv)was coevaporated with toluene twice, then taken up in anhydrous CH8β was obtained  as a single isomer from the reaction of donor 7a  and acceptor by following the general procedure GP5 for glycosylation(eluting with 10% ethyl acetate in hexanes).Compound D20 = −14.0 . 1H NMR  δ 7.50 , 7.39–7.21, 7.21–7.14 , 5.12 , 5.03–4.94 , 4.91 , 4.80 , 4.77–4.70 , 4.70–4.66 , 4.63 , 4.54 , 4.40 , 4.34 , 4.25 ,4.18 , 4.05–3.99, 3.97–3.91 , 3.87 , 3.73 , 3.58–3.50 , 3.38 , 3.32 , 3.13 . 13C{1H} NMR  δ 139.04 (C), 139.02 (C), 138.8(C), 138.7 (C), 138.5 (C), 138.4 (C), 138.3 (C), 137.1 (C), 134.8(Ar), 132.5 (Ar), 131.9 (Ar), 128.8 (Ar), 128.6 (Ar), 128.5 (Ar),128.45 (Ar), 128.43 (Ar), 128.40 (Ar), 128.3 (Ar), 128.28 (Ar), 128.24(Ar), 128.13 (Ar), 128.11 (Ar), 128.08 (Ar), 128.04 (Ar), 128.00 (Ar),127.9 (Ar), 127.65 (Ar), 127.60 (Ar), 127.58 (Ar), 127.53 (Ar), 127.51(Ar), 127.46 (Ar), 127.40 (Ar), 104.3 (C1′), 98.1 (C1), 87.0(C6′), 82.7 (CH), 82.2 (CH), 80.0 (CH), 79.4 (CH), 78.4 (CH),75.8 (CH2), 75.3 (CH2), 75.1 (CH2), 74.7 (CH2), 74.3 (CH), 73.5 (CH), 73.4 (CH2), 73.0 (CH2), 70.3 (CH2), 70.0 (CH), 68.9(C6), 55.3 (OCH3). HRMS (ESI) m/z: [M + Na]+ calcd for C68H70O11SNa 1117.4531; found 1117.4495.Colorless syrup.[α]9β was obtained as a single isomer from the reaction of donor 7a  and acceptor by following the general procedure GP5 for glycosylation(eluting with 10% ethyl acetate in hexanes).Compound D21 = −45.9 . 1H NMR  δ 7.56–7.51 , 7.42–7.36 , 7.35–7.16 , 5.57 , 5.12 , 5.02–4.97 ,4.94 , 4.74–4.66, 4.65–4.57 , 4.40 , 4.31, 4.29–4.21 , 4.19 , 4.11 ,4.01 , 3.79 , 3.73 , 3.35 , 3.08 ,1.50–1.46 , 1.34 , 1.30 ; 13C{1H} NMR  δ 139.06 (C), 139.0 (C), 138.7 (C), 137.1 (C), 135.2(Ar), 131.4 (Ar), 128.8 (Ar), 128.66 (Ar), 128.60 (Ar), 128.4 (Ar),128.3 (Ar), 128.26 (Ar), 128.20 (Ar), 128.1 (Ar), 128.0 (Ar), 127.8(Ar), 127.46 (Ar), 127.40 (Ar), 127.38 (Ar), 109.4 (CMe2), 108.7 (CMe2), 104.7 (C1′), 96.5 (C1), 86.7 (C6′),82.3 (C3′), 79.1 (C2′), 74.71 (CH2), 74.68(CH2), 74.4 (C4′), 73.2 (C5), 71.6 (CH), 70.9 (CH),70.6 (CH), 70.2 (C6), 70.1 (CH2), 67.8 (CH), 26.16 (CH3), 26.12 (CH3), 25.2 (CH3), 24.6 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C52H58O11SNa 913.3592; found 913.3571.Colorless syrup.[α]10β was obtained  as a single isomer from the reaction of donor 7a  and acceptor by following the general procedure GP5 for glycosylation(eluting with 10% ethyl acetate in hexanes).Compound D21 = −6.0 . 1H NMR  δ 7.55–7.48 , 7.35–7.17 , 5.08–5.02 , 4.98 , 4.94 , 4.74–4.69 , 4.66 , 4.56 , 4.44 , 4.19 , 4.07 , 3.79 , 3.47–3.31 , 2.13 , 1.95–1.88 , 1.88–1.79, 1.67–1.55 . 13C{1H} NMR  δ 139.1 (C), 138.9 (C), 138.7 (C), 137.1(C), 134.0 (Ar), 132.7 (Ar), 128.7 (Ar), 128.5 (Ar), 128.35 (Ar),128.30 (Ar), 128.27 (Ar), 128.21 (Ar), 128.1 (Ar), 127.9 (Ar), 127.6(Ar), 127.5 (Ar), 127.48 (Ar), 127.45 (Ar), 127.29 (Ar), 127.23 (Ar),96.67 (C1), 87.87 (C6), 82.93 (C3), 79.63 (C2), 75.27 (CH2), 75.23 (CH2), 74.73 (C4), 74.69 (CH2), 74.09(C5), 73.20 (CH2), 70.37 (CH2), 42.79 (Ada),42.36 (Ada), 36.43 (Ada), 30.82 (Ada), 30.74 (Ada). HRMS (ESI) m/z: [M + Na]+ calcd for C50H54O6SNa 805.3533;found 805.3537.Colorless syrup.[α]11α and 11β were obtained as a mixture of anomers  from the reaction of donor 7a  and acceptor  by following thegeneral procedure GP5 for glycosylation (eluting with10% ethyl acetate in hexanes). Repeated column chromatography gavepure samples of α- and β-isomers for full characterization.Compounds D20 = +12.5 . 1H NMR  δ7.57–7.52, 7.99–7.06 , 5.86 , 5.25 ,5.03 , 4.99 , 4.94 , 4.83–4.74 , 4.73–4.66, 4.50–4.42 , 4.38 , 4.24 , 4.20–4.14 , 4.13–4.05 , 4.02–3.94 , 3.89–3.83 , 1.47 , 1.40 , 1.24 , 1.22 . 13C{1H} NMR  δ 139.0(C), 138.7 (C), 138.6 (C), 137.0 (C), 135.1 (C), 132.5 (Ar), 129.1(Ar), 128.5 (Ar), 128.4 (Ar), 128.38 (Ar), 128.28 (Ar), 128.22 (Ar),128.1 (Ar), 127.6 (Ar), 127.59 (Ar), 127.57 (Ar), 127.46 (Ar), 127.4(Ar), 127.3 (Ar), 111.8 (CMe2), 108.9(CMe2),105.3 (C1), 99.3 (C1′), 90.1 (C6′), 83.3 (C2), 82.1(C4), 81.3 (CH), 78.7 (CH), 76.5 (CH), 75.6 (CH), 74.7 (CH2), 73.5 (CH2), 73.14 (CH2), 73.1 (CH), 72.6(CH), 70.7 (CH2), 66.9 (CH2), 26.94 (CH3), 26.9 (CH3), 26.4 (CH3), 25.4 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C52H58O11SNa 913.3592; found 913.3566.Colorlesssyrup. [α]D21 = −22.9. 1H NMR  δ 7.54–7.49, 7.35–7.21 , 5.79 , 5.12 , 5.01 ,4.96 , 4.73, 4.67–4.62, 4.51–4.45 , 4.42–4.38 , 4.25 , 4.19 , 4.11–4.05 , 4.03 , 3.69, 3.37 , 3.11 , 1.51 , 1.45, 1.37 , 1.25 . 13C{1H} NMR  δ138.9 (C), 138.39 (C), 138.3 (C), 137.0 (C), 134.5 (Ar), 132.1 (Ar),129.0 (Ar), 128.6 (Ar), 128.51 (Ar), 128.50 (Ar), 128.48 (Ar), 128.45(Ar), 128.3 (Ar), 128.18 (Ar), 128.17 (Ar), 127.84 (Ar), 127.8 (Ar),127.7 (Ar), 127.5 (Ar), 127.3 (Ar), 111.8 (CMe2), 108.3(CMe2), 105.2 (C1), 101.4 (C1′), 86.9 (C6′), 82.6(C3′), 82.3 (CH), 80.4 (CH), 80.2 (CH), 79.4 (C2′),75.4 (CH2), 74.7 (CH2), 74.3 (C4′), 74.0(CH), 73.9 (C5′), 73.0 (CH2), 70.4 (CH2), 65.4 (CH2), 26.9 (CH3), 26.6 (CH3), 26.3 (CH3), 25.5 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C52H58O11SNa 913.3592;found 913.3564.Colorless syrup. [α]12β was obtained  as a single isomer from the reaction of donor 7a  and acceptor by following the general procedure GP5 for glycosylation(eluting with 10% ethyl acetate in hexanes).Compound D21 = −26.6 . 1H NMR  δ 7.56–7.49 , 7.39 , 7.34–7.17 ,5.09–5.03 , 4.99 , 4.92 , 4.86 , 4.78 , 4.74–4.69 , 4.66 , 4.42 , 4.26, 4.20 , 4.11 , 4.04 ,3.77 , 3.67, 3.49 ,3.39 , 3.38–3.31 , 1.54 , 1.40 , 1.34 . 13C{1H} NMR  δ 139.2 (C), 138.9 (C), 138.7 (C), 137.1 (C), 133.6(Ar), 133.5 (Ar), 133.2 (Ar), 128.9 (Ar), 128.5 (Ar), 128.3 (Ar),128.29 (Ar), 128.25 (Ar), 128.0 (Ar), 127.6 (Ar), 127.57 (Ar), 127.53(Ar), 127.52 (Ar), 127.47 (Ar), 127.42 (Ar), 127.37 (Ar), 127.34 (Ar),109.3 (CMe2), 102.9 (C1′), 98.2 (C1), 88.0 (C6′), 82.6(C3′), 82.4 (CH), 80.2 (CH), 79.8 (CH), 78.4 (CH), 75.9 (CH),75.2 (CH2), 74.8 (C4′), 74.6 (CH2), 74.0(CH), 73.2 (CH2), 70.3 (CH2), 64.5 (CH2), 54.9 (OCH3), 28.2 (CH3), 26.2 (CH3), 18.1 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C50H56O10SNa 871.3486; found 871.3470.Colorless syrup.[α]13α and 13β were obtained as a mixture of anomers  from the reaction of donor 7a  and acceptor  by following thegeneral procedure GP5 for glycosylation . Repeated column chromatographygave pure samples of α- and β-isomers for full characterization.Compounds D21 = +18.0 . 1H NMR  δ 7.59–7.53, 7.35–7.15 , 5.68 , 5.07–4.99 , 4.96–4.89 , 4.82 , 4.67 , 4.65–4.61 , 4.61–4.55 , 4.55–4.49, 4.45 , 4.37 , 4.17 , 4.07 , 4.04–3.94, 3.90–3.81 , 3.76–3.68, 3.66 , 3.55 , 3.38 . 13C{1H} NMR  δ 139.2 (C), 139.0 (C), 138.8 (C), 138.4 (C), 138.2 (C),138.2 (C), 137.4 (C), 135.6 (C), 132.7 (Ar), 129.0 (Ar), 128.5 (Ar),128.4 (Ar), 128.37 (Ar), 128.35 (Ar), 128.32 (Ar), 128.31 (Ar), 128.29(Ar), 128.27 (Ar), 127.99 (Ar), 127.96 (Ar), 127.94 (Ar), 127.7 (Ar),127.59 (Ar), 127.53 (Ar), 127.4 (Ar), 127.3 (Ar), 127.2 (Ar), 127.1(Ar), 127.0 (Ar), 97.8 (C1), 97.1 (C1′), 90.4 (C6′),82.0 (CH), 79.9 (CH), 79.1 (CH), 76.0 (CH), 75.7 (C4′), 74.9(CH2), 74.3 (CH2), 73.9 (CH), 73.5 (CH2), 73.4 (CH2), 73.1 (CH2), 72.6 (CH), 70.6(CH2), 70.1 (C6), 69.6 (CH), 55.2 (OCH3). HRMS (ESI) m/z: [M + Na]+ calcd for C68H70O11SNa 1117.4531;found 1117.4503.Colorless syrup. [α]D21 = −22.0. 1H NMR  δ 7.59–7.55, 7.47–7.41 , 7.34–7.16 , 7.16–7.06 , 7.05–6.98 , 5.17 ,5.03 , 4.99, 4.84 , 4.80 , 4.79–4.70 , 4.69–4.64 , 4.57 , 4.38 , 4.33 , 4.21–4.14, 4.07 , 4.04 ,3.87 , 3.80–3.72 , 3.61 , 3.49 , 3.44 , 3.37 ,3.26–3.14 . 13C{1H} NMR  δ 139.8 (C), 139.3 (C), 139.0(C), 138.7 (C), 138.6 (C), 138.0 (C), 137.3 (C), 134.5 (Ar), 133.6(Ar), 128.78 (Ar), 128.7 (Ar), 128.6 (Ar), 128.48, (Ar) 128.41 (Ar),128.38 (Ar), 128.30 (Ar), 128.27 (Ar), 128.24 (Ar), 128.2 (Ar), 127.97(Ar), 127.94 (Ar), 127.93 (Ar), 127.74 (Ar), 127.7 (Ar), 127.6 (Ar),127.5 (Ar), 127.4 (Ar), 127.34 (Ar), 127.30 (Ar), 127.2 (Ar), 127.0(Ar), 102.3 (C1′), 98.8 (C1), 88.8 (C6′), 82.6 (C3′),80.6 (CH), 80.2 (CH), 78.5 (CH), 75.9 (CH), 75.7 (CH2),75.2 (CH2), 74.7 (CH2), 74.5 (C4′), 74.4(C5′), 73.8 (CH2), 73.5 (CH2), 72.8 (CH2), 70.26 (CH2), 70.2 (CH), 67.9 (CH2), 55.4 (OCH3). HRMS (ESI) m/z: [M + Na]+ calcd for C68H70O11SNa 1117.4531; found 1117.4529.Colorless syrup.[α]14α and 14β were obtained as a mixture of anomers  from the reaction of donor 7b  and acceptor  by followingthe general procedure GP5 for glycosylation (elutingwith 10% ethyl acetate in hexanes). Repeated column chromatographygave pure samples of the α- and β-isomers for full characterization.Compounds D20 = +23.2 . 1H NMR  δ 7.42–7.12, 5.14 , 5.05 , 4.95 , 4.85 , 4.82–4.67 , 4.64–4.54 , 4.49 , 4.45 , 4.36 , 4.07 , 3.94 , 3.91 , 3.84–3.81, 3.71 , 3.50 , 3.38 , 3.23 . 