HETEROCYCLES

Paper | Special issue | Vol. 90, No. 1, 2015, pp. 563-578
Received, 30th June, 2014, Accepted, 30th July, 2014, Published online, 4th August, 2014.
DOI: 10.3987/COM-14-S(K)61

■ Synthesis of Model Compounds Related to Linear β-D-(1→6)-Galactosyl Side-Chains of Polysaccharides from Astragalus mongholicus Bunge

Noriyasu Hada, Ryo Shimura, Kyoko Hakamata, Hiroaki Kiyohara, Haruki Yamada, Tadahiro Takeda, and Fumiyuki Kiuchi*

Faculty of Pharmacy, Keio University, 1-5-30 Shiba-Kouen, Minato-ku, Tokyo, Japan

Abstract

Stereocontrolled efficient syntheses of β-(1→6)-linked di-, tetra- and octa-galactans as model compounds of arabino-3,6-galactans isolated from Astragalus mongholicus are described. The syntheses consisted of simple glycosylation cycles: an acceptor and a donor prepared from a common compound were coupled, and the glycosylation product was converted to the acceptor and donor of the next glycosylation.

INTRODUCTION
Many plants have long been used as traditional herbal medicines, and many pharmacological activities such as immuno-stimulating, anti-tumor, anti-virus, anti-oxidation, hypoglycemic, and anti-radiation activities have been observed for polysaccharides isolated from several medicinal plants.1 Among the pharmacological activities of plant polysaccharides, immuno-modulating activity seems to be most essential because of the accumulated evidence. Yamada et al.2 reported that several plant polysaccharides are involved in modulation of several immune systems, and affect the complement system, lymphocyte proliferation, antibody production, macrophage function, and intestinal immunity. Peyer’s patches are unique islands of lymphoid tissue in gut-associated lymphoid tissue (GALT), and known as an inductive site for the intestinal immune system. Regulative molecules of this organ have potential as new immuno-modulators of mucosal and systemic immune systems. Certain arabino-3,6-galactans isolated from an extract of the aerial part of Astragalus mongholicus Bunge were demonstrated to have potent enhancing activity on Peyer’s patch immunocompetent cells.2e Arabino-3,6-galactans generally consist of a backbone of (1→3)-linked β-D-galactopyranose (Galp) residues, some of which are substituted at 6-position with side chains such as (1→6)-linked β-D-Galp chain. The side chains are often branched or terminated with α-L-arabinofuranose or β-D-glucuronic acid. However, detailed understanding of the functions of such natural molecules is limited. Chemical synthesis has been expected to be an important tool to provide pure glycans, for elucidation of biological roles of glycans. As β-(1→6)-linked galactooligosaccharides are shown to play important role in the biological functions of arabino- 3,6-galactans from A. mongholicus, we systematically synthesized β-(1→6)-linked di-, tetra- and octa- galactooligosaccharides (A—C, Figure 1). Kong et al. reported earlier the synthesis of β-(16)-branched β-(13)-linked D-galactans found from the rhizome of Atractylodes lancea DC, but their products had 4-methoxyphenyl group at the reducing end.3 In this paper we synthesized free galactooligosaccharides of even number galactose units by an orthogonal synthesis.

RESULTS AND DISCUSSIONS
The synthetic routes for the oligosaccharide derivatives are outlined in Scheme 1. For the synthesis of these compounds, we adopted the synthetic method reported by Pathak et al.4 They synthesized linear α-(1→6)-linked octamannans using glycosyl fluoride donor and thiotolyl glycoside acceptor, which were prepared from a common intermediate. We selected monosaccharide glycosyl acceptor 15 as the first common derivative for the synthesis of the oligosaccharides A—C, and levulinoyl (Lev) group for the protection of 6-position in 1. The elongation of the carbohydrate chain consists of three reactions: glycosylation of acceptor with donor, conversion of the product to the next acceptor (deprotection of Lev group) and donor (displacement of the phenylthio group to fluorine atom6). For the β-glycosidic bond formation, we adopted O-benzoyl protecting group expecting the neighboring group participation of the 2-O-benzoyl group in the bond formation.7
The synthetic routes to the protected octasaccharide via the di- and tetra-saccharide derivatives are shown in Scheme 1. Compound 1 was levulinoylated with levulinic acid to give 2 and its phenylthio group was converted to fluoride by using N-bromosuccinimide (NBS) and diethylaminosulfur trifluoride (DAST) to afford 3 in nearly quantitative yield (α/β=56:44).6 Several conditions used for the glycosylation of 1 with 3 are given in Table 1. Glycosylation using AgOTf and SnCl28 gave no glycoside (entries 1 and 2). Combination of AgOTf and Cp2HfCl29 gave good results and the best result was obtained under the condition in which the reaction was heated to 60°C using toluene as a solvent (entry 5) to give desired disaccharide 4 in 94% yield. β-Configuration of the new glycosidic linkage was determined by the

coupling constant of the anomeric proton (H-1 of Gal, δ = 4.91 ppm, J = 7.9 Hz). Compound 4 was converted to 1-fluoro donor 5 in 95%. Selective removal of the Lev group of 4 with hydrazine acetate afforded disaccharide acceptor 6 in 97%. Coupling of 5 and 6 gave tetrasaccharide 7 which was then converted to corresponding donor 8 and acceptor 9, and coupled to give octasaccharide 10.
Next, removal of the protecting groups of the oligosaccharides 4, 7 and 10 were examined to obtain free oligosaccharides A—C. Deprotection of 4 was attempted through two routes: first deprotection of the phenylthio group at the anomeric position followed by deacylation (route A), and the other way round (route B). Unfortunately, neither route proceeded successfully (Scheme 2). Although the first deprotection steps were successful to give 11 and 12, respectively, 10 the second steps gave complicated mixtures in both routes. These results suggested that acidic or basic conditions should be avoided in the last step. Therefore, phenylthio group in the acceptor was converted to benzyl group, which can be removed by catalytic hydrogenation (route C). Although this needed more steps, it was possible to obtain compound A without by-products (Scheme 2).

