Synthesis of the Non Reducing End Oligosaccharides of Glycosphingolipids from Ascaris suum

Abstract

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the non reducing end oligosaccharides of glycosphingolipids from Ascaris suum have been accomplished. Galα1→3GalNAcβ1→OR (1), Galβ1→3Galα1→3GalNAcβ1→OR (2), Galβ1→6Galα1→3GalNAcβ1→OR (3), Galβ1→6(Galβ1→3)Galα1→3GalNAcβ1→OR (4) and GlcNAcβ1→6Galβ1→6(Galβ1→3)Galα1→3GalNAcβ1→OR (5) (R = biotinylated probe) were synthesized by stepwise condensation (1–4) and block synthesis (5) using 5-(methoxycarbonylpentyl) 2-O-benzoyl-3-O-2-napthylmethyl-4,6-O-di-tert-butylsilylene-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (12) as a common precursor. Compound 12 was converted into two kinds of glycosyl acceptors and was condensed with suitable galactosyl donors, respectively.

Introduction

Over the past years, we have been studying the structure activity relationships of glycosphingolipids from invertebrates animal species and have synthesized oligosaccharides from various Protostomia phyla.^1–10) Geyer et al. identified glycosphingolipids having arthro-series carbohydrate structure (GlcNAcβ1→3Manβ1→4Glcβ1→) from a parasitic nematode Ascaris suum.¹¹⁾ In our previous paper,¹²⁾ we reported the total synthesis of two glycosphingolipids, GlcNAcβ1→3Manβ1→4Glcβ1→Cer and GalNAcβ1→4GlcNAcβ1→3Manβ1→4Glcβ1→Cer. Moreover, we also synthesized arthro-type trisaccharides bearing a phosphocholine (PC) related structure in order to investigate the immunomodulatory functions of zwitterionic oligosaccharides.¹³⁾

In the course of our studies on novel glycoconjugates found in parasites, we have synthesized various glycosphingolipids and carbohydrate portions of glycoproteins with the aim of elucidating the mechanisms of host–parasite interactions.^7–10) In recent studies we have synthesized unusual carbohydrates from the parasites Echinococcus multilocularis and Toxocara canis and researched their antigenicity in infected patient sera.^8,9,14,15) In this paper we describe the synthesis of the carbohydrate portion of nonreducing end oligosaccharides from A. suum.

Although it is possible to diagnose larva migrans caused by A. suum and Toxocara canis by serologic test with protein antigen, it is difficult to distinguish the causative helminthes by the method. Hiromatsu and colleagues found that combined glycosphingolipids from A. suum exhibited immunoglobulin G (IgG) immunological response toward the serum of A. suum infected mice, whereas they did not react with that from T. canis infected mice.¹⁶⁾ This finding indicated that glycosphingolipids from A. suum can be used to distinguish larva migrans caused by A. suum and by T. canis. Based on this information we synthesized non-reducing end fragments of novel carbohydrate structures A–E from A. suum previously reported by Geyer and colleagues,¹⁷⁾i.e. biotin-tagged oligosaccharides (1–5) (Figs. 1, 2).

Fig. 1. Some of Structures Found from Ascaris suum

Fig. 2. Target Oligosaccharides 1–5

Results and Discussion

We developed a synthetic strategy for the synthesis of target oligosaccharides 1–5 (Fig. 2) on Chart 1. Compounds 12, 18, 22, 27 and 35 were selected as precursors of target compounds 1–5. Intermediates 18, 22, 27 and 35 are derived from common intermediate 12. In all cases the 5-(methoxycarbonyl)pentyl group was selected at the reducing end, because this group can be conveniently used for conjugation with biotin for use in enzyme-linked immunosorbent assay (ELISA) as previously described by us.^8,9) The galactosyl imidate donor 17 was selected to react with acceptors 13, 24 and 34 while the thiophenyl donor 21 was selected for the reaction with acceptor 14 as the glycosylation of 14 with 17 resulted in poor yield. The synthetic routes for target compounds 1–5 are outlined in Charts 2–8.