13C{1H} NMR  δ 138.9 (C), 138.8 (C), 138.4(C), 138.2 (C), 137.9 (C), 132.9 (Ar), 128.9 (Ar), 128.4 (Ar), 128.36(Ar), 128.34 (Ar), 128.3 (Ar), 128.19 (Ar), 128.13 (Ar), 128.0 (Ar),127.97 (Ar), 127.94 (Ar), 127.9 (Ar), 127.8 (Ar), 127.6 (Ar), 127.58(Ar), 127.53 (Ar), 127.50 (Ar), 127.37 (Ar), 127.34 (Ar), 97.7 (C1′),97.6 (C1), 87.5 (C6′), 82.0 (CH), 80.1 (CH), 78.6 (CH), 78.1(CH), 77.2 (CH), 76.36 (CH), 76.34 (CH), 75.7, 75.0, 74.9, 73.4 (CH2), 73.0 (CH2), 72.5 (CH2), 71.8 (CH2), 70.4 (CH2), 70.1 (CH2), 66.0 (CH2), 54.9 (OCH3). HRMS (ESI) m/z: [M + Na]+ calcd for C68H70O11SNa 1117.4531; found 1117.4532.Colorless syrup. [α]D21 = +27.4 . 1H NMR  δ 7.39–7.15, 7.11–7.06 , 5.14–5.06 , 4.96–4.89 , 4.84 , 4.80–4.69 , 4.68–4.55, 4.45 , 4.29 ,4.21–4.12 , 3.94 , 3.86 , 3.77 , 3.61 , 3.54–3.44, 3.35 , 3.33–3.27 . 13C{1H} NMR  δ 139.0 (C), 138.9(C), 138.8 (C), 138.52 (C), 138.5 (C), 138.3 (C), 137.5 (C), 133.7(Ar), 131.9 (Ar), 129.0 (Ar), 128.53 (Ar), 128.50 (Ar), 128.41 (Ar),128.39 (Ar), 128.34 (Ar), 128.31 (Ar), 128.23 (Ar), 128.19 (Ar), 128.03(Ar), 128.01 (Ar), 127.99 (Ar), 127.97 (Ar), 127.96 (Ar), 127.8 (Ar),127.78 (Ar), 127.74 (Ar), 127.71 (Ar), 127.57 (Ar), 127.52 (Ar), 127.44(Ar), 127.41 (Ar), 104.2 (C1′), 98.1 (C1), 86.6 (C6′),82.5 (C3′), 82.1 (-CH), 79.9 (CH), 79.2 (C2′), 77.9(CH), 75.9 (C5′), 75.7 (CH2), 75.2 (C4′),75.1 (CH2), 74.9 (CH2), 74.8 (-CH2), 73.4 (CH2), 73.3 (CH2), 71.0 (CH2), 69.9 (C5), 68.5 (C6), 55.2 (OCH3). HRMS (ESI) m/z: [M + Na]+ calcd for C68H70O11SNa 1117.4531;found 1117.4541.Colorless syrup. [α]15α and 15β were obtained as a mixture of anomers  from the reaction of donor 7b  and acceptor  by following thegeneral procedure GP5 for glycosylation . The α-isomer was not obtainedpure and was characterized in the mixture of anomers by the followingdiagnostic signals.Compounds 1H NMR δ 7.42–7.39 ,7.38–7.31, 7.30–7.19 , 5.48 , 5.15–5.10 , 5.07 , 4.88–4.82 , 4.78–4.67 , 4.63–4.57 , 4.51, 4.39–4.35, 4.27 ,4.14 , 4.07 , 3.99 , 3.94–3.90 , 3.85 , 3.77 , 3.67 , 1.51 , 1.37 , 1.31 , 1.25 .Repeated column chromatography gave a pure sample of the β-isomerfor full characterization.D21 = +23.5 . 1H NMR  δ 7.48–7.43, 7.41–7.18 , 5.56 , 5.15–5.10 ,5.05 , 4.91, 4.86 , 4.75 , 4.65 , 4.51 , 4.34 , 4.32 ,4.29 , 4.18 , 4.14 , 4.09 , 3.86 ,3.73 , 3.40–3.34, 1.50 , 1.41, 1.32 , 1.29 . 13C{1H} NMR  δ139.1 (C), 138.9 (C), 138.8 (C), 137.6 (C), 133.8 (Ar), 131.9 (Ar),129.1 (Ar), 128.7 (Ar), 128.49 (Ar), 128.46 (Ar), 128.3 (Ar), 128.23(Ar), 128.21 (Ar), 128.1 (Ar), 128.0 (Ar), 127.8 (Ar), 127.7 (Ar),127.48 (Ar), 127.44 (Ar), 109.3 (CMe2), 108.7 (CMe2), 104.9 (C1′), 96.5 (C1), 86.9 (C6′), 82.0(C3′), 78.9 (C2′), 75.8 (C5′), 75.3 (C4′),75.0 (CH2), 74.6 (CH2), 73.5 (CH2), 71.5 (CH2), 71.1 (CH2), 70.8 (CH2), 70.6 (C2), 69.9 (C6), 67.7 (C5), 26.2 (CH3), 26.1 (CH3), 25.2 (CH3), 24.5 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C52H58O11SNa 913.3592;found 913.3575.Colorless syrup. [α]16α and 16β were obtained as a mixture of anomers  from the reaction of donor 7b  and adamantanol  by followingthe general procedure GP5 for glycosylation (elutingwith 10% ethyl acetate in hexanes). Repeated column chromatographygave pure samples of α- and β-isomers for full characterization.Compounds D20 = −6.6. 1H NMR  δ 7.46–7.42, 7.41–7.38 , 7.38–7.33 , 7.32–7.19 , 5.33 , 5.13 , 5.06 , 4.95 , 4.86 , 4.76 , 4.72 , 4.68 , 4.61 , 4.48 , 4.42, 4.08–4.00 , 3.85 , 2.00, 1.81–1.74 , 1.66 , 1.59–1.52, 1.51–1.45 . 13C{1H} NMR  δ 139.18 (C), 139.14 (C), 138.9 (C),137.9 (C), 133.5 (Ar), 132.7 (Ar), 128.9 (Ar), 128.4 (Ar), 128.3 (Ar),128.23 (Ar), 128.18 (Ar), 128.07 (Ar), 128.04 (Ar), 127.7 (Ar), 127.6(Ar), 127.58 (Ar), 127.55 (Ar), 127.50 (Ar), 127.42 (Ar), 127.39 (Ar),90.6 (C1), 87.9 (C6), 79.7 (C3), 77.3 (-CH2), 76.4 (C2),76.3 (C4), 74.9 (CH2), 74.5 (CH2), 73.2 (CH2), 72.9 (CH2), 71.5 (CH2), 71.3 (C5),42.4 (Ada), 36.3 (Ada), 30.7 (Ada). HRMS (ESI) m/z: [M + Na]+ calcd for C50H54O6SNa 805.3533; found 805.3520.Colorless syrup. [α]D21 = +21.6 . 1H NMR  δ 7.40–7.18, 5.15–5.08 , 4.95, 4.87 , 4.78 , 4.74–4.68 , 4.60 , 4.56 , 4.51, 4.29 , 3.78 , 3.37 ,3.33 , 2.02 , 1.85 , 1.75 , 1.61–1.47 . 13C{1H} NMR  δ 139.04 (C), 139.0 (C), 138.8 (C), 137.6 (C), 133.5(Ar), 132.3 (Ar), 129.0 (Ar), 128.44 (Ar), 128.39 (Ar), 128.34 (Ar),128.30 (Ar), 128.27 (Ar), 128.1 (Ar), 128.0 (Ar), 127.8 (Ar), 127.7(Ar), 127.65 (Ar), 127.60 (Ar), 127.54 (Ar), 127.51 (Ar), 127.4 (Ar),96.7 (C1), 87.3 (C6), 82.8 (C3), 79.4 (CH2), 75.9 (C5),75.5 (C4), 75.16 (CH2), 75.1 (CH2), 74.9 (CH2), 73.5 (CH2), 71.5 (CH2), 42.8 (Ada),36.3 (Ada), 30.7 (Ada). HRMS (ESI) m/z: [M + Na]+ calcd for C50H54O6SNa 805.3533; found 805.3525.Colorless syrup. [α]17α and 17β were obtained as a mixture of anomers  from the reaction of donor 7b  and acceptor  by following thegeneral procedure GP5 for glycosylation (eluting with10% ethyl acetate in hexanes). Repeated column chromatography gavepure samples of the α- and β-isomers for full characterization.Compounds D23 = +36.3 . 1H NMR  δ 7.43–7.20, 5.20 , 5.13, 5.08 , 5.01 , 4.96 , 4.87 , 4.76–4.64 , 4.62 , 4.53 , 4.44 ,4.36 , 4.12 , 4.05–3.97 , 3.89 , 3.63, 3.57, 1.39 , 1.38 , 1.19 , 0.86 . 13C{1H} NMR  δ 138.85 (C), 138.79 (C), 138.75 (C), 137.3 (C), 134.5(C), 130.9 (Ar), 129.0 (Ar), 128.57 (Ar), 128.54 (Ar), 128.43 (Ar),128.40 (Ar), 128.3 (Ar), 128.1 (Ar), 128.0 (Ar), 127.80 (Ar), 127.78(Ar), 127.66 (Ar), 127.63 (Ar), 127.61 (Ar), 127.5 (Ar), 111.5 (CMe2),108.8 (CMe2), 105.0 (C1), 99.7 (C1′), 86.2 (C6′),82.8 (CH), 82.4 (CH), 80.9 (CH), 78.8 (CH), 76.6 (CH), 76.1 (CH),75.1 (CH2), 73.5 (CH2), 73.2 (CH2), 72.8 (CH), 71.9 (CH2), 71.3 (CH2), 66.5(C6), 26.9 (CH3), 26.8 (CH3), 25.8 (CH3), 25.3 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C52H58O11SNa 913.3592; found 913.3596.Colorless syrup. [α]D21 = +26.1 . 1H NMR  δ 7.38–7.23, 7.23–7.18 , 5.72 , 5.10 , 5.05 , 4.85, 4.79–4.72, 4.71–4.64 , 4.61 , 4.45–4.37, 4.33 , 4.31–4.25, 4.04–3.97 , 3.70 , 3.35 , 3.29 , 1.44 , 1.36 , 1.26 , 1.18 . 13C{1H} NMR  δ 138.9(C), 138.5 (C), 138.3 (C), 137.3 (C), 133.8 (Ar), 131.6 (Ar), 129.1(Ar), 128.5 (Ar), 128.4 (Ar), 128.3 (Ar), 128.27 (Ar), 128.1 (Ar),127.88 (Ar), 127.83 (Ar), 127.77 (Ar), 127.72 (Ar), 127.6 (Ar), 127.4(Ar), 111.7 (CMe2), 108.4 (CMe2), 105.2 (C1), 101.7 (C1′),86.2 (C6′), 82.8 (CH), 82.3 (C3′), 80.40 (CH), 80.3(CH), 79.3 (C2′), 76.3 (C5′), 75.2(CH2),75.1 (CH2), 74.8 (CH), 73.7 (CH), 73.3 (CH2),71.3 (CH2), 65.7 (C6), 26.8 (CH3), 26.6 (CH3), 26.2 (CH3), 25.4 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C52H58O11SNa 913.3592;found 913.3589.Colorless syrup. [α]18α and 18β were obtained as a mixture of anomers  from the reaction of donor 7b  and acceptor  by following thegeneral procedure GP5 for glycosylation (eluting with10% ethyl acetate in hexanes). Repeated column chromatography gavepure samples of α- and β-isomers for full characterization.Compounds D21 = +31.0 . 1H NMR  δ 7.48–7.44, 7.38–7.18 , 5.23 , 5.15 , 5.03 , 4.84 , 4.81–4.77 , 4.74 , 4.67 , 4.50 , 4.37 ,4.22–4.16 , 4.13 , 3.95 , 3.86 ,3.62 , 3.37 , 3.33 ,1.33 , 1.29 , 1.13 . 13C{1H} NMR  δ 139.1 (C), 138.8 (C), 138.7(C), 137.8 (C), 132.8 (Ar), 128.9 (Ar), 128.4 (Ar), 128.39 (Ar), 128.33(Ar), 128.09 (Ar), 128.07 (Ar), 128.01 (Ar), 127.7 (Ar), 127.6 (Ar),127.5 (Ar), 127.4 (Ar), 127.3 (Ar), 127.2 (Ar), 109.0 (CMe2), 98.3 (C1′), 98.1 (C1), 89.7 (C6′), 81.4 (C4), 79.3(C3′), 77.0 (C3), 76.5 (C2′), 75.7 (C2), 75.4 (C4′),74.6 (CH2), 73.8 (CH2), 72.9 (CH2), 71.5 (C5′), 71.2, 64.6 (C5), 54.8 (OCH3), 28.2(CH3), 26.3 (CH3), 17.9 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C50H56O10SNa 871.3486;found 871.3478.Colorless syrup. [α]D21 = +18.6 . 1H NMR  δ7.39–7.21, 7.20–7.15 , 5.13–5.08 , 4.93–4.83 , 4.80 , 4.75 , 4.73–4.67, 4.61 , 4.25 ,4.17 , 4.04 , 3.78–3.70 , 3.62, 3.42–3.36 ,3.31–3.26 , 1.33 , 1.29–1.22 . 13C{1H} NMR  δ 139.2 (C),138.8 (C), 137.4 (C), 133.6 (Ar), 132.1 (Ar), 128.9 (Ar), 128.5 (Ar),128.26 (Ar), 128.24 (Ar), 128.21 (Ar), 128.1 (Ar), 127.9 (Ar), 127.86(Ar), 127.80 (Ar), 127.73 (Ar), 127.69 (Ar), 127.46 (Ar), 127.41 (Ar),109.3 (CMe2), 102.2 (C1′), 98.2 (C1), 86.3 (C6′),82.5 (C3′), 79.7 (C4), 78.5 (C3), 78.1 (C2′), 76.0 (C2),75.8 (C5′), 75.6 (C4′), 75.0 (CH2), 74.9(CH2), 73.6 (CH2), 70.7 (CH2), 64.5(C5), 54.9 (OCH3), 27.7 (CH3), 26.4 (CH3), 18.0 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C50H56O10SNa 871.3486; found 871.3484.Colorless syrup.[α]19α and 19β were obtained as a mixture of anomers  from the reaction of donor 7b  and acceptor  by following thegeneral procedure GP5 for glycosylation (eluting with10% ethyl acetate in hexanes). Repeated chromatography gave a puresample of the α-isomer for full characterization.Compounds D21 = +35.8 . 1H NMR  δ 7.44–7.40 , 7.25 , 5.60 , 5.10–5.03 ,4.90–4.84 , 4.82 , 4.72 , 4.68–4.61 ,4.59–4.54 , 4.53–4.49 , 4.47 , 4.37–4.32 , 4.22 , 4.00 , 3.94 , 3.82 , 3.80–3.75 , 3.76–3.70 , 3.53 , 3.43, 3.37 , 3.32 . 