Based on this result, conversions of the phenylthio group of 7 and 10 to benzyl group were carried out, but the yields were moderate (60% and 63%, data not shown). Furthermore, the yield of the glycosylation using glycosyl fluoride donor and thioglycosyl acceptor decreased markedly when the number of sugars increased (Scheme 1). Therefore, a benzyl glycosyl acceptor and a thioglycosyl donor were used in the following glycosylations.
The new synthetic routes for the oligosaccharide derivatives and the free oligosaccharides A—C are outlined in Schemes 3 and 4. Compound 4 was converted to benzyl glycoside 13 using benzyl alcohol in the presence of NIS and TfOH.11 The anomeric proton of the reducing end galactose unit appeared as a doublet (J = 7.9 Hz) at δ 4.62, indicating the formation of the β-linkage. Selective removal of the Lev group in 13 with hydrazine acetate in THF/MeOH gave disaccharide acceptor 14. Glycosylation of the disaccharide donor 4 with acceptor 14 in the presence of NIS, TfOH and MS 4Å in CH2Cl2 afforded desired tetrasaccharide 15 in 68% yield. The β-configuration of the new glycosidic linkage was determined by the coupling constant of the anomeric proton (δ = 4.56 ppm, J = 7.3 Hz). Removal of the Lev group in 15 with hydrazine acetate gave tetrasaccharide acceptor 16. Glycosylation of the tetrasaccharide donor 7 with acceptor 16 afforded desired octasaccharide 17 in 58% yield. Deacylation followed by removal of the benzyl protecting groups of 15 and 17 with catalytic hydrogenation over 10% Pd-C in MeOH-AcOH provided tetrasaccharide B and octasaccharide C, respectively, which were purified by gel filtration on Sephadex LH-20 (Schemes 3 and 4).

CONCLUSION
In conclusion, we achieved the synthesis of di-, tetra- and octa-galactans by a different method from Pathak's. We introduced benzyl group in place of the phenylthio group as a protecting group of the reducing end of the glycosyl acceptor at the stage of disaccharide and the resulted benzyl glycosides were used in the following steps, which enabled clean removal of the protecting groups in the final step to obtain free oligosaccharides. These oligosaccharides can be used as probes to elucidate the effects of arabino-3,6-galactans on immune systems.

EXPERIMENTAL
General
Optical rotations were measured with a Jasco P-1020 digital polarimeter. 1H and 13C NMR spectra were recorded with a Varian 500 FT NMR spectrometer. Me4Si and acetone were used as internal standards for CDCl3 and D2O, respectively. MALDI-TOFMS was recorded on an AB SCIEX Voyager RP mass spectrometer. High-resolution mass spectra were recorded on a JEOL JMS-700 under FAB conditions. TLC was performed on Silica Gel 60 F254 (E. Merck) with detection by quenching of UV fluorescence and by charring with 10% H2SO4. Column chromatography was performed on columns of silica gel 60 (Kanto Kagaku, 63-210 µm). Gel filtration was performed on Sephadex LH-20 (Pharmacia). Phenyl 2,3,4-tri-O-benzoyl-1-thio-β-D-galactopyranoside (1)5 was prepared as reported.

Phenyl 2, 3, 4-tri-O-benzoyl-6-O-levulinoyl-1-thio-β-D-galactopyranoside (2)
To a solution of 1 (359.5 mg, 0.61 mmol) in CH2Cl2 (2.0 mL) was added levulinic acid (68.6 μL, 0.68 mmol), DCC (228.4 mg, 1.11 mmol) and DMAP (19.2 mg, 0.16 mmol), and the reaction mixture was stirred for 3 h at 0 ºC. Toluene was added and evaporated. To a solution of this compound in pyridine was added benzoyl chloride, and the mixture was stirred for 5 h at room temperature. The reaction mixture was poured into ice-water and extracted with CHCl3. The extract was washed with 5% HCl, aq. NaHCO3 and water, dried (MgSO4), and concentrated. The product was purified by column chromatography using 20:1 toluene-acetone as eluent to give 2 (387.6 mg, 94.7%). [α]D25 +113.4 (c 0.9, CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 7.98—7.20 (20H, m, Ph), 5.89 (1H, d, J3, 4=3.7 Ｈｚ, H-4), 5.71 (1H, t, J1, 2= J2, 3=9.8 Hz, H-2), 5.56 (１H, dd, H-3), 5.01 (1H, d, H-1), 4.34 (1H, dd, J5, 6=6.1 Hz, H-6a), 4.27—4.19 (2H, m, H-5, H-6b), 2.73 (2H, t, J = 6.1 Hz, -COCH2CH2-), 2.58 (2H, t, -COCH2CH2-), 2.17 (3H, s, COCH3); 13C-NMR (125 MHz, CDCl3) δ: 206.3, 172.1, 165.4, 165.3, 165.1, 134.3, 133.5, 133.3, 133.2, 130.7, 129.9, 129.8, 129.7, 129.3, 128.8, 128.7, 128.5, 128.4, 128.2, 85.4 (C-1), 74.9, 73.0, 68.1, 67.7, 62.0, 37.8, 29.7, 27.8; HR-FABMS: Calcd for C38H34O10SNa: m/z 705.1770. Found: 705.1731 [M+Na]+.