Chart 1. Synthetic Strategy

Chart 2. Preparation of Monosaccharide Acceptor 9 and Donor 11

Chart 3. Synthesis of Disaccharide 1

Chart 4. Synthesis of Trisaccharide 2

Chart 5. Synthesis of Trisaccharide 3

Chart 6. Synthesis of Tetrasaccharide 4

Chart 7. Synthesis of Tetrasaccharide Acceptor 34

Chart 8. Synthesis of Pentasaccharide 5

Initially, monosaccharide acceptor 9 was prepared as follows: Glycosylation of imidate donor 6¹⁸⁾ with alcohol 7¹⁹⁾ in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf)²⁰⁾ and 4 Å molecular sieves (MS-4 Å) in CH₂Cl₂ afforded desired monosaccharide 8 in 88% yield. Zemplén deacetylation followed by protection of the 4- and 6-hydroxy groups as a benzylidine group provided 9. Next, to prepare the Galα1-2GalN sequence with high α-stereoselectivity, we selected galactopyranosyl donor 11. Regioselective introduction²¹⁾ of the naphthylmethyl (NAP) group at the 3-position of 4,6-O-di-tert-butylsilylene-1-thio-β-D-galactopyranoside (10),²²⁾ mediated by Bu₂SnO in the presence of tetra-n-butylammonium bromide (TBAB) and naphthylmethyl bromide in dry toluene, followed by benzoylation afforded thioglycoside donor 11 (Chart 2).

Glycosylation of acceptor 11 with donor 9 in the presence of N-iodosuccinimide (NIS)/trifluoromethanesulfonic acid (TfOH)²³⁾ and MS AW-300 in CH₂Cl₂ afforded desired disaccharide (12) in α-selective and 80% yield. The nature of the new glycosidic linkage was determined by the vicinal coupling constant of the anomeric proton (H-1′ of Gal, δ = 5.50 ppm, J = 3.7 Hz). Disacccharide 12 was deprotected using two different conditions to afford partially deprotected compounds 13 and 14. Selective removal of the 3′-O-NAP group from 12 by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone removal of the di-tert-butylsilylene (DTBS) group in 13 was achieved with tetra-n-butylammonium fluoride (TBAF) and benzylidene acetal was removed by acidic hydrolysis. Next, the phthalimido-group was converted to an acetamido moiety by ethylenediamine in the presence of BuOH and acetylation afforded 15 in 28% yield (4 steps). After deacylation by Zemplén-based method, the 5-(methoxycarbonyl)pentyl glycoside 16 was converted into an ethylenediamine monoamide²⁴⁾ by exposure to ethylenediamine followed by conjugation to N-hydroxy succinimide ester of biotin (biotin-NHS) to afford target biotinylated disaccharide 1 after column chromatographic purification on Sephadex LH-20 (Chart 3). On the other hand, 4,6-diol disaccharide acceptor 14 was obtained by removal of the DTBS group using TBAF.

The synthesis of the trisaccharide 2 is outlined in Chart 4. Glycosylation of acceptor alcohol 13 with the trichloroacetimidate donor 17 was carried out in the presence of TMSOTf and MS 4 Å in CH₂Cl₂ to afford desired β-glycoside 18 in 74% yield as a single anomer. The nature of the new glycosidic linkage was determined by the vicinal coupling constant of the anomeric proton (H-1″ of Gal, δ = 4.68 ppm, J = 7.9 Hz). Deprotection and biotinylation were performed as described for compound 1 to provide target trisaccharide 2 (Chart 4).

Glycosylation of thioglycoside donor 21²⁵⁾ with the 4,6-diol acceptor 14 in the presence of NIS, TfOH and MS AW-300 in CH₂Cl₂ afforded desired trisaccharide 22 in 86% yield. The nature of the new glycosidic linkage was determined from the coupling constant of the anomeric proton (H-1″ of Gal, δ: 4.80 ppm, J = 7.9 Hz). ¹³C-NMR data of 22 showed that the C-6 signal of the inner galactose residue was shifted down field from 62.2 ppm (C-6′, 14) to 67.2 ppm (C-6′, 22), suggesting that the new galactose residue was linked to C-6 according to the glycosylation shift rule. Compound 22 was converted into 24 by benzoylation (23) followed by removal of NAP group with DDQ in order to prepare to the acceptor for the synthesis of tetrasaccharide 4. On the other hand, conventional removal of the protecting groups from 24 afforded the unprotected oligosaccharide 26. Finally, 5-(methoxycarbonyl)pentyl glycoside 26 was converted into the ethylenediamine monoamide by exposure to ethylenediamine and conjugated to biotin to afford tetrasaccharide–biotin conjugate 3 in 43% yield (Chart 5).

Glycosylation of the common imidate donor 17 with the trisaccharide acceptor 24 was carried out in the presence of TMSOTf and MS AW-300 in CH₂Cl₂ to afford desired tetrasaccharide 27 in 63% yield as a single anomer. The nature of the new β-glycosidic linkage was determined from the coupling constant of the anomeric proton (H-1 of Galc, δ: 4.76 ppm, J = 7.9 Hz). Deprotection and biotinylation were performed as described for compound 3 to provide target tetrasaccharide 4 (Chart 6).