13C{1H} NMR  δ 139.2 (C),138.8 (C), 138.7 (C), 138.6 (C), 138.4 (C), 138.2 (C), 137.8 (C),133.09 (Ar), 133.06 (C), 129.1 (Ar), 128.48 (Ar), 128.47 (Ar), 128.45(Ar), 128.33 (Ar), 128.29 (Ar), 128.25 (Ar), 128.23 (Ar), 127.96 (Ar),127.93 (Ar), 127.89 (Ar), 127.81 (Ar), 127.66 (Ar), 127.61 (Ar), 127.58(Ar), 127.54 (Ar), 127.50 (Ar), 127.4 (Ar), 127.1 (Ar), 127.0 (Ar),97.6 (C1′), 97.4 (C1), 88.3 (C6′), 81.9 (CH2), 80.0 (CH2), 79.2 (CH2), 76.2 (CH), 75.8(CH2), 75.0 (CH2), 74.6 (CH2), 74.5(CH2), 73.4 (CH2), 73.3 (CH2), 73.2(CH2), 73.1 (CH2), 72.9 (C5′), 71.4 (CH),70.2 (CH), 69.5 (C6), 55.0 (OCH3). HRMS (ESI) m/z: [M + Na]+ calcd for C68H70O11SNa 1117.4531; found 1117.4504.Colorless syrup. [α]:The β-isomer was not obtained pure and was characterizedin the mixture of anomers by the following diagnostic signals1H NMR  δ 4.94 ,4.56 , 3.25 ; 13C NMR  δ 139.4 (C), 139.1 (C), 139.0 (C), 138.69(C), 138.67 (C), 138.2 (C), 137.9 (C), 133.1 (Ar), 132.8 (Ar), 129.0(Ar), 128.97 (Ar), 128.91 (Ar), 128.49 (Ar), 128.48 (Ar), 128.45 (Ar),128.43 (Ar), 128.33 (Ar), 128.30 (Ar), 128.26 (Ar), 128.23 (Ar), 128.1(Ar), 128.09 (Ar), 128.06 (Ar), 127.97 (Ar), 127.94 (Ar), 127.91 (Ar),127.90 (Ar), 127.7 (Ar), 127.69 (Ar), 127.67 (Ar), 127.64 (Ar), 127.63(Ar), 127.59 (Ar), 127.56 (Ar), 127.55 (Ar), 127.51 (Ar), 127.4 (Ar),127.2 (Ar), 127.1 (Ar), 127.07 (Ar), 127.01 (Ar), 102.4 (C1′),98.7 (C1), 87.3 (C6′), 82.7 (CH), 80.2 (CH), 80.0 (CH), 79.1(CH), 76.5 (CH2), 75.9 (CH), 75.7 (CH), 75.6 (CH2), 75.2 (CH2), 75.1 (CH2), 73.9 (CH2), 73.3 (CH2), 73.1 (CH2), 72.3 (CH2), 70.2 (CH2), 67.9 (C6), 55.3 (OCH3).20α and 20β were obtained as a mixture of anomers  from the reaction of donor 7c  and acceptor  by following thegeneral procedure GP5 for glycosylation (eluting with10% ethyl acetate in hexanes). Repeated column chromatography gavea pure sample of β-isomer for full characterization.Compounds D20 = +7.1 . 1H NMR  δ 7.48–7.45, 7.34–7.14 , 7.06 , 5.39 , 4.97 , 4.89 , 4.83 , 4.78 , 4.76–4.69 , 4.64 , 4.60, 4.55–4.51 , 4.38–4.30 , 4.23 ,4.00 , 3.86 , 3.75 , 3.71–3.64 , 3.61 , 3.54–3.45 , 3.32 . 13C{1H} NMR  δ 139.0(C), 138.5 (C), 138.46 (C), 138.43 (C), 138.3 (C), 138.2 (C), 137.3(C), 136.3 (C), 131.6 (Ar), 129.2 (Ar), 128.57 (Ar), 128.51 (Ar),128.49 (Ar), 128.44 (Ar), 128.40 (Ar), 128.3 (Ar), 128.2 (Ar), 128.08(Ar), 128.05 (Ar), 128.02 (Ar), 127.9 (Ar), 127.8 (Ar), 127.79 (Ar),127.75 (Ar), 127.73 (Ar), 127.72 (Ar), 127.69 (Ar), 127.64 (Ar), 127.1(Ar), 104.6 (C1′), 98.1 (C1), 88.0 (C6′), 84.8 (CH),82.1 (CH), 82.0 (CH), 80.0 (CH), 79.4 (CH), 78.3 (CH), 78.2 (CH),77.4 (CH2), 77.1 (CH2), 76.9 (CH2), 75.9 (CH2), 75.7 (CH), 75.0 (CH2), 74.9(CH2), 74.9 (CH2), 73.4 (CH2), 70.0(CH), 69.8 (CH2), 68.9, 55.3 (CH3). HRMS(ESI) m/z: [M + Na]+ calcd for C68H70O11NaS 1117.45310;found 1117.4521.Colorless syrup. [α]1H NMR  δ 5.31 , 5.15 , 3.48–3.42 , 3.38 .The α-isomer was identified in the mixtureby the following diagnostic signals: 21α and 21β were obtained as a mixture of anomers  from the reactionof donor 7c  and acceptor  by following the general procedure GP5 for glycosylation (eluting with 10% ethyl acetate in hexanes). Repeatedcolumn chromatography gave a pure sample of β-isomer for fullcharacterization.Compounds 7c  and 0.416 g of 1,2:3,4-di-O-isopropylidene-α-d-galactopyranose  was coevaporated with toluene twice, then taken up in 8.8 mLof anhydrous CH2Cl2 (0.15 M), and stirred for1 h with 2.6 g of activated 4 Å AWMS (2 g/mmol of the donor)at room temperature under argon before cooling to −78 °C.The reaction mixture was treated with 48 μL of TMSOTf  and stirred for 2 h at −78 °C beforeit was quenched with triethylamine (0.4 mL). The reaction mixturewas diluted with dichloromethane (50 mL), filtered through a pad ofcelite, and washed with 50 mL of saturated aqueous NaHCO3. The organic layer was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purificationby flash column chromatography on silica gel eluting with 10% ethylacetate in hexanes afforded 1.096 g of 21α and 21β in a 92% yield with a 1:3 α/β; 98 mgof 21α (8%) and 602 mg of 21β (51% yield) were isolated after repeated silica gel column chromatographyeluting with 10% ethyl acetate in hexanes. The anomeric ratio of theproducts was determined by integration of the H6′ signals inthe1H NMR spectra.A mixture of 1.056 g of donor D20 = −16.8. 1H NMR  δ 7.55–7.49, 7.46–7.40 , 7.39–7.07 , 7.04–6.99 , 5.57 , 5.36 ,5.07 , 4.95 , 4.86 , 4.77–4.67 , 4.60 , 4.52, 4.47 , 4.31 , 4.25 , 4.22 , 4.15–4.04 , 3.74 , 3.63 , 3.49 ,1.50 , 1.47 , 1.33 , 1.30 . 13C{1H} NMR  δ 138.8 (C), 138.6 (C), 138.3(C), 137.1 (C), 136.3 (C), 131.9 (Ar), 129.2 (Ar), 128.78 (Ar), 128.74(Ar), 128.5 (Ar), 128.4 (Ar), 128.39 (Ar), 128.36 (Ar), 128.30 (Ar),128.0 (Ar), 127.9 (Ar), 127.8 (Ar), 127.68 (Ar), 127.66 (Ar), 127.5(Ar), 127.2 (Ar), 109.4 (CMe2), 108.6 (CMe2),105.0 (C1′), 96.5 (C1), 88.0 (C6), 84.7 (C3′), 81.5(C2′), 79.3 (C4′), 78.2 (C5′), 75.8 (CH2), 74.7 (CH2), 74.3 (CH2), 71.5 (C4), 70.9(CH2), 70.6 (C3), 70.1 (CH2), 69.9 (CH2), 67.6 (CH3), 26.17 (CH3), 26.14 (CH3), 24.5 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C52H58O11NaS 913.3592; found 913.3573.Colorless syrup.[α]D20 = +2.1 . 1H NMR  δ 7.55–7.50, 7.41–7.38 , 7.37–7.16 , 7.14–7.10 , 7.05 , 5.56 , 5.37 , 5.17, 5.01 , 4.88–4.73 , 4.69 , 4.62 , 4.44–4.37, 4.34 , 4.24–4.18 ,4.11 , 4.07–4.02, 3.84 , 3.72 ,3.64 , 1.54, 1.44 , 1.32 , 1.30 . 13C{1H} NMR  δ 138.9 (C), 138.5 (C), 138.4(C), 136.9 (C), 136.6 (C), 132.0 (Ar), 129.2 (Ar), 128.6 (Ar), 128.51(Ar), 128.50 (Ar), 128.49 (Ar), 128.42 (Ar), 128.41 (Ar), 128.2 (Ar),128.0 (Ar), 127.9 (Ar), 127.8 (Ar), 127.74 (Ar), 127.70 (Ar), 127.3(Ar), 109.4 (C), 108.7 (C), 96.7 (C1′), 96.5 (C1), 88.5 (C6′),82.1 (C3′), 79.8 (C2′), 78.4 (C4′), 75.9 (CH2), 75.5 (C5′) 74.8 (CH2), 72.2 (CH2), 70.9 (C4), 70.8 (C3), 70.78 (C2), 70.6 (CH2), 65.9(C6), 65.4 (C5), 26.3 (CH3), 26.2 (CH3), 25.1(CH3), 24.9 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C52H58O11NaS 913.3592; found 913.3584.Colorless syrup. [α]22α and 22β were obtained as a mixture of anomers  from the reaction of donor 7c  and acceptor  by following thegeneral procedure GP5 for glycosylation (eluting with10% ethyl acetate in hexanes). Repeated column chromatography gavepure samples of α- and β-isomers for full characterization.Compounds D20 = +46.5 . 1H NMR  δ 7.55–7.50, 7.41–7.15 , 7.11–7.07 , 7.05–7.01 , 5.42 , 5.39 , 5.00 , 4.82–4.75 , 4.71 , 4.41 , 4.37 ,4.13 , 4.06 , 3.69 , 3.58 , 2.20–2.17 , 2.02–1.98, 1.88 , 1.65 . 13C{1H} NMR  δ138.8 (C), 138.3 (C), 138.2 (C), 136.9 (C), 136.8 (C), 131.8 (Ar),129.1 (Ar), 128.6 (Ar), 128.4 (Ar), 128.3 (Ar), 128.2 (Ar), 128.1(Ar), 128.0 (Ar), 127.9 (Ar), 127.8 (Ar), 127.7 (Ar), 127.6 (Ar),127.5 (Ar), 127.0 (Ar), 90.0 (C1′), 88.6 (C6′), 81.8(C4′), 80.0 (C2′), 78.7 (C3′), 75.6 (CH2), 75.1 (C5′), 74.9 (CH2), 74.7 (CH2), 73.0 (CH2), 70.3 (CH2), 42.5 (CH2), 36.3 (CH2), 30.7 (CH). HRMS (ESI) m/z: [M + Na]+ calcd for C50H54O6NaS 805.3533; found 805.3537.Colorless syrup. [α]D20 = +5.7 . 1H NMR  δ 7.49 , 7.37–7.33 ,7.32–7.11 , 7.05 , 5.43 , 5.02 , 4.90 , 4.83 , 4.77–4.66 , 4.56 , 4.32, 3.78–3.69, 3.63 , 3.48 , 2.22–2.16 , 1.99 , 1.91 , 1.66 . 13C{1H} NMR  δ 138.7 (C), 138.6 (C), 138.4 (C), 137.5 (C), 136.9 (C),131.4 (Ar), 129.1 (Ar), 128.45 (Ar), 128.44 (Ar), 128.36 (Ar), 128.34(Ar), 128.31 (Ar), 128.0 (Ar), 127.76 (Ar), 127.70 (Ar), 127.68 (Ar),127.63 (Ar), 127.0 (Ar), 97.2 (C1′), 88.2 (C6′), 85.1(C3′), 82.3 (C2′), 79.3 (C5′), 78.4 (C4′),75.8 (CH2), 75.6 (C), 75.0 (CH2), 74.8 (CH2), 69.6 (CH2), 42.9 (CH2), 36.4 (CH2), 30.8 (CH). HRMS (ESI) m/z: [M + Na]+ calcd for C50H54O6NaS 805.3533; found 805.3537.Colorless syrup. [α]23α and 23β were obtained as a mixture of anomers  from the reaction of donor 7c  and acceptor  by followingthe general procedure GP5 for glycosylation (elutingwith 10% ethyl acetate in hexanes). Repeated column chromatographygave pure samples of α- and β-isomers for full characterization.Compounds D20 = +34.9 .1H NMR  δ 7.53–7.46, 7.37–7.11 , 7.02–6.97 , 5.87 , 5.34–5.30, 4.97–4.91 , 4.86 , 4.76–4.72, 4.68 , 4.47 , 4.42 , 4.31 , 4.17 , 4.15–4.10 ,4.06–4.04 , 3.92 , 3.68 ,3.58 , 1.49, 1.41 , 1.25 . 13C{1H} NMR  δ138.5 (C), 138.2 (C), 138.1 (C), 138.0 (C), 136.7 (C), 136.2 (C),131.8 (Ar), 129.3 (Ar), 128.9 (Ar), 128.7 (Ar), 128.68 (Ar), 128.65(Ar), 128.5 (Ar), 128.49 (Ar), 128.47 (Ar), 128.2 (Ar), 128.1 (Ar),127.86 (Ar), 127.8 (Ar), 127.7 (Ar), 127.6 (Ar), 111.9 (CMe2), 109.1 (CMe2), 105.4 (C1), 98.4 (C1′), 88.5 (C6′),83.7 (CH), 81.6 (CH), 81.4 (CH), 81.3 (CH), 80.0 (CH), 78.2 (CH),76.3 (CH), 75.9 (CH2), 75.1 (CH2), 73.2 (CH2), 72.5 (CH), 70.7 (CH2), 67.2 (CH2),27.1 (CH3), 26.9 (CH3), 26.3 (CH3), 25.6 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C52H58O11NaS 913.3592; found 913.3596.Colorless syrup. [α]D20 = +6.2 .1H NMR  δ 7.50–7.44, 7.35–7.18 , 7.13–7.06 , 5.78 , 5.40 , 4.89 , 4.84 , 4.79–4.72 , 4.59 , 4.48–4.45 , 4.44–4.40, 4.27 , 4.06 , 3.80–3.70 , 3.63 ,3.38 , 1.50, 1.45 , 1.28 , 1.25 . 