2, 3, 4-Tri-O-benzoyl-6-O-levulinoyl-α,β-D-galactopyranosyl fluoride (3a, 3b)
To a solution of 2 (200 mg, 0.29 mmol) in CH2Cl2 (5.0 mL) was added DAST (115.3 μL, 0.44 mmol) at 0 ºC and the reaction mixture was stirred for 10 h at 40 ºC. DAST (57.6 μL, 0.22 mmol, 1.5 eq) was added and the mixture was stirred for 12 h at 40 ºC. Then, NBS (52.1 mg, 0.29 mmol, 1.0 eq) was added and stirring was continued for 4 h. The mixture was extracted with AcOEt, and the extract was washed with aq. NaHCO3, dried (MgSO4), and concentrated to give 3a (95.9 mg, 56%) and 3b (74.5 mg, 44%). 3a: [α]D25 +181.6 (c 1.2, CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 8.06—6.94 (15H, m, Ph), 6.09 (1H, dd, J1, 2=2.4 Hz, J1, F=53.1 Hz, H-1a), 6.01 (1H, d, J3, 4=3.1 Hz, H-4a), 5.95 (1H, dd, J2, 3=10.4 Hz, H-3a), 5.73 (1H, ddd, J2, F=23.8 Hz, H-2a), 4.68 (1H, t, J5, 6=6.7 Hz, H-5a), 4.35—4.24 (2H, m, H-6aa, H-6ba), 2.63 (2H, t, J = 6.7 Hz, -COCH2CH2-), 2.47 (2H, t, -COCH2CH2-), 2.05z (3H, s, COCH3); 13C-NMR (125 MHz, CDCl3) d: 206.2, 188.6, 172.1, 168.7, 165.8, 165.4, 165.3, 149.0, 142.2, 139.9, 133.71, 133.66, 133.3, 129.89, 129.86, 129.7, 129.0, 128.8, 128.7, 128.6, 128.5, 128.3, 127.0, 125.3, 104.6 (d, JC-1, F= 229.65 Hz, C-1α), 69.3, 68.5, 68.3, 67.8, 61.7, 46.6, 46.1, 37.8, 29.7, 27.8; HR-FABMS: Calcd for C32H29FO10Na: m/z 615.1642. Found: 615.1615 [M+Na]+. 3b: [α]D25 +154.6 (c 0.7, CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 7.98—7.14 (15H, m, Ph), 5.85 (1H, br-s, H-4β), 5.81—5.75 (1H, m, H-2β), 5.552 (1H, dd, J2, 3= 9.8 Hz, J3, 4= 3.1 Hz, H-3β), 5.545 (1H, dd, J1, 2= 6.7 Hz, J1, F= 51.9 Hz, H-1β), 4.33—4.26 (3H, m, H-5β, H-6aβ, H-6bβ), 2.63 (2H, t, J = 6.7 Hz, -COCH2CH2-), 2.47 (2H, t, -COCH2CH2-), 2.05 (3H, s, COCH3); 13C-NMR (125 MHz, CDCl3) δ: 206.3, 172.0, 165.2, 165.0, 144.2, 133.6, 133.4, 133.3, 130.4, 129.8, 129.7, 129.6, 128.7, 128.62, 128.56, 128.4, 128.3, 128.2, 126.2, 107.1 (d, JC-1, F=219 Hz, C-1β), 71.6, 70.6, 70.5, 69.8, 69.6, 67.2, 61.7, 37.7, 29.5, 27.7; HR-FABMS: Calcd for C32H29FO10Na: m/z 615.1642. Found: 615.1602 [M+Na]+.

Phenyl 2,3,4-tri-O-benzoyl-6-O-levulinoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-thio-β-D-galactopyranoside (4)
To a solution of 1 (124.0 mg, 0.21 mmol), 3 (138.3 mg, 0.23 mmol) in dry toluene (2.0 mL) was added, and the mixture was stirred with MS4Å (500 mg) for 5 h at room temperature. Cp2HfCl2 (132.9 mg, 0.35 mmol) and AgOTf (179.9 mg, 0.70 mmol) were added, and the mixture was stirred for 18 h at 60 °C, then neutralized with Et3N. The precipitates were filtered off and washed with CHCl3. The combined filtrate and washings were successively washed with water, dried (MgSO4), and concentrated. The product was purified by column chromatography (30:1 toluene-acetone) to give 4 (229 mg, 94.3%). [α]D25 +141.7 (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 8.09—7.17 (35H, m, Ph), 5.85 (1H, d, J3’, 4’=3.7 Hz, H-4’), 5.84 (1H, d, J3, 4=3.7 Hz, H-4), 5.78 (1H, dd, J1’, 2’=7.9 Hz, J2’, 3’=10.4 Hz, H-2’), 5.68 (1H, t, J1, 2= J2, 3 =9.8 Hz, H-2), 5.52 (1H, dd, H-3’), 5.48 (1H, dd, H-3), 4.93 (1H, d, H-1), 4.91 (1H, d, H-1’), 4.33 (1H, dd, J5, 6a=4.3 Hz, J5, 6b=7.3 Hz, H-5), 4.14 (3H, s, H-5’, H-6’a, H-6’b), 4.06 (1H, dd, J6a, 6b=11.6 Hz, H-6a), 3.98 (1H, dd, H-6b), 2.73 (2H, t, J = 6.1 Hz, -COCH2CH2-), 2.52 (2H, t, -COCH2CH2-), 2.14 (3H, s, COCH3); 13C-NMR (125 MHz, CDCl3) δ: 206.7, 172.3, 165.2, 133.8, 133.5, 133.4, 133.2, 131.3, 130.0, 129.9, 129.8, 128.8, 128.6, 128.5, 128.34, 128.25, 128.18, 101.6 (C-1’), 85.4 (C-1), 77.1, 73.0, 71.8, 71.2, 69.6, 68.7, 68.6, 68.0, 67.9, 61.8, 38.0, 29.6, 27.9; HR-FABMS: Calcd for C65H56O18SNa: m/z 1179.3085. Found: 1179.3125 [M+Na]+.