The synthesis of the pentasaccharide 5 is outlined in Charts 7 and 8. Branched pentasaccharide 5 was synthesized by glycosylation of the previously mentioned disaccharide accepter 14 with the disaccharide thioglycoside donor 32. Donor 32 was prepared by glycosylation of known imidate donor 30²⁶⁾ with known acceptor alcohol 31²⁷⁾ using TMSOTf as a promotor in CH₂Cl₂. Glycosylation of 14 with 32 in the presence of NIS/TfOH and MS AW-300 in CH₂Cl₂ afforded desired tetrasaccharide (33) in 73% yield. The presence of the β-galactosidic linkage in 33 was indicated by a doublet at δ 4.56 ppm showing homonuclear coupling constant of 7.9 Hz in the ¹H-NMR spectrum. Compound 33 was converted into 34 by benzoylation and removal of NAP group. Subsequently, tetrasaccharide acceptor 34 was converted into pentasaccharide 35 by TMSOTf-promoted glycosylation with imidate donor 17 in 45% yield. Deacylation and biotinylation were performed as described for compound 3 to give target pentasaccharide 5. The yield was not satisfactory in some case of deprotections, especially hydrazinolysis of phthalimido group. It was supposed that the ethylenediamine monoamide from the methyl ester of the aglycon was formed.

Conclusion

In summary, a systematic and integrated approach for the first synthesis of novel five oligosaccharides 1–5 found in the parasite, Ascaris suum was developed. It is expected that these compounds will find use in future studies designed to distinguish larva migrans caused by A. suum and by T. canis.

Experimental

General

Optical rotations were measured with a Jasco P-1020 digital polarimeter (Tokyo, Japan). ¹H- (500 MHz) and ¹³C-NMR (125 MHz) spectra were recorded with a Varian 500 FT NMR spectrometer (CA, U.S.A.). Me₄Si and acetone were used as internal standards for CDCl₃ and D₂O, respectively. Electrospray ionization-high resolution (ESI-HR)-MS was recorded on a JEOL MS T-100 mass spectrometer. Matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF)-MS was recorded on an AB SCIEX Voyager RP mass spectrometer (MS, U.S.A.). TLC was performed on Silica Gel 60 F254 (E. Merck, Darmstadt, Germany) with detection by quenching of UV fluorescence and by charring with 10% H₂SO₄. Column chromatography was carried out on Silica Gel 60. 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-D-galactopyranosyl trichloroacetimidate (6),¹⁸⁾ methyl 6-hydroxyhexanoate (7),¹⁹⁾ phenyl 4,6-O-di-tert-butylsilylene-1-thio-β-D-galactopyranoside (10),²²⁾ phenyl 2,3,4,6-tetra-O-benzoyl-1-thio-β-D-galactopyranoside (21),²⁵⁾ 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-D-glucopyranosyl trichloroacetimidate (30),²⁶⁾ phenyl 2,3,4-tri-O-benzoyl-1-thio-β-D-galactopyranoside (31)²⁷⁾ were prepared as reported.

5-(Methoxycarbonyl)pentyl 3,4,6-Tri-O-acethyl-2-deoxy-2-phthalimido-β-D-galactopyranoside (8)

A mixture of 6 (1.84 g, 3.17 mmol), 7 (0.71 g, 4.86 mmol) and MS 4 Å (500 mg) in dry CH₂Cl₂ (20 mL) was stirred for 2 h at room temperature, then cooled to −40°C. TMSOTf (58.7 µL, 0.32 mmol) was added, and the mixture was stirred for 2 h at −40°C, then neutralized with Et₃N. The precipitates were filtrated off and washed with CHCl₃. The combined filtrate and washings were washed with water, dried (MgSO₄), and concentrated. The product was purified by silica gel column chromatography (4 : 1 toluene–EtOAc) to give 8 (1.57 g, 88%). [α]_D²⁴ −4.4 (c = 2.5, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 7.86–7.72 (4H, m, Phth), 5.79 (1H, dd, J_2,3 = 11.5 Hz, H-3), 5.48 (1H, br s, H-4), 5.29 (1H, d, J_1,2 = 8.4 Hz, H-1), 4.54 (1H, dd, H-2), 4.25–4.15 (2H, m, H-6a, 6b), 4.07 (1H, br s, H-5), 3.87–3.82 (1H, m, –OCH₂–), 3.63 (3H, s, OCH₃), 3.45–3.40 (1H, m, –OCH₂–), 2.05–1.95 (2H, m, –CH₂COOMe), 1.50–1.36 (4H, m, –OCH₂CH₂CH₂CH₂–), 1.17–1.08 (2H, m, –OCH₂CH₂CH₂–), 2.20, 2.07, 1.86 (9H, each s, 3 × COCH₃). HR-FAB-MS: Calcd for C₂₇H₃₃NO₁₂Na, m/z 586.1900. Found: m/z 586.1925 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 4,6-O-Benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (9)