13C{1H} NMR  δ 138.4 (C), 138.2 (C), 138.1(C), 137.5 (C), 136.2 (C), 131.3 (Ar), 129.2 (Ar), 128.59 (Ar), 128.51(Ar), 128.47 (Ar), 128.42 (Ar), 128.2 (Ar), 127.89 (Ar), 127.84 (Ar),127.80 (Ar), 127.7 (Ar), 127.1 (Ar), 112.0 (CMe2), 108.4(CMe2), 105.2 (C1), 102.0 (C1′), 87.5 (CH), 84.6(CH), 82.8 (CH), 82.1 (CH), 80.8 (CH), 80.2 (CH), 79.6 (CH), 78.2(CH), 77.3 (CH), 77.1 (CH), 76.8 (CH), 75.8 (CH2), 75.0(CH2), 73.8 (CH), 69.4 (CH2), 65.6 (CH2), 26.6 (CH3), 26.2 (CH3), 25.2 (CH3). HRMS (ESI) m/z:[M + Na]+ calcd for C52H58O11NaS 913.3592; found 913.3596.Colorless syrup. [α]24α and 24β were obtained as a mixtureof anomers  from the reaction of donor 7c  and acceptor  by following thegeneral procedure GP5 for glycosylation (eluting with10% ethyl acetate in hexanes). Repeated column chromatography gavepure samples of α- and β-isomers for full characterization.Compounds D20 = +20.6 . 1H NMR  δ 7.54–7.48, 7.35–7.12 , 7.11–7.06 , 7.05–6.98 , 5.31 , 5.28 , 4.92 , 4.83–4.70 , 4.65 , 4.48 , 4.38 , 4.32 ,4.19 , 4.10 , 3.97 , 3.72 , 3.57 , 3.42 , 3.34 , 1.48 , 1.35 , 1.28 . 13C{1H} NMR  δ 138.7 (C), 138.4 (C), 138.3 (C), 137.0(C), 136.9 (C), 132.1 (Ar), 129.1 (Ar), 128.6 (Ar), 128.54 (Ar), 128.50(Ar), 128.3 (Ar), 128.1 (Ar), 127.94 (Ar), 127.90 (Ar), 127.7 (Ar),127.6 (Ar), 127.1 (Ar), 109.1 (CMe2), 98.4 (C1), 97.5 (C1′),89.5 (C6′), 82.8 (C3), 81.7 (C3′), 80.5 (C2′),78.7 (CH), 77.0 (CH), 75.7 (CH2), 75.6 (CH), 75.5 (CH),74.8 (CH2), 73.5 (CH2), 70.5 (CH2), 64.4 (CH), 54.8 (OCH3), 28.1 (CH3), 26.1(CH3), 18.1 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C50H56O10NaS 871.3486; found 871.3477.Colorless syrup. [α]D20 = −27.6. 1H NMR  δ 7.47–7.43, 7.40–7.36 , 7.35–7.15 , 7.13–7.09 , 5.48 , 5.01–4.86 ,4.85–4.65 , 4.48 , 4.24 , 4.11 , 3.82–3.62, 3.44 , 3.40 , 1.59 , 1.42 , 1.35 . 13C{1H} NMR  δ 138.7 (C), 138.6(C), 138.4 (C), 137.8 (C), 136.7 (C), 130.9 (Ar), 129.1 (Ar), 128.44(Ar), 128.41 (Ar), 128.3 (Ar), 128.1 (Ar), 127.9 (Ar), 127.8 (Ar),127.7 (Ar), 127.6 (Ar), 126.8 (Ar), 109.6 (CMe2), 102.9(C1′), 98.1 (C1), 87.8 (CH), 84.8 (CH), 82.4 (CH), 79.3 (CH),79.2 (CH), 78.5 (CH), 78.4 (CH), 76.0 (CH), 75.8 (CH2),74.9 (CH2), 74.8 (CH2), 68.9 (CH2), 64.4 (CH), 54.9 (CH3), 28.2 (CH3), 26.5(CH3), 17.8 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C50H56O10NaS 871.3486; found 871.3477.Colorless syrup. [α]25α and 25β were obtained as a mixture of anomers  from the reaction of donor 7c  and acceptor  by following thegeneral procedure GP5 for glycosylation (eluting with10% ethyl acetate in hexanes). Repeated column chromatography gavea pure sample of α-isomer for full characterization.Compounds D20 = +29.5 . 1H NMR  δ 7.46–7.41, 7.32–7.12 , 7.11–7.07 , 7.04–7.00 , 5.65 , 5.34 , 4.97 , 4.86 , 4.82 , 4.78, 4.74 , 4.70–4.65 , 4.60–4.57 , 4.59–4.51 , 4.45–4.41 , 4.17  4.13 , 4.06 , 3.96–3.82 , 3.63 , 3.58 , 3.45 , 3.37 . 13C{1H} NMR  δ 139.2 (C), 138.6 (C), 138.5(C), 138.3 (C), 138.2 (C), 138.0 (C), 136.9 (C), 136.7 (C), 131.2(Ar), 129.2 (Ar), 128.7 (Ar), 128.5 (Ar), 128.46 (Ar), 128.42 (Ar),128.40 (Ar), 128.33 (Ar), 128.29 (Ar), 128.24 (Ar), 128.0 (Ar), 127.9(Ar), 127.7 (Ar), 127.6 (Ar), 127.4 (Ar), 127.09 (Ar), 127.05 (Ar),126.9 (Ar), 97.7 (C1), 96.9 (C1′), 88.0 (C6′), 81.9(CH), 81.8 (CH), 79.9 (CH), 79.5 (CH), 78.4 (CH), 76.5 (CH), 75.7(CH2), 74.9 (CH2), 74.8 (CH2), 74.5(CH), 73.4 (CH2), 73.3 (CH2), 73.1 (CH2), 70.1 (CH2), 69.7 (CH), 55.1 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C68H70O11NaS 1117.4531;found 1117.4506.Colorless syrup. [α]The β-isomer was not obtained pure andwas characterized in the mixture of anomers by the following diagnosticsignals.1H NMR  δ 5.35, 4.46 , 3.38 .26α and 26β were obtained as a mixture of anomers  from the reaction of donor 7d  and acceptor  by following thegeneral procedure GP5 for glycosylation (eluting with10% ethyl acetate in hexanes). Repeated column chromatography gavepure samples of α- and β-isomers for full characterization.Compounds D20 = +3.2 . 1H NMR  δ 7.39–7.11, 7.01 , 5.52 , 5.36 , 5.08 , 5.00 , 4.86 , 4.76 , 4.68–4.62, 4.57 ,4.39 , 4.34 , 4.31–4.27 , 4.07, 3.90 , 3.82, 3.78 , 3.61 , 1.54 , 1.42 , 1.32 , 1.29 . 13C{1H} NMR  δ 138.8(C), 138.4 (C), 138.3 (C), 137.4 (C), 135.9 (C), 131.8 (Ar), 129.1(Ar), 128.5 (Ar), 128.47 (Ar), 128.41 (Ar), 128.39 (Ar), 128.03 (Ar),128.00 (Ar), 127.83 (Ar), 127.79 (Ar), 127.6 (Ar), 127.1 (Ar), 109.3(CMe2), 108.7 (CMe2), 96.8 (C1′), 96.4(C1), 89.3 (CH), 81.8 (CH), 80.0 (CH), 79.1 (CH), 75.6 (CH2), 74.8 (CH), 74.0 (CH2), 72.4 (CH2), 70.9(CH), 70.8 (CH), 70.7 (CH), 70.1 (CH), 66.3 (CH), 65.6 (CH2), 26.3 (CH3), 26.2 (CH3), 25.0 (CH3), 24.8 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C52H58O11NaS 913.3592; found 913.3573.Colorless syrup. [α]D20 = −17.5. 1H NMR  δ 7.44–7.39, 7.32–7.15 , 7.02–6.98 , 5.56 , 5.40 , 5.05 , 4.96 , 4.84–4.77 ,4.75–4.69 , 4.57 , 4.51 , 4.41 , 4.30 , 4.24 , 4.13 , 4.08 , 3.87–3.79 , 3.73 , 3.68 , 3.49 , 1.51 , 1.42 , 1.30 . 13C{1H} NMR  δ 138.76 (C), 138.7 (C), 138.1 (C), 137.7(C), 136.0 (C), 131.5 (Ar), 129.1 (Ar), 128.7 (Ar), 128.44 (Ar), 128.41(Ar), 128.38 (Ar), 128.3 (Ar), 128.0 (Ar), 127.89 (Ar), 127.8 (Ar),127.65 (Ar), 127.6 (Ar), 127.0 (Ar), 109.4 (C), 108.7 (C), 104.2 (C1′),96.5 (C1), 88.7 (C6′), 84.5 (CH), 81.7 (CH), 78.8 (CH), 78.4(CH), 77.35 (CH), 77.30 (CH), 77.1 (CH), 76.8 (CH), 75.6 (CH2), 75.0 (CH2), 74.4 (CH), 71.5 (CH), 70.9 (CH), 70.6 (CH2), 69.8 (CH2), 69.6 (CH2), 67.6 (CH),26.2 (CH3), 26.1 (CH3), 25.1 (CH3), 24.5 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C52H58O11NaS 913.3592; found 913.3573.Colorless syrup. [α]27α and 27β were obtained as a mixture of anomers  from the reaction of donor 7d  and acceptor  by followingthe general procedure GP5 for glycosylation (elutingwith 10% ethyl acetate in hexanes). Repeated column chromatographygave a pure sample of α-isomer for full characterization.Compounds D20 = +46.2 . 1H NMR  δ 7.46–7.14, 7.08 , 5.33 ,5.02 , 4.90 , 4.84–4.75 , 4.71–4.67 , 4.55–4.45, 4.39 , 4.10 , 3.83 ,3.58 , 2.08, 1.89 , 1.83 , 1.64–1.54 . 13C{1H} NMR  δ 138.9(C), 138.4 (C), 138.3 (C), 137.6 (C), 136.1 (C), 131.8 (Ar), 129.1(Ar), 128.5 (Ar), 128.4 (Ar), 128.3 (Ar), 128.2 (Ar), 128.0 (Ar),127.9 (Ar), 127.8 (Ar), 127.7 (Ar), 127.68 (Ar), 127.62 (Ar), 127.5(Ar), 90.4 (C1′), 89.9 (C6′), 82.0 (C3′), 80.4(C2′), 79.5 (C4′), 75.5 (CH2), 75.0 (CH2), 74.9 (CH2), 73.3 (CH2), 73.0 (C5′),71.0 (CH2), 42.5 (CH2), 36.3 (CH2), 30.7 (CH). HRMS (ESI) m/z: [M + Na]+ calcd for C50H54O6NaS 805.3533; found 805.3519.Colorless syrup. [α]The β-isomerwas not obtained pure and was characterized in the mixture of anomersby the following diagnostic signals.1H NMR  δ 5.40 , 3.71 , 3.50 .28α and 28β were obtained as a mixtureof anomers  from the reaction of donor 7d and acceptor  by following the general procedure GP5 for glycosylation (eluting with 10% ethyl acetate in hexanes).Repeated column chromatography gave a pure sample of α-isomerfor full characterization.Compounds D20 = +7.2  1H NMR  δ 7.44–7.38, 7.37–7.11 , 5.32 , 5.12 , 4.95 , 4.85 , 4.83–4.79 , 4.77 , 4.69 , 4.64 , 4.58 , 4.51 ,4.21 , 4.06 , 3.99 , 3.89 , 3.75–3.68 , 3.57 , 3.38 , 3.34 , 1.41 , 1.33 , 1.16 . 13C{1H} NMR  δ 138.7 (C), 138.3 (C), 138.1 (C), 137.8 (C), 136.8(C), 131.5 (Ar), 129.0 (Ar), 128.53 (Ar), 128.50 (Ar), 128.3 (Ar),128.0 (Ar), 127.95 (Ar), 127.93 (Ar), 127.8 (Ar), 127.7 (Ar), 127.6(Ar), 127.56 (Ar), 126.9 (Ar), 109.2 (C), 98.1 (C1′), 98.0(C1), 89.8 (C6′), 82.3 (C4), 81.8 (C3′), 80.6 (C2′),78.9 (C4′), 77.2 (C3), 75.80 (CH2), 75.6 (CH2), 75.0 (CH2), 74.9 (CH), 73.9 (CH2),70.6 (CH2), 64.6 (CH5), 54.7 (CH3), 28.0 (CH3), 26.2 (CH3), 17.8 (CH3). HRMS(ESI) m/z: [M + Na]+ calcd for C50H56O10NaS 871.3486;found 871.3455.Colorless syrup. [α]The β-isomer was not obtained pure andwas characterized in the mixture of anomers by the following diagnosticsignals.1H NMR  δ 5.35, 4.99, 3.38 .2SO4, filtered through cotton, and concentrated under vacuum toafford a crude residue, which was purified by silica gel chromatography(0→10% CH3OH in CH2Cl2) togive the desired deprotected glycoside.A suspensionof glycoside (1.0 equiv) in ethanol (0.1 M) was treated with Raneynickel (1 g/mmol) that had been previously washed with ethanol. Thereaction mixture was stirred overnight under 1 atm of hydrogen atroom temperature. The reaction mixture was diluted with ethanol andfiltered through celite. The filter cake was washed with additionalethanol, and the combined filtrate was dried over Na9β (15 mg) using the generalprocedure for desulfurizationeluting from silica gel with 10% MeOH/ in CH2Cl2 to give 5.3 mg for a 75% yield, and spectral data consistent withthe literature.51Obtained from thedeprotection of compound D23 = −36.5 1HNMR  δ 5.49 , 4.60 ,4.35 , 4.28 , 4.21 , 4.07–3.98 , 3.80 , 3.75–3.59 , 3.54–3.47 ,3.44 , 1.49 , 1.37, 1.32 , 1.30 . 13C {1H} NMR  δ 109.1 (C), 108.7 (C), 104.0 (CH), 96.4 (CH),75.4 (CH), 73.4 (CH), 71.1 (CH), 70.65 (CH), 70.61 (CH), 68.9 (CH),68.4 (CH), 67.6 (CH), 61.1 (CH2), 24.9 (CH2),23.7 (CH3), 23.1 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C18H30O11Na 445.1680; found 445.1674.Colorless syrup. [α]10β (9.0 mg) using general procedure for desulfurization eluting fromsilica gel with 10% MeOH in CH2Cl2 to give 2.7mg for a 76% yield.Obtained from the deprotection of compound D23 = +1.4 . 