2,3,4-Tri-O-benzoyl-6-O-levulinoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-α,β-d-galactopyranosyl fluoride (5)
To a solution of 4 (356.3 mg, 0.31 mmol) in CH2Cl2 (5.0 mL) was added DAST (148.9 μL, 1.14 mmol) and NBS (54.8 mg, 0.31 mmol), and the reaction mixture was refluxed for 4 h at 45 ºC. DAST (57.6 μL, 0.22 mmol, 1.5 eq) was added and the mixture was stirred for 12 h at 40 ºC. The reaction mixture was poured into iced water and extracted with AcOEt, washed with aq. NaHCO3 and brine, dried (MgSO4), and concentrated. The product was purified by column chromatography using 20:1 toluene-acetone as eluent to give 5 (313.2 mg, 95.3%) as a mixture of α and β isomers. [α]D25 +171.0 (c 6.0, CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 8.08—7.13 (60H, m, Ph), 6.00 (2H, d, J3, 4=3.1 Hz, H-4α, H-4β), 5.94—5.89 (3H, m, H-3α, H-4’α, β), 5.92 (0.6H, dd, J1α, 2=2.4 Hz, J1α, F =53.1 Hz, H-1α), 5.85—5.74 (3H, m, H-2β, H-2’α, β), 5.66 (1H, ddd, J2, 3=10.4 Hz, J2, F=23.8 Hz, H-2α), 5.56—5.51 (3H, m, H-3β, H-3’α, β), 5.40 (0.4H, dd, J1, 2=7.3 Hz, J1, F=51.8 Hz, H-1β), 4.92 (2H, t, J1’, 2’=8.5 Hz, H-1’α, β), 4.72 (1H, t, J5, 6= 6.1 Hz, H-5α), 4.38—4.35 (1H, m, H-5β), 4.18—3.93 (8H, m, H-5’a, b, H-6aα, H-6bα, H-6’aα, H-6’bα, H-6aβ, H-6bβ, H-6’aβ, H-6’bβ), 2.70 (2H, t, J = 6.7 Hz, -COCH2CH2-), 2.49 (2H, t, -COCH2CH2-), 2.13 (3H, s, COCH3); 13C-NMR (125 MHz, CDCl3) δ: 206.5, 172.1, 165.8, 165.5, 165.2, 165.1, 137.8, 133.5, 133.3, 133.2, 133.1, 130.0, 129.9, 129.84, 139.79, 129.7, 129.3, 129.1, 129.0, 128.7, 128.59, 128.56, 128.41, 128.38, 128.31, 128.25, 128.2, 125.2, 107.2 (d, JC-1, F=219Hz, C-1β), 104.5 (d, JC-1, F= 230 Hz, C-1α), 101.9 (C-1’β), 101.56 (C-1’α), 73.7, 71.7, 71.2, 71.13, 71.11, 71.06, 71.04, 70.8, 70.7, 70.0, 69.8, 69.6, 69.51, 69.50, 68.64, 68.58, 68.5, 68.3, 68.0, 67.9, 67.84, 67.75, 67.71, 67.69, 61.8, 61.6. 37.9, 29.6, 27.8; HR-FABMS: Calcd for C59H51O18FNa: m/z 1089.2957. Found: 1089.2949 [M+Na]+.

Phenyl 2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-thio-β-D-galactopyranoside (6)
To a solution of 4 (370.6 mg, 0.32 mmol) in THF : MeOH = 10:1 (5.5 mL) was added hydrazine acetate (88.5 mg, 0.96 mmol), and the reaction mixture was stirred for 7 h at room temperature. The mixture was diluted with CHCl3, washed with, aq. NaHCO3 and brine, dried (MgSO4), and concentrated. The product was purified by column chromatography using 10:1 toluene-acetone as eluent to give 6 (327.7 mg, 96.6%). [α]D25 +163.3 (c 2.8, CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 8.13—7.17 (35H, m, Ph), 5.97 (1H, d, J3, 4=3.1 Hz, H-4), 5.82 (1H, dd, J1’, 2’= 7.9 Hz, J2’, 3’=10.4 Hz, H-2’), 5.79 (1H, d, J3’, 4’=3.1 Hz, H-4’), 5.67 (1H, t, J1, 2=J2, 3=9.8 Hz, H-2), 5.55 (1H, dd, H-3’), 5.50 (1H, dd, H-3), 4.87 (1H, d, H-1), 4.87 (1H, d, H-1’), 4.20 (1H, t, J5, 6a=5.5 Hz, H-5), 4.03 (1H, dd, J6a, 6b=11.0 Hz, H-6a), 3.97—3.93 (2H, m, H-6b, H-5’), 3.64 (1H, dd, J5’, 6’=6.7 Hz, J6a’, 6b’=12.2 Hz, H-6’a), 3.47 (1H, dd, H-6’b); 13C-NMR (125 MHz, CDCl3) δ: 166.6, 165.5, 165.44, 165.2, 165.0, 134.4, 133.5, 133.3, 133.2, 133.1, 130.7, 130.1, 129.8, 129.7, 129.3, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 101.4 (C-1), 85.3 (C-1’), 76.8, 74.2, 73.1, 71.8, 69.9, 68.8, 68.5, 67.9, 67.8, 60.6; HR-FABMS: Calcd for C60H50O16SNa: m/z 1081.2717. Found: 1081.2660 [M+Na]+.

Phenyl 2,3,4-tri-O-benzoyl-6-O-levulinoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-thio-β-D-galactopyranoside (7)
Compound 7 was prepared from 5 (130.5 mg, 0.12 mmol) and 6 (117.8 mg, 0.11 mmol) as described for preparation of 4. The product was purified by column chromatography (20:1 toluene-acetone) to give 7 (152.5 mg, 65.1%) as an amorphous powder. [α]D25 +78.3 (c 1.1 CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 8.13—7.06 (65H, m, Ph), 4.85 (1H, d, J1, 2=9.8 Hz, H-1a), 4.84 (1H, d, J1, 2=7.3 Hz, H-1d), 4.59 (1H, d, J1, 2=7.9 Hz, H-1b), 4.56 (1H, d, J1, 2=7.9 Hz, H-1c), 2.69 (2H, t, J = 6.7 Hz, -COCH2CH2-), 2.50-2.42 (2H, m, -COCH2CH2-), 2.15 (3H, s, COCH3); 13C-NMR (125 MHz, CDCl3) δ: 206.8, 172.1, 165.6, 165.4, 165.3, 165.2, 165.1, 164.9, 134.1, 133.3, 133.2, 133.1, 133.0, 130.9, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.3, 129.0, 128.8, 128.4, 128.3, 128.2, 128.1, 125.3, 101.2 (C-1c), 101.0 (C-1d), 100.9 (C-1b), 85.4 (C-1a), 76.6, 73.0, 12.3, 72.1, 71.9, 71.60, 71.59, 71.2, 69.98, 69.96, 69.84, 69.79, 68.5, 68.0, 67.8, 67.4, 67.3, 67.2, 66.7, 61.5, 38.0, 29.6, 27.9; MALDI-TOFMS: Calcd for C119H100NaO34S: m/z 2127.6. Found: m/z 2127.5 [M+Na]+.

2,3,4-Tri-O-benzoyl-6-O-levulinoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galacto-pyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-α,β-D-galactopyranosyl fluoride (8)
Compound 8 was prepared from 7 (145.8 mg, 69.2 μmol) as described for preparation of 5. The product was purified by column chromatography (1:1 hexane-EtOAc) to give an α, β mixture of 7 (122.9 mg, 88.0%) as syrup. [α]D25 +96.4 (c 3.1 CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 8.07—7.16 (60H, m, Ph), 5.74 (0.6H, dd, J1α, 2=3.1 Hz, J1α, F=51.3 Hz, H-1aα), 5.30 (0.4H, dd, J1β, 2=6.7 Hz, J1α, F=52.5 Hz, H-1aβ), 4.89 (1H, t, J1, 2=2.4 Hz, H-1d of Gal), 4.64—4.57 (3H, m, H-1 Galb, H-1c of Gal); 13C-NMR (125 MHz, CDCl3) δ: 215.4, 206.9, 198.9, 191.3, 185.1, 173.7, 165.8, 165.6, 165.4, 165.3, 165.2, 165.1, 165.0, 164.8, 160.1, 159.2, 133.6, 133.4, 133.3, 133.2, 133.1, 130.1, 130.0, 129.94, 129.89, 129.8, 129.7, 129.6, 129.5, 129.33, 129.27, 129.0, 128.9, 128.7, 128.5, 128.43, 128.36, 128.2, 128.1, 128.0, 126.9, 122.2, 108.5, 107.2 (d, JC-1β, F= 219 Hz, C-1aβ), 104.4 (d, JC-1α, F= 231 Hz, C-1aα), 101.4 (C-1b), 101.1 (C-1d), 100.9 (C-1c), 79.2, 73.23, 73.20, 72.4, 72.2, 72.1, 72.0, 71.8, 71.6, 71.4, 70.3, 70.2, 70.01, 69.98, 69.9, 69.83, 69.81, 69.8, 69.0, 68.8, 68.3, 68.1, 67.9, 67.8, 67.7, 67.5, 67.2, 67.1, 67.0, 66.3, 62.8, 60.1, 45.4, 38.1, 38.0, 29.6, 27.9, 21.3, 14.5; MALDI-TOFMS: Calcd for C113H95FNaO34: m/z 2037.6. Found: m/z 2037.6 [M+Na]+.

Phenyl 2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galacto-pyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-thio-β-D-galactopyranoside (9)
Compound 9 was prepared from 7 (120.7 mg, 57.3 μmol) as described for preparation of 6. The product was purified by column chromatography (10:1 toluene-acetone) to give 9 (106.9 mg, 92.9%) as syrup. [α]D25 +82.9 (c 2.7 CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 8.00—7.11 (65H, m, Ph), 4.85 (1H, d, J1, 2= 9.86 Hz, H-1a), 4.84 (1H, d, J1, 2= 7.3 Hz, H-1d), 4.59 (1H, d, J1, 2= 7.9 Hz, H-1b), 4.56 (1H, d, J1, 2= 7.9 Hz, H-1); 13C-NMR (125 MHz, CDCl3) δ: 166.5, 165.5, 165.4, 165.3, 165.11, 165.06, 164.9, 134.1, 133.5, 133.4, 133.2, 133.1, 130.9, 130.1, 129.8, 129.7, 129.64, 129.55, 129.3, 129.2, 129.0, 128.9, 128.8, 128.5, 128.4, 128.3, 128.2, 128.1, 100.99 (C-1c), 100.95 (C-1d), 100.7 (C-1b), 85.4 (C-1a), 76.7, 74.1, 73.0, 72.1, 72.0, 71.74, 71.72, 71.70, 71.6, 69.9, 69.78, 69.76, 68.7, 68.4, 67.9, 67.7, 67.23, 67.18, 66.50, 66.49, 66.1, 66.0, 60.68, 60.66; MALDI-TOFMS: Calcd for C114H94NaO32S: m/z 2029.5. Found: m/z 2028.9 [M+Na]+.

Phenyl 2,3,4-tri-O-benzoyl-6-O-levulinoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-thio-β-D-galactopyranoside (10)
Compound 10 was prepared from 8 (74.8 mg, 37.1 μmol) and 9 (67.7 mg, 33.8 μmol) as described for preparation of 4. The product was purified by Bio Beads S-X1 column chromatography (5:1 toluene-acetone) to give 10 (8.1 mg, 14.5 μmol, 43.1%) as an amorphous powder. [α]D25 －10.1 (c 0.9 CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 7.95—7.11 (125H, m, Ph), 4.76 (1H, d, J1, 2=9.8 Hz, H-1), 4.66 (1H, d, J1, 2=7.9 Hz, H-1), 4.39 (2H, d, J1, 2=7.3 Hz, H-1), 4.29 (2H, d, J1, 2=7.3 Hz, H-1), 4.26 (2H, d, J1,2 =7.9 Hz, H-1), 2.58 (2H, t, J = 6.7 Hz, -COCH2CH2-), 2.34-2.26 (2H, m, -COCH2CH2-), 2.05 (3H, s, COCH3); 13C-NMR (125 MHz, CDCl3) δ: 223.7, 206.6, 194.2, 172.1, 171.8, 165.6, 165.4, 165.2, 165.0, 164.9, 134.1, 133.4, 133.3, 133.1, 131.0, 130.0, 129.2, 129.8, 129.7, 129.4, 129.3, 129.22, 129.17, 129.1, 128.94, 128.8, 128.5, 128.4, 128.1, 101.1 (C-1), 101.0 (C-1), 100.8 (C-1), 100.71 (C-1), 100.69 (C-1), 100.66 (C-1), 100.64 (C-1), 85.4 (C-1), 73.0, 72.2, 72.1, 71.8, 71.7, 71.6, 71.0, 70.0, 69.8, 68.5, 67.9, 67.7, 67.6, 67.4, 67.2, 66.8, 65.9, 65.6, 61.3, 57.1, 44.5, 37.9, 31.9, 29.7, 29.3, 27.8; MALDI-TOFMS: Calcd for C227H118NaO66S: m/z 4024.1. Found: 4024.7 [M+Na]+.