1H NMR  δ 4.52–4.43 , 3.82–3.77 , 3.72–3.61 ,3.49–3.39 , 2.10 , 1.93–1.87, 1.83–1.74 , 1.70–1.59 . 13C {1H} NMR  δ 96.41 (C1), 74.91 (CH), 74.55 (CH), 73.76 (CH), 71.24 (CH),68.84 (C4), 61.02 (C6), 42.31 (Ada), 36.07 (Ada), 30.87 (Ada). HRMS (ESI) m/z: [M + Na]+ calcd for C16H26O6Na 337.1621;found 337.1613.Colorless syrup. [α]12β (12 mg) usingthe general procedure for desulfurizationeluting from silica gel with 10% MeOH in CH2Cl2 to give 5.0 mg for a 93% yield.Obtained fromthe deprotection of compound D23 = −27.6. 1HNMR  δ4.77 , 4.73–4.65 , 4.23 , 4.07 , 3.84–3.80 , 3.75–3.57, 3.45–3.39 ,3.33 , 1.47 , 1.30, 1.24 . 13C {1H} NMR  δ 109.0 (CMe2), 101.6 (C1′), 98.0 (C1),78.1 (CH), 77.9 (CH), 75.9 (CH), 75.3 (CH), 73.7 (CH), 71.5 (CH),68.8 (CH), 64.0 (CH), 60.8 (C6′), 53.8 (OCH3), 27.0(CH3), 26.9 (CH3), 25.25 (CH3), 16.7(CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C16H28O10Na 403.1574; found 403.1567.Colorless syrup. [α]11β (15 mg) using thegeneral procedure for desulfurizationeluting from silica gel with 10% MeOH in CH2Cl2 to give 6.5 mg for a 91% yield.Obtained from thedeprotection of compound D23 = −1.8. 1HNMR  δ5.91 , 4.68 , 4.46 , 4.36 , 4.34–4.29, 4.09 , 4.02 , 3.84–3.75 , 3.70 , 3.56–3.49 ,3.49–3.44 , 1.46 , 1.40 , 1.33 , 1.31 ; 13C {1H} NMR  δ 111.68(CMe2), 108.31 (CMe2), 105.24 (C1), 102.32 (C1′),83.13 (CH), 80.36 (CH), 80.12 (CH), 75.66 (CH), 73.71 (CH), 73.59(CH), 70.67 (CH), 68.93 (CH), 65.45 (CH2), 61.25 (CH2), 25.77 (CH3), 25.41 (CH3), 25.11 (CH3), 24.08 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C18H30O11Na 445.1680; found 445.1673.Colorless syrup. [α]13α (15 mg) using thegeneral procedure for desulfurization. The crude residue obtainedwas triturated with CHCl3 (3 × 2 mL), filtered, andconcentrated to afford pure product; yield , and spectraldata consistent with the reported literature.52Obtained from thedeprotection of compound D23 = +30.7 . 1H NMR δ 5.33 , 4.74 , 3.95–3.73 , 3.70-3.66, 3.59–3.49 , 3.35 . 13C{1H} NMR δ 99.9 (C1), 99.0 (C1′), 77.0 (CH), 73.5 (CH), 71.7 (CH),71.0 (CH), 70.1 (CH), 69.3 (CH), 69.1 (CH), 68.6 (CH), 61.1 (CH2), 60.5 (CH2), 55.0 (OCH3). HRMS (ESI) m/z: [M + Na]+ calcd for C13H24O11Na 379.1210;found 379.1206.Colorless syrup. [α]13β (16 mg) using thegeneral procedure for desulfurization. The crude residue obtainedwas triturated with CHCl3 (3 × 2 mL), filtered, andconcentrated to afford pure product; yield .54Obtained from thedeprotection of compound D23 = +31.6 . 1H NMR  δ 4.84 , 4.47 ,3.99–3.93 , 3.90–3.73 , 3.69 , 3.66–3.62, 3.57 , 3.45 . 13C{1H} NMR  δ 102.9 (C1′), 99.1 (C1), 78.46(CH), 75.44 (CH), 72.63 (CH), 71.82 (CH), 71.05 (CH), 70.99 (CH),70.34 (CH), 68.65 (CH), 61.12 (CH2), 59.99 (CH2), 55.18 (OMe). HRMS (ESI) m/z: [M + Na]+ calcd for C13H24O11Na 379.1210; found 379.1201.Colorless syrup. [α]18α (14 mg) usingthe general procedure for desulfurizationeluting from silica gel with 10% MeOH in CH2Cl2 to give 5.9 mg for a 94% yield.Obtained fromthe deprotection of compound D23 = +46.6 . 1H NMR  δ 4.91 , 4.77 , 4.12–4.06 , 4.06–4.01 , 3.96 , 3.77 , 3.72 ,3.69–3.64 , 3.34 , 1.48, 1.36–1.25 . 13C{1H} NMR  δ 109.00 (CMe2),100.46 (C1′), 97.95 (C1), 81.24 (CH), 77.11 (CH), 75.97 (CH),70.21 (CH), 69.99 (CH), 69.76 (C4′), 69.06 (CH), 64.80 (CH),61.03 (CH2), 53.81 (OMe), 27.02 (CH3), 25.15(CH3), 16.67 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C16H28O10Na 403.1574; found 403.1567.Colorless syrup. [α]21β (20 mg) using the general procedure for desulfurizationeluting from silica gel with 10% MeOH in CH2Cl2 to give 6.4 mg for a 67% yield.Obtained from the deprotection of compound 26β (12 mg) using the general procedurefor desulfurization eluting from silica gel with 10% MeOH in CH2Cl2 to give 5.1 mg for an 83% yield.Obtained from the deprotectionof compound 21β in 2 mL of ethanolwhile stirring was added 1 g of Raney nickel that had been previouslywashed with ethanol. The reaction mixture was stirred for 36 h under1 atm of hydrogen at room temperature. The reaction mixture was dilutedwith 50 mL of ethanol and filtered through a pad of celite; the filtercake was washed with additional 50 mL of ethanol. The combined filtratewas dried over Na2SO4, filtered through cotton,and concentrated under reduced pressure to afford a crude residue.Two hundred and fifty-two milligrams were obtained after silica gelcolumn chromatography eluting with 10% MeOH in CH2Cl2 for an 88% yield. The spectral data is consistent with thereported literature.56To asuspension of 603 mg of D20 = −41.8 1H NMR  δ 5.44 , 4.58 , 4.31 ,4.28–4.22 , 3.97–3.88 , 3.71 , 3.63–3.51 , 3.43 , 3.35 , 3.30 , 3.27–3.23, 3.20 , 3.07 , 2.80 , 1.47 , 1.35 , 1.28 . 13C{1H} NMR  δ 108.9 (CMe2), 108.4 (CMe2), 103.4 (CH), 96.3 (CH), 76.7 (CH),76.3 (CH), 73.7 (CH), 71.0 (CH), 70.6 (CH), 70.5 (CH), 68.4 (CH2), 67.2 (CH), 61.9 (CH2), 25.4 (CH3),25.3 (CH3), 24.2 (CH3), 23.7 (CH3). HRMS (ESI) m/z:[M + Na]+ calcd for C18H30O11Na 445.1680; found 445.1680.Colorless syrup. [α]22β (25 mg) using the general procedurefor desulfurization elutingfrom silica gel with 10% MeOH in CH2Cl2 to give4.7 mg for a 46% yield. The spectral data is consistent with the literature.57Obtained from the deprotectionof compound D20 = −12.4 1H NMR  δ 4.53 , 3.79 , 3.61 , 3.33 , 3.24 , 3.26–3.19 , 3.09 , 2.11 , 1.93–1.87 , 1.81–1.76, 1.69–1.60 . 13C{1H} NMR  δ 95.8 (C1), 76.8 (CH), 76.2 (CH), 74.7(CH), 73.7 (CH), 70.4 (CH), 61.5 (CH2), 42.2 (Ada), 36.0(Ada), 30.8 (Ada). HRMS (ESI) m/z: [M + Na]+ calcd for C16H26O6Na 337.1622; found 337.1612.Colorless syrup. [α]24α (23.5 mg) usingthe general procedure for desulfurization eluting from silica gelwith 10% MeOH in CH2Cl2 to give 8.6 mg for a76% yield.Obtained fromthe deprotection of compound 28α (9.7 mg) using the general procedure for desulfurizationeluting from silica gel with 10% MeOH in CH2Cl2 to give 3.8 mg for an 87% yield. The spectral data is consistentwith the reported literature.58Obtained from the deprotection of compound D20 = +41.7  1H NMR  δ 4.92 ,4.80 , 4.15–4.06 ,3.84 , 3.77, 3.70 ,3.65–3.60 , 3.47–3.35 ,3.36 , 3.35–3.32  1.51 , 1.33 , 1.31 . 13C{1H} NMR  δ 110.3 (CMe2), 101.5 (C1′),99.3 (C1), 82.54 (CH), 78.5 (CH), 77.3 (CH), 74.9 (CH), 73.7 (CH),73.3 (CH), 71.3 (CH), 66.2 (CH), 62.1 (CH3), 55.1 (CH2), 28.4 (CH3), 26.5 (CH3), 17.9 (CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C16H28O10Na 403.1575; found 403.1572.Whiteamorphous solid. [α]27α (16.7 mg) using the general procedurefor desulfurization elutingfrom silica gel with 10% MeOH in CH2Cl2 to give4.3 mg for a 64% yield.Obtained from the deprotectionof compound D20 = +19.1  1H NMR  δ 5.17 , 3.75–3.70 , 3.65 , 3.62–3.58 , 3.30 , 3.26 , 2.15–2.08 ,1.94–1.84 , 1.85–1.78 , 1.65. 13C{1H} NMR  δ 91.6 (C1), 74.0 (CH), 73.9 (CH), 72.1(CH), 71.8 (CH), 70.7 (CH), 61.4 (CH2), 42.2 (Ada), 36.1(Ada), 30.8 (Ada). HRMS (ESI) m/z: [M + Na]+ calcd for C16H26O6Na 337.1622; found 337.1608.White amorphous solid. [α]


Significant (p < 0.1) univariate relationships existed between PFS and ER%, AR%, and PIK3CA and/or PTEN mutations. Baseline levels of phospho-proteins in the mTOR pathway were more highly expressed in biopsies of patients with shorter PFS. Fulvestrant plus enzalutamide had manageable side effects. The primary endpoint of CBR24 was 25% in heavily pretreated metastatic ER+/HER2− BC. Short PFS was associated with activation of the mTOR pathway, and PIK3CA and/or PTEN mutations were associated with an increased hazard of progression. Thus, a combination of fulvestrant or other SERD plus AKT/PI3K/mTOR inhibitor with or without AR inhibition warrants investigation in second-line endocrine therapy of metastatic ER+ BC.This clinical trial combined fulvestrant with the anti-androgen enzalutamide in women with metastatic ER+/HER2− breast cancer (BC). Eligible patients were women with ECOG 0–2, ER+/HER2− measurable or evaluable metastatic BC. Prior fulvestrant was allowed. Fulvestrant was administered at 500 mg IM on days 1, 15, 29, and every 4 weeks thereafter. Enzalutamide was given at 160 mg po daily. Fresh tumor biopsies were required at study entry and after 4 weeks of treatment. The primary efficacy endpoint of the trial was the clinical benefit rate at 24 weeks (CBR24). The median age was 61 years (46–87); PS 1 (0–1); median of 4 prior non-hormonal and 3 prior hormonal therapies for metastatic disease. Twelve had prior fulvestrant, and 91% had visceral disease. CBR24 was 25% . Median progression-free survival (PFS) was 8 weeks (95% CI: 2–52). Adverse events were as expected for hormonal therapy.  