2,3,4-Tri-O-benzoyl-6-O-levulinoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri–O-benzoyl-β-D-galacto-pyranose (11)
Entry 1: To a solution of 4 (100.0 mg, 86.4 μmol) in MeCN-H2O (10:1, 2.2 mL) was added NISac (106.8 mg, 0.34 mmol). The mixture was stirred for 3.5 h at room temperature. After completion of the reaction, the mixture was filtered and extracted with CHCl3. The solution was washed with water, dried (MgSO4), and concentrated. The residue was purified by column chromatography using 10:1 toluene-acetone as eluent to give 11 (72.1 mg, 78.3%) as syrup.
Entry 2: To a solution of 4 (100.0 mg, 86.4 μmol) in CH2Cl2 (1.0 mL) and H2O (3.1 μL, 0.17 mmol, 2.0 eq.) was added NIS (25.3 mg, 0.11 mmol, 1.3 eq) and TfOH (1.9 μL, 21.6 μmol, 0.25 eq) and the reaction mixture was refluxed for 18 h at 0 ºC, then neutralized with Et3N. The reaction mixture was filtered and extracted with CHCl3. The combined filtrate and washings were successively washed with saturated aqueous Na2S2O3 and water, dried (MgSO4), and concentrated. The residue was purified by silica gel column chromatography using 10:1 toluene-acetone as eluent to give 11 (77.6 mg, 84.3%) as syrup. HR-FABMS: Calcd for C59H52O19Na: m/z 1087.3000. Found: 1087.3031 [M+Na]+.

Phenyl β-D-galactopyranosyl-(1→6)-1-thio-β-D-galactopyranoside (12)
To a solution of 4 (203.4 mg, 0.18 mmol) in MeOH (2.0 mL) was added dioxane (2.0 mL) and NaOMe (20 mg) and the mixture was stirred for 18 h at 40 °C, then neutralized with Amberlite IR 120 [H+]. The mixture was filtered and concentrated, and the product was purified by a gel filtration with MeOH to give 12 as white solid (76.4 mg, quant.). HR-FABMS: Calcd for C18H26O10SNa: m/z 434.1247. Found: 434.1252 [M+Na]+.

Benzyl 2,3,4-tri-O-benzoyl-6-O-levulinoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranoside (13)
To a solution of 4 (298.5 mg, 0.26 mmol) and BnOH (66.5 μL, 0.64 mmol) in dry CH2Cl2 (2.0 mL) was added powdered MS4Å (600 mg), and the mixture was stirred under Ar atmosphere for 2 h at room temperature. NIS (145.1 mg, 0.64 mmol) and TfOH (18.4 μL, 0.21 mmol) were added to the mixture, which was stirred for 1 h at room temperature, then neutralized with Et3N. The precipitates were filtered off and washed with CHCl3. The combined filtrate and washings were successively washed with saturated aqueous Na2S2O3 and water, dried (MgSO4), and concentrated. The product was purified by column chromatography (30:1 toluene–acetone) to give 13 (258.1 mg, 86.0%) as an amorphous powder. [α]D25 +130.7 (c 4.2, CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 7.99—6.99 (35H, m, Ph), 5.77—5.68 (4H, m, H-4, H-4’, H-2, H-2’), 5.49 (1H, dd, J2’, 3’=10.4 Hz, J3’, 4’=3.1 Hz, H-3’), 5.36 (1H, dd, J2, 3’=9.4 Hz, J3, 4=3.7 Hz, H-3), 4.82 (1H, d, J1’, 2’=7.9 Hz, H-1’), 4.62 (1H, d, J1, 2=7.9 Hz, H-1), 4.54 and 4.35 (2H, each d, Jgem=12.2 Hz, benzyl methylene), 4.15—4.05 (5H, m, H-6a, b, H-5, H-6’a, H-5’), 3.84 (1H, dd, J5’, 6’=7.3 Hz, 9.8 Hz, H-6’b), 2.60 (2H, t, J = 6.7 Hz, -COCH2CH2-), 2.45 (2H, m, -COCH2CH2-), 2.15 (3H, s, COCH3); 13C-NMR (125 MHz, CDCl3) δ: 206.3, 172.1, 165.43, 165.36, 135.3, 135.1, 136.5, 133.5, 133.4, 133.1, 133.0, 130.1, 129.9, 129.6, 129.3, 129.2, 129.0, 128.9, 128.8, 128.7, 128.5, 128.4, 128.3, 128.2, 128.1, 127.8, 127.7, 101.3 (C-1), 99.2 (C-1’), 73.1, 71.7, 71.6, 71.1, 70.0, 69.7, 68.8, 68.5, 67.9, 61.6, 38.6, 37.8, 30.3, 29.5, 28.8; HR-FABMS: Calcd for C66H58NaO19: m/z 1177.3470. Found: 1177.3446 [M+Na]+.

Benzyl 2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranoside (14)
Compound 14 was prepared from 13 (159.0 mg, 0.14 mmol) as described for preparation of 6. The product was purified by column chromatography (25:1 toluene-acetone) to give 14 (136.2 mg, 94%) as syrup. [α]D25 +152.6 (c 1.6, CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 8.13—7.09 (35H, m, Ph), 5.93 (1H, d, J3, 4=3.4 Hz, H-4), 5.87 (1H, dd, J1’, 2’=7.9 Hz, J2’, 3’=10.3 Hz, H-2’), 5.83 (1H, d, J3’, 4’=3.2 Hz, H-4’), 5.67 (1H, dd, J1, 2=8.0 Hz, J2, 3=10.3 Hz, H-2), 5.60 (1H, dd, J2’, 3’=10.5 Hz, J3’, 4’=3.4 Hz, H-3’), 5.47 (1H, dd, J2, 3=10.3 Hz, J3, 4=3.4 Hz, H-3), 4.89 (1H, d, J1’, 2’=7.9 Hz, H-1’), 4.69 (1H, d, J1, 2=8.0 Hz, H-1), 4.65 and 4.45 (2H, each d, Jgem=12.2 Hz, benzyl methylene), 4.19—4.14 (2H, m, H-6a, H-5), 3.99 (1H, t, J5’, 6’a= J5’, 6’ｂ=6.5 Hz, H-5’), 3.91 (1H, dd, J5, 6ｂ=5.6 Hz, J6a, 6b=9.0 Hz, H-6b), 3.68 (1H, dd, J5’, 6’ｂ=6.6 Hz, J6’a, 6’b=11.9 Hz, H-6’a), 3.52 (1H, dd, J5’, 6’b=6.5 Hz, J6a’, 6b’=12.0 Hz, H-6’b); 13C-NMR (125 MHz, CDCl3) δ: 166.5, 165.5, 165.43, 165.38, 165.2, 165.1, 136.5, 133.6, 133.5, 133.2, 133.1, 130.0, 129.9, 129.8, 129.68, 129.66, 129.63, 129.61, 129.5, 129.2, 129.0, 128.7, 128.6, 128.5, 128.33, 128.29, 128.26, 128.19, 128.17, 128.1, 127.8, 127.7, 127.6, 101.2 (C-1’), 99.3 (C-1), 74.0 (C-5’), 73.0 (C-5), 71.73 (C-3), 71.71 (C-3’), 70.1 (benzyl methylene), 69.9 (C-2’), 69.7 (C-2), 68.8 (C-4’), 68.6 (C-4), 68.2 (C-6), 60.5 (C-6’); HR-FABMS: Calcd for C61H52NaO17: m/z 1079.3102. Found: 1079.3133[M+Na]+.

β-D-Galactopyranosyl-(1→6)-β-D-galactopyranose (A)
To a solution of 13 (75.5 mg, 65.4 μmol) in dioxane (1.0 mL) was added MeOH (1.0 mL) and NaOMe (10 mg) at 40 °C. The mixture was stirred for 21 h and then neutralized with Amberlite IR 120 [H+]. The mixture was filtered and concentrated. The residue in MeOH (1.0 mL) was hydrogenolysed in the presence of Pd/C (100 mg) for 5 h at room temperature. The mixture was filtered and concentrated, and the residue was purified by gel filtration in 1:1 MeOH-H2O to give A as white solid (15.9 mg, 71.0%). [α]D25 6.9 (c 0.3, H2O); 1H-NMR (500 MHz, D2O) δ: 5.08 (0.4H, br-s, H-1α), 4.41 (0.6H, d, J1, 2=7.9 Hz, H-1β), 4.27 (1H, d, J1, 2=7.3 Hz, H-1’); 13C-NMR (125 MHz, D2O) δ: 102.9 (C-1’β), 102.7 (C-1’α), 96.0 (C-1β), 91.9 (C-1α), 74.7, 73.4, 72.4, 72.23, 72.19, 71.6, 70.8, 70.43, 70.35, 69.7, 69.3, 69.0, 68.9, 68.8, 68.7, 68.5, 68.4, 68.21, 68.18, 68.1, 67.8, 62.8, 62.1, 60.6; HR-FABMS: Calcd for C12H22NaO11: m/z 365.1060. Found: 365.1051 [M+Na]+.

Benzyl 2,3,4-tri-O-benzoyl-6-O-levulinoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranoside (15)
To a solution of 14 (192.9 mg, 0.18 mmol) and 4 (253.4 mg, 0.22 mmol) in dry CH2Cl2 (2.0 mL) was added powdered MS4Å (600 mg), and the mixture was stirred under Ar atmosphere for 2 h at room temperature. NIS (98.5 mg, 0.44 mmol) and TfOH (3.9 μL, 0.18 mmol) were added to the mixture, which was stirred for 1 h at 0 ºC, then neutralized with Et3N. The precipitates were filtered off and washed with CHCl3. The combined filtrate and washings were successively washed with saturated aqueous Na2S2O3 and water, dried (MgSO4), and concentrated. The product was purified by column chromatography (25:1 toluene–acetone) to give 15 (255.7 mg, 67.5%) as an amorphous powder. [α]D25 +88.6 (c 2.1 CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 7.95-6.97 (65H, m, Ph), 4.77 (1H, d, J1, 2=7.9 Hz, H-1 d), 4.58 (1H, d, J1, 2=7.9 Hz, H-1b), 4.56 (1H, d, J1, 2=7.3 Hz, H-1c), 4.49 and 4.29 (2H, each d, Jgem=12.5 Hz, benzyl methylene), 4.29 (1H, d, J1, 2=7.9 Hz, H-1a); 13C-NMR (125 MHz, CDCl3) δ: 206.7, 172.1, 165.6, 165.4, 165.34, 165.31, 165.28, 135.23, 165.18, 165.0, 164.9, 140.7, 136.6, 133.31, 133.25, 133.2, 133.13, 133.07, 133.02, 132.97, 129.99, 122.96, 129.73, 129.69, 129.6, 129.5, 129.41, 129.38, 129.3, 129.2, 129.0, 128.94, 128.85, 128.7, 128.4, 128.3, 128.21, 128.18, 128.10, 128.10, 127.8, 127.7, 101.3 (C-1c), 101.1 (C-1d), 100.7 (C-1b), 99.4 (C-1a), 72.8, 72.3, 72.1, 71.8, 71.72, 71.65, 71.6, 71.2, 70.07, 69.96, 69.9, 69.83, 69.76, 68.6, 68.0, 67.9, 67.8, 67.5, 66.5, 61.5, 38.0, 31.9, 29.6, 29.3, 27.9; MALDI-TOFMS: m/z 2125.9 [M+Na]+ (C120H102NaO35).