Androgen receptors (AR) are expressed in most BC, and AR positivity (nuclear staining by immunohistochemistry (IHC) in 10% or more of cells) was observed in 77% of 3093 invasive breast tumors of all subtypes, including 91% of ER+ BC2. The predominately nuclear localization of AR by IHC2 indicates that it is in the liganded state. In primary ER+ BC compared to patient-matched metastatic disease, AR is commonly maintained in metastases, while ER often decreases3.Breast cancer (BC) is a genetically heterogeneous and biologically diverse disease. We currently subdivide BC by estrogen receptor alpha (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2/neu) status, in part because these markers represent important predictive biomarkers that guide treatment with discernible survival benefits. Endocrine therapies, such as tamoxifen, fulvestrant, and aromatase inhibitors (AI), target ER directly or the production of estrogen and play a critical role in the treatment of patients with ER+ disease4, with confusion arising from the fact that when estradiol is present, androgens decrease ER-mediated proliferation in cell lines and xenograft models5. In contrast, in the absence of estrogen, androgens stimulate proliferation, and AR is associated with resistance to tamoxifen and aromatase inhibitors (AI) in ER+ BC10. Tumors that respond to tamoxifen express similar percent cells positive for ER and AR protein, as does adjacent uninvolved epithelium10. However, tamoxifen-resistant breast tumors have a high ratio of percent cells positive for nuclear AR versus ER. A ratio of AR:ER ≥ 2.0 in primary tumors is associated with an over four-fold increased risk for failure while on adjuvant tamoxifen and overall disease-free survival, with an independent effect on risk for relapse beyond ER positivity alone9. Similar findings have been reported for patients treated with adjuvant AI therapy13.The functional role of AR in ER+ BC remains controversial17, resulting in AR activation and resistance to AI therapy. In the absence of estradiol (E2)  dihydrotestosterone (DHT) (which cannot be aromatized to estrogen) increased proliferation of ER+ cell lines and patient-derived xenografts19. On the other hand, the selective androgen receptor modulator enobosarm decreased tumor growth in preclinical models5. A recent trial NCT02007512 using the AI exemestane, with or without enzalutamide, in patients with ER+ advanced/metastatic disease found that high levels of AR and low levels of ESR1 were associated with the significantly greater benefit of enzalutamide20. These complexities emphasize the importance of clinical context and hormonal milieu when considering AR action in BC21. Clearly, AR does influence BC biology, and high AR relative to ER levels can serve as an independent predictor of response to anti-estrogen therapies, perhaps by identifying tumors poised to escape ER-directed therapies and switch to survival dependent on androgens and AR. Thus, we postulated that when ER+ BC becomes resistant to ER-targeting therapies, agents targeting AR may provide clinical benefits.While AIs effectively block the conversion of androgens to estrogens to decrease ER-stimulated tumor growth, over time, circulating and intra-tumoral androgens can increase as an unintended consequence22, and it is now under investigation in women with ER+ BC. An extended phase I trial of enzalutamide identified 160 mg/day as the recommended phase 2 dose, and the PK and safety profile in women was similar to that observed in men23. Enzalutamide is a potent CYP3A4 inducer and reduced the areas under the concentration-time curve (AUC) of anastrozole and exemestane by 80% and ~50%, respectively. However, when combined with fulvestrant, no significant PK interaction or new safety signals were found25. Preclinical modeling showed synergistic inhibitory effects of fulvestrant plus enzalutamide on tumor cell growth3. Therefore, we hypothesized that enzalutamide, combined with fulvestrant, would be effective in patients with ER+ AR+ metastatic BC resistant to traditional therapeutic strategies targeting ER or estrogen production (AI therapy). The primary objective of the current trial was to determine CBR24 of the combination of enzalutamide and fulvestrant in metastatic BC originally diagnosed as ER+ disease. The secondary objectives were to confirm the safety profile of the combination, response rate, and progression-free survival (PFS) at 24 weeks. Serial biopsies were obtained during pretreatment and at the end of the fourth week of treatment (hence referred to as week 5) to evaluate the effects of treatment on the tumor and its relationship to clinical outcomes.The use of the anti-androgen enzalutamide showed efficacy in women with TNBCOf the 32 eligible participants, 28 were evaluable for response Table . The medTreatment-emergent adverse events (AEs) were consistent with what would be expected with hormonal therapy. Fatigue, nausea, and achiness were most common. Cognitive dysfunction described as “difficulty concentrating” was reported in 5 patients and resolved upon completion of treatment. AEs greater than 20% included fatigue (53.1%), nausea (50.0%), vomiting (28.1%), constipation (31.2%), anorexia (28.1%), headache (34.4%), achiness (43.8%), and tumor-associated pain (31.3%). Most AEs were low-grade. G3 toxicity was uncommon and, in most cases, was unrelated to protocol treatment. There were no G4 or G5 toxicities  (95% CI: 10.7–44.9) participants had stable disease, and no participants had a partial response Fig. . The medp = 0.003) units lower at week 5 than at baseline, and Ki67 was 10.9  percentage points lower at week 5 than at baseline. No other IHC variable, including PR and glucocorticoid receptor (GR), had a significant difference between baseline and week 5 of treatment  times the hazard for participants with AR ≥ 10% positive after controlling for ER percent positive (p = 0.057). The hazard of disease progression for participants with ER < 10% positive was 4.69 (95% CI: 1.53–14.35) times the hazard for participants with ER ≥ 10% positive, after controlling for AR percent positive (p = 0.007)  Table .Table 3UA Kaplan–Meier curve of PFS stratified by AR and ER protein by IHC suggested that those with biopsies with <10% positive cells for both ER and AR had the shortest PFS, and those with ≥10% of both receptors received the longest PFS, albeit sample size was too limited to perform statistical tests Fig. .Significant (p < 0.2) univariate relationships were observed between continuous PFS and the following variables: ER and AR percent positivity by IHC and PIK3CA and/or PTEN mutations. Specifically, the average hazard for progression was 4.32 (95% CI: 1.46–12.83) times higher for participants with ER < 10% positive compared to participants with ER ≥ 10% positive (p = 0.008). This association remained significant when ER was considered continuous (p = 0.024). Additionally, the average hazard of progression was 2.28 (95% CI: 0.93–5.62) times higher for participants with AR < 10% positive compared to participants with AR ≥ 10% positive (p = 0.073). The hazard of disease progression for participants with AR and/or ER < 10% positive was 2.35  times the hazard for participants with AR and/or ER ≥ 10% positive. Finally, the hazard of disease progression for patients with PTEN and/or PIK3CA mutated tumor biopsies was 2.02  times the hazard for participants with neither molecular event. This is illustrated in a swimmer plot of PFS, prior fulvestrant, and prior everolimus with activating mutations in the PIK3CA or potential loss of function PTEN mutations, as well as ESR1 mutations, indicated , everolimus (28%), or cdk4/6 inhibitors (83%) had significant univariate associations with PFS  to those who experienced “Long PFS” defined as PFS > 24 weeks (168 days) (n = 7), the mTOR pathway had higher baseline expression levels in the tumors of those with Short PFS. According to robust moderated t-tests, baseline mTOR S2448 (p = 0.008), eNOS/NOSIII S116 (p = 0.029), S6RP S240/S244 (p = 0.031), eIF4G S118 (p = 0.037), and p7056K T389 (p = 0.043) were significantly higher in the Short PFS group compared to the Long PFS group . This suggests mTOR activation in the pre-treatment tumor biopsies from patients with a short time to progression  proteins that had significant baseline associations with total ER in the RPPA data  proteins had significant baseline associations with AR, and 80 (51.0%) proteins had significant fold change associations with AR. Respectively, 48 (100%) and 79 (98.8%) were positively correlated , but a lower level of amino acids  and purine breakdown and deamination products  at baseline , and glutathione metabolism are elevated in the plasma of the Short PFS group.Metabolomics analyses of patient plasma revealed baseline and post-treatment differences in acyl-carnitines, amino acid, and purine metabolism in patients with Short versus Long PFS. Given the role of mTOR as a master sensor and regulator of metabolismine Fig. . Followiine Fig. . Howeverine