Benzyl 2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranoside (16)
Compound 16 was prepared from 15 (283.6 mg, 0.13 mmol) as described for preparation of 6. The product was purified by column chromatography (10:1 toluene-acetone) to give 16 (208.5 mg, 77%) as syrup. [α]D25 +88.9 (c 7.1, CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 8.10—7.07 (65H, m, Ph), 4.79 (1H, d, J=7.8 Hz, H-1), 4.66 (1H, d, J1, 2=8.0 Hz, H-1a), 4.59 and 4.40 (2H, each d, Jgem= 12.6 Hz, benzyl methylene), 4.57 (1H, d, J=7.8 Hz, H-1), 4.47 (1H, d, J=7.8 Hz, H-1); 13C-NMR (125 MHz, CDCl3) δ: 166.4, 165.4, 165.35, 165.30, 165.27, 165.23, 165.15, 165.09, 165.0, 164.89, 164.86, 136.5, 133.5, 133.33, 133.28, 133.2, 133.1, 130.0, 129.9, 129.7, 129.6, 129.5, 129.22, 129.18, 129.1, 128.9, 128.84, 128.79, 128.7, 128.6, 128.52, 128.48, 128.4, 128.33, 128.30, 128.2, 128.1, 127.8, 127.7, 125.2, 101.0 (C-1c), 100.9 (C-1d), 100.5 (C-1b), 99.2 (C-1a), 74.0, 72.7, 72.0, 71.9, 71.6, 70.0 (benzyl methylene), 69.8, 69.6, 68.6, 68.5, 67.7, 67.6, 66.5, 65.8, 60.6; MALDI-TOFMS: m/z 2027.7 [M+Na]+ (C115H96NaO33).

Benzyl 2,3,4-tri-O-benzoyl-6-O-levulinoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranoside (17)
Compound 17 was prepared from 16 (200.0 mg, 0.10 mmol) and 7 (252.0 mg, 0.12 mmol) as described for preparation of 15. The product was purified by ULTRA PACK SI-40B column chromatography (15:1 toluene–acetone) to give 17 (230.9 mg, 58%) as an amorphous powder. [α]D25 +59.5 (c 0.6 CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 8.02—7.06 (125H, m, Ph), 4.74 (1H, d, J1, 2=8.0 Hz, H-1), 4.62 (1H, d, J1, 2=8.0 Hz, H-1 of Gal a), 4.58 (1H, d, benzyl methylene), 4.53 (1H, d, J1, 2=8.0 Hz, H-1), 4.48 (1H, d, J1, 2=8.0 Hz, H-1), 4.39—4.33 (5H, m, H-1, benzyl methylene, H-1, H-1, H-1); 13C-NMR (125 MHz, CDCl3) δ: 206.7, 172.1, 165.6, 165.5, 165.42, 165.39, 165.37, 165.34, 165.3, 165.22, 165.17, 165.1, 164.98, 164.95, 164.9, 164.8, 136.6, 133.39, 133.35, 133.2, 133.1, 133.0, 130.14, 130.08, 130.04, 130.01, 129.98, 129.8, 129.7, 129.6, 129.5, 129.44, 129.40, 129.36, 129.3, 129.2, 129.07, 129.05, 128.9, 128.83, 128.77, 128.51, 128.47, 128.39, 128.39, 128.36, 128.24, 128.20, 128.17, 128.1, 127.8, 127.7, 101.2 (C-1), 101.0 (C-1), 100.7 (C-1x3), 100.63 (C-1), 100.62 (C-1), 99.3 (C-1 of Gal a), 72.8, 72.0, 71.9, 71.75, 71.69, 71.6, 71.0, 70.1 (benzyl methylene), 70.0, 69.9, 69.8, 69.7, 68.6, 67.8, 67.7, 67.6, 67.4, 61.3, 37.9, 29.7, 27.8; MALDI-TOFMS: m/z 4022.2 [M+Na]+ (C228H190NaO67).

β-D-Galactopyranosyl-(1→6)-β-D-galactopyranosyl-(1→6)-β-D-galactopyranosyl-(1→6)-β-D-galactopyranose (B)
Compound B was prepared from 15 (55.9 mg, 26.6 μmol) as described for preparation of A. The product was purified by gel filtration (1:1 MeOH-H2O) to give B (16.4 mg, 92.6%) as syrup. [α]D25 6.9 (c 0.3, H2O); 1H-NMR (500 MHz, D2O) δ: 5.08 (0.4H, d, J1α, 2=2.4 Hz, H-1aα), 4.41 (0.6H, d, J1β, 2=7.9 Hz, H-1bβ), 4.28—4.26 (3H, m, H-1bα, H-1cα, H-1dα, H-1bβ, H-1cβ, H-1dβ); 13C-NMR (125 MHz, D2O) δ: 102.9 (C-1), 102.8 (C-1), 102.7 (C-1), 96.0 (C-1), 74.7, 73.3, 72.3, 72.2, 72.1, 71.4, 70.3, 70.2, 69.1, 68.9, 68.84, 68.77, 68.5, 68.4, 68.2, 67.8, 60.6, 48.4; HR-FABMS: Calcd for C24H42NaO21: m/z 689.2116. Found: 689.2137 [M+Na]+.

β-D-Galactopyranosyl-(1→6)-β-D-galactopyranosyl-(1→6)-β-D-galactopyranosyl-(1→6)-β-D-galactopyranosyl-(1→6)-β-D-galactopyranosyl-(1→6)-β-D-galactopyranosyl-(1→6)-β-D-galactopyranosyl-(1→6)-D-galactopyranose (C)
Compound C was prepared from 17 (85.0 mg, 21.2 μmol) as described for preparation of A. The product was purified by gel filtration (1:1 MeOH-H2O) to give C (14.8 mg, 53.3%) as syrup. [α]D25 +2.7 (c 0.2, H2O); 1H-NMR (500 MHz, D2O) δ: 5.08 (0.25H, d, J1α, 2=3.6 Hz H-1aα), 4.41 (0.75H, d, J1, 2=8.0 Hz, H-1aβ), 4.31—4.26 (7H, m, H-1b-h); 13C-NMR (125 MHz, D2O) δ: 102.98 (C-1x2), 102.96 (C-1x2), 102.9 (C-1), 102.8 (C-1), 102.7 (C-1), 96.0 (C-1 β of Gal a), 91.9 (C-1 α of Gal a), 74.7, 73.3, 72.3, 72.2, 72.1, 71.4, 71.3, 70.3, 69.0, 68.9, 68.8, 68.5, 68.3, 68.2, 67.8, 60.6, 60.0; MALDI-TOFMS: m/z 1337.9. [M+Na]+ (C48H82NaO41).

ACKNOWLEDGEMENTS
This work was supported by a Grant-in-Aid for Scientific Research (No. 25460131) and by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). The authors are grateful to Ms. J. Hada for measurements of HR-FABMS data.

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