Fig. . This is30, and this was observed in 13% of the patients in this study. In primary tumors versus patient-matched metastases, we previously found that nuclear AR was often maintained even when ER was reduced or absent3. These data emphasize the need to consider fresh biopsy to confirm the presence of the therapeutic targets of interest.The current phase II clinical trial investigated the combination of fulvestrant with enzalutamide in a heavily pretreated population of women with metastatic ER+/HER2− BC. The regimen achieved CBR24 in 25% of the patients, with generally low-grade toxicity with no new safety signals. It is notable that 29% (2 of 7) patients in the Long PFS (PFS > 24 weeks) group had prior exposure to fulvestrant. Analyses of tumor biopsies demonstrated that patients with ≥10% positive cells for ER or AR by IHC had longer PFS than patients with ER and AR < 10% positive. Metastatic disease evolves under the selective pressure of treatments and can lose expression of ER31. In addition, patients with Long PFS are characterized by preservation of circulating levels of lipid oxidation markers (acyl-carnitines) compared to patients with Short PFS, suggestive of preserved mitochondrial function32, in the former group following treatment with fulvestrant with enzalutamide. This suggests potential utility for adding an mTOR inhibitor such as everolimus to enzalutamide, particularly for patients with activated mTOR or PIK3CA/PTEN mutations, to potentially enhance response rate as has been suggested previously by preclinical and clinical studies33. PIK3CA and PTEN mutations are frequent in metastatic ER+ BC35. In this study, we find that PIK3CA activating and or PTEN inactivating mutations were significantly associated with a greater hazard of PFS.Patients with Short PFS (PFS ≤ 60 days) had increased mTOR pathway activation in the pre-treatment biopsies compared to patients with PFS > 24 weeks. Interestingly, these findings were supported by plasma metabolomics data, showing significantly lower levels of multiple amino acids in patients with Short PFS (<24 weeks) compared to Long PFS (>24 weeks)—an observation that would be consistent with mTOR activation via sensing of systemic amino acid depletion20. Overall, 247 patients with ER+ BC were randomized into two cohorts (one with no prior endocrine therapy for metastatic disease and the other with one prior endocrine therapy in that setting). In this study, only the cohort without prior endocrine therapy had a numerical advantage in PFS for the addition of enzalutamide in patients with high AR (above the median), particularly in combination with low (below median) levels of ESR1 mRNA in their primary tumors. These patients had a reduced risk of progression or death compared to the control arm [HR, 0.24 (95% CI: 0.10–0.60); p = 0.001, with the median PFS extended by 10.2 months 20. These data may suggest that primary tumors with higher-than-average AR but lower-than-average ESR1 are poised to become resistant to AI therapy and may benefit from anti-androgen therapy. This is in line with the high AR to ER percent cells positive data summarized in the introduction36. Interestingly, enobosarm, a selective androgen receptor modulator (SARM) with both agonist and antagonist activity depending on tissue type, showed activity in preclinical37 and clinical studies38. In women with endocrine-sensitive ER+ BC on adjuvant endocrine therapy (ET) for at least 3 years or who responded to the most recent ET for metastatic disease for at least 6 months, CBR24 was 32% for those treated with enobosarm (9 mg daily), and 29% for those in the 18 mg cohort. Responses were observed in patients with AR+ BC (>10% nuclear AR staining)38. Another recent trial examined the efficacy and safety of enzalutamide with trastuzumab in patients with HER2+/AR+ locally advanced or metastatic BC in a single-arm phase II study in heavily pretreated patients with advanced HER2+ AR+ BC and observed a 24% CBR2439.Recently, a randomized phase II trial comparing the aromatase inhibitor exemestane alone (25 mg daily) to exemestane (50 mg daily) plus enzalutamide was completedPTEN or PIK3CA mutations, there were no clear indicators of response. Limitations include a heavily pretreated population with substantial heterogeneity of prior treatment. The sample size is limited; thus, all correlations are by nature exploratory. Future research must be conducted to evaluate the reproducibility of the results. Regarding the interpretation of the plasma metabolomics, we acknowledge that system-wide metabolomics analyses cannot distinguish between tumor versus organ response to drugs; however, the differences between the Short and Long PFS groups are intriguing. These analyses were performed at a steady state in vivo, and while more relevant than ex vivo studies, they are confounded by factors such as dietary or other physiological effects of the treatment, depending on patient-to-patient heterogeneity in response to the regimen.A major strength of NCT02953860 includes paired tumor biopsies pre-treatment and on-treatment to assess mutations, phospho-protein expression, and associations with PFS. We find that 7 (25%) of patients reached CBR24, including 2 who were progressing on prior fulvestrant, suggesting possible benefit for this subset. The study lends insight into the pathways that might be of importance regarding resistance, such as mTOR activation. Other than ER ≥ 10%, AR ≥ 10%, and the absence of 41. We recently completed accrual to a companion phase II trial (NCT02955394) of fulvestrant with or without enzalutamide in the neoadjuvant setting in women with T2 or greater ER+/HER2− BC, also with serial biopsies. The neoadjuvant trial will not have the confounding factor of the various prior treatments inherent to this metastatic trial. In conclusion, the present phase II study demonstrates that enzalutamide can be given safely in combination with fulvestrant for ER+ metastatic disease and that this combination warrants additional investigation. Future trials could be done in second-line endocrine therapy (progressing after AI and cdk4/6 inhibition) and in PI3K wild-type tumors to evaluate fulvestrant plus everolimus with or without an AR signaling blocker.If mTOR activation is confirmed as a potential mechanism of de novo resistance to enzalutamide, the addition of mTOR inhibitors to ER/AR blockade could be a feasible trial since combinations of enzalutamide and AR inhibitors with mTOR or PI3K inhibitors have proven clinical safetyNCT02953860 (COMIRB 16–1001) was an open-label, non-randomized trial combining fulvestrant plus enzalutamide. All patients received fulvestrant 500 mg IM on days 1, 15, 29, and then every 4 weeks plus enzalutamide 160 mg po daily until disease progression or unacceptable toxicity. Pre- or peri-menopausal women received concurrent ovarian suppression with a gonadotropin-releasing hormone agonist.The study was conducted at the University of Colorado and the University of Tennessee. The study protocol and its amendments were approved by the respective Institutional Review Boards, including the Colorado Multiple Institutional Review Board (COMIRB), the Office of Human Research Oversight (OHRO), and the University of Tennessee Institutional Review Board (UTHSC IRB). All patients provided written informed consent prior to participating in the study. The study was conducted under the principles of the World Medical Association, the Declaration of Helsinki, and the Good Clinical Practice guidelines of the International Conference on Harmonization. The study did not require an Investigational New Drug Application. Drug support  was provided by Astellas and Pfizer as part of this investigator-sponsored research study.Eligible patients were women ≥18 years of age with adequate organ and bone marrow function and an ECOG performance score (PS) of 2 or less. All had metastatic BC determined to be ER-positive and HER2-negative. Prior anti-androgen treatment was not allowed. Prior fulvestrant was allowed if the treating physician felt that retreatment with fulvestrant was clinically indicated. The measurable or evaluable disease was required. Men were excluded due to potential confounding from androgenic stimuli. Central nervous system (CNS) metastases or a history of seizures were exclusionary due to the toxicity profile of enzalutamide. Determination of AR expression was not a requirement as it was expected that ~90% of tumors would stain for AR, and the assay has not yet been validated for clinical decision-making. Concomitant medications with substantial pharmacokinetic (PK) interaction with enzalutamide were avoided.All patients who received at least one dose of enzalutamide were assessed for safety biweekly for the first 4 weeks, then every 4 weeks until 30 days after the last dose of enzalutamide or prior to the initiation of a new treatment, whichever occurred first. Safety and tolerability were determined by assessment of AEs, physical examinations, ECOG PS, vital signs, and laboratory tests. The severity of abnormal laboratory values and AEs were classified using the National Cancer Institute Common Terminology Criteria for AEs (CTCAE), version 4.03. SAEs were also evaluated by Astellas Pharma Global Development—United States. A monthly teleconference was held among the institutional investigators to review patients and AEs. An institutional Data Safety and Monitoring Committee at the University of Colorado also had oversight for monitoring.Radiographic assessments of disease status were performed at baseline and every 8 weeks thereafter. Tumor responses were defined using Response Evaluation Criteria in Solid Tumors (RECIST 1.1) version 1.1 criteria. Patients evaluable for response, PFS, and CBR had a follow-up scan or withdrew from the study because of toxicity or clinical progression.g), separated plasma was transferred to a 15 mL conical tube, centrifuged a second time for 15 min (1500×g), aliquoted into 500 µL aliquots, and stored at −80 °C until analysis.Fresh tumor biopsies (punch biopsies for skin lesions or core needle biopsies for other sites) were required at study entry (baseline), and after 4 weeks on therapy  and these were termed “WK5” biopsy. Archival tissue from the primary or prior biopsies of metastatic disease was obtained if available. A tumor biopsy at the time of progression was requested but is optional. Lithium-heparin (LiHep) plasma samples were obtained at the same intervals. After collection LiHep vacutainers were centrifuged for 20 min  were eligible and evaluable, and 28 (74%) were considered evaluable for the CBR endpoint because they had at least one post-baseline tumor assessment. The 4 patients who withdrew early did not withdraw due to disease progression or for reasons of toxicity. Three (9.3%) participants had a fresh biopsy that stained negative for both AR and ER; they were thus excluded from the AR:ER ratio analysis. All missing observations were eliminated from the respective univariate analyses . A Kaplan–Meier survival curve was used to determine the median time to progression.ESR1, and PIK3CA/PTEN mutation status, AR, ER, PR , and the AR:ER ratio (<2 vs. ≥2). Additionally, the following lines of treatment were assessed: adjuvant chemotherapy, neoadjuvant chemotherapy, adjuvant endocrine therapy, chemotherapy for metastatic disease, endocrine therapy for metastatic disease, prior fulvestrant, the number of prior agents, the number of hormonal prior agents, and the number of non-hormonal prior agents.To identify the demographic and clinical risk factors of progression, the univariate relationships between PFS and each of the following risk factors were assessed with Cox Proportional Hazard models (CoxPH): age, ECOG PS, metastatic sites, bone metastatic sites , baseline and change in IHC Ki67, p < 0.2) univariate associations with PFS were included in a multivariate CoxPH model. Analyses were performed using R version 4.0.2.To assess the multivariate associations between risk factors and PFS, prior fulvestrant and all factors that had moderately significant  sections were analyzed for mutations in ESR1 exon 8 and 67 other gene hotspots in genes frequently altered in cancer using a modified Archer VariantPlex Solid Tumor Assay through the CMOCO Laboratory . The majority (60%) of biopsies were from the liver. The other sites included lymph nodes (16%), other soft tissue (9%), bone (9%), skin (3%), and breast (3%). Patients’ original primary tumors consisted of invasive ductal carcinomas (63%), invasive lobular carcinoma (18.5%) and invasive mammary carcinoma (14.8%), and unknown (3.7%).Core needle biopsies were acquired from patients who gave informed written consent, with ER7. Briefly, biopsies from patients were formalin-fixed, paraffin-embedded, and 5 µm sections were heat immobilized onto charged slides. Slides were deparaffinized and rehydrated in a series of xylenes, and ethanols and antigens were heat retrieved using either citrate pH 6.0  or 10 mM Tris/1 mM EDTA pH 9.0 buffer  before probing with primary antibodies. Antibodies were detected using either the Vectastain Elite ABC Universal kit #PK-7200  or the ImmPRESS Goat Anti-Rabbit IgG Polymer kit #MP-7451  from Vector Laboratories  followed by DAB detection and counterstained with dilute hematoxylin.IHC was performed for ER , PR , AR , and GR  as well as Ki67  and cleaved caspase 3  as described previously42. Approximately 10,000 epithelial cells were captured for each sample. Microdissected material was lysed in extraction buffer composed of 1:1 Tissue Protein Extraction Reagent  and 2× SDS-PAGE Sample Buffer (ThermoFisher) plus 2.5% beta-mercaptoethanol (BME) at a concentration of approximately 500–600 cells per 1 µL Samples were heated at 100 °C for 5 min, briefly centrifuged and stored at −20 °C until printed. Lysates were printed in triplicate spots (approx. 10 nL per spot) onto nitrocellulose coated slides  using a Quanterix 2470 Arrayer . Standard curves of control cell lysates were also included for quality assurance purposes43. Antibodies used on the arrays were validated before use44. Immunostaining was performed as previously described45. Each slide was probed with one primary antibody targeting each of the 158 proteins of interest  using an Arcturus Pixcell IIe Laser Capture Microdissection system  as described2-transformed baseline abundance of each protein. An empirical Bayes method was employed to shrink sample variances toward a pooled estimate, allowing for a powerful and stable inference to detect significant differences in baseline expression between the Long PFS and Short PFS groups. The analysis was then repeated to compare differences in the change in abundance between baseline and the beginning of week 5 of treatment across PFS groups. Proteins in the mTOR pathway with significant baseline differences between “Long PFS” and “Short PFS” were identified and analyzed with previously described moderated t-tests to compare patients with PTEN and/or PIK3CA mutations (PTEN/PIK3CA mutants) to PTEN/PIK3CA wildtypes at baseline. Baseline and fold change associations between each RPPA phospho-protein, AR, and ER were then assessed. Each log2 autoscaled protein and AR were separately assessed using cell means linear regression models with baseline AR or ER as the outcome and the baseline protein of interest as the primary predictor. Each model was adjusted for post-treatment measurements. The association between the fold change of each protein and the fold change of AR or ER was similarly assessed, with the change in AR or ER as the outcome and the change in the protein of interest as the predictor.To assess differences in phospho-protein abundance in pre-treatment tumor biopsies from patients who experienced “Short PFS”  compared to those who experienced “Long PFS” (PFS > 24 weeks), robust moderated t-tests by response were performed on the log46, and proteins without annotations (18%) were eliminated. Candidate proteins were identified separately for each analysis  based on significance (p < 0.05). Gene Ontology (GO) enrichment analysis was then performed using the topGO R package47. Fisher’s exact tests with >2 proteins per node were used to test for enrichment.We then evaluated whether proteins associated with AR and ER at baseline and with the change with treatment were enriched relative to all analyzed proteins. Proteins were first annotated with the biomaRt R package48. Briefly, plasma (20 µl) was extracted in 980 μL of methanol:acetonitrile:water . After vortexing at 4 °C for 30 min, extracts were separated from the protein pellet by centrifugation for 10 min at 18,000 g at 4 °C and stored at −80 °C until analysis. Analyses were performed using a Vanquish UHPLC coupled online to a Q Exactive mass spectrometer . Samples were analyzed using a 5 min gradient as described50. Solvents were supplemented with 0.1% formic acid for positive mode runs and 1 mM ammonium acetate for negative mode runs. MS acquisition, data analysis, and elaboration were performed as described50.Plasma metabolomics analyses were performed via UHPLC-MS , as previously described51 by comparing autoscale normalized data for “Short PFS” and “Long PFS” groups at baseline and after treatment. In addition, Log2-transformed autoscaled metabolomic data were analyzed analogously to RPPA data to compare patients who experienced “Short PFS”  with those who experienced “Long PFS” (PFS > 24 weeks) with the change following treatment.Baseline and week 5 Metabolomics data analysis was performed via MetaboAnalyst 5.0Further information on research design is available in the Supplementary MaterialsReporting Summary