To a solution of 8 (1.57 g, 2.79 mmol) in MeOH (20 mL) was added NaOMe (60 mg) and the mixture was stirred for 30 min at room temperature. After completion of the reaction, the reaction mixture was neutralized with Amberlite IR 120 [H⁺]. After filtration, the solution was concentrated, and the residue was dissolved in dry acetonitrile (30 mL). To the stirred solution was added benzaldehyde dimethylacetal (0.59 mL, 3.93 mmol) and (±)-camphor-10-sulfonic acid (305 mg, 1.31 mmol), and the mixture was stirred for 3 h at 40°C, then neutralized with Et₃N. The mixture was poured into iced-water and extracted with CHCl₃. The extract was successively washed with brine, dried (MgSO₄), and concentrated. The product was purified by silica gel column chromatography (8 : 1 toluene–acetone) to give 9 (1.25 g, 85%, 2 steps). [α]_D²⁴ +45.2 (c = 3.0, CHCl₃).¹H-NMR (500 MHz, CDCl₃) δ: 7.84–7.39 (9H, m, Phth, Ph), 5.59 (1H, s, PhCH), 5.23 (1H, d, J_1,2 = 8.3 Hz, H-1), 4.52 1H, (dt, J_2,3 = 11.0 Hz, J_3,4 = 3.5 Hz, H-3), 4.38 (1H, d, J_6a,6b = 12.1 Hz, H-6a), 4.28 (1H, d, H-4), 4.12 (1H, d, H-6b), 3.63 (1H, s, H-5), 3.60 (3H, s, CH₃), 3.46–3.41 (1H, m, –OCH₂–), 2.57 (1H, br s, OH), 2.06–1.95 (2H, m, –CH₂COOMe), 1.51–1.39 (4H, m, –OCH₂CH₂CH₂CH₂–), 1.17–1.08 (2H, m, –OCH₂CH₂CH₂–). ¹³C-NMR (125 MHz, CDCl₃) δ: 173.8, 168.7, 168.2, 137.3, 133.9, 131.7, 131.7, 129.2, 128.2, 126.4, 123.5, 123.0, 101.5, 98.2 (C-1), 77.2, 75.1 (C-4), 69.2 (C-6), 69.1 (–OCH₂–), 67.9 (C-3), 66.7 (C-5), 54.7 (C-2), 51.3 (OMe), 33.7, 28.8, 25.3, 24.4. MALDI-TOF-MS: Calcd for C₂₈H₃₁NO₉Na, m/z 548.2. Found, m/z 548.0 [M + Na]⁺. HR-FAB-MS: Calcd for C₂₈H₃₁NO₉Na, m/z 548.1897. Found: m/z 548.1859 [M + Na]⁺.

Phenyl 2-O-Benzoyl-3-O-(2-napthylmethyl)-4,6-O-di-tert-butylsilylene-1-thio-β-D-galactopyranoside (11)

A solution of 10 (0.69 g, 1.67 mmol) and dibutyltin oxide (0.50 g, 2.01 mmol) in 30 mL of dry toluene was stirred under reflux for 5 h. Toluene was distilled off, the stannylidene derivative was dissolved in toluene (15 mL) and Bu₄NBr (647 mg, 2.00 mmol) and NapBr (743 mg, 3.34 mmol) were added at room temperature. After being stirred for 14 h at 60°C, the solution was concentrated. The residue was dissolved with EtOAc, washed with aq NaCl, dried (MgSO₄), and concentrated. The residue was purified by silica gel column chromatography using 4 : 1 hexane–EtOAc to give intermediate. To a solution of this compound in pyridine (10 mL) was added benzoyl chloride (0.82 mL, 7.08 mmol), and the reaction mixture was stirred for 16 h at room temperature. Toluene was added and evaporated, then the residue was dissolved in CHCl₃, washed with 5% HCl, aq NaHCO₃ and water, dried (MgSO₄), and concentrated. The product was purified by silica gel column chromatography using 2 : 1 hexane–EtOAc as eluent to give 11 (0.69 g, 63% 2 steps). [α]_D²² +48.1 (c = 1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 8.06–7.19 (17H, m, 2 × Ph, naphtyl), 5.74 (1H, t, J_1,2 = J_2,3 = 9.7 Hz, H-2), 4.88 and 4.74 (2H, each d, J_gem = 13.1 Hz, naphtyl methylene), 4.77 (1H, d, H-1), 4.62 (1H, d, J_3,4 = 2.6 Hz, H-4), 4.26 (2H, ddd, J_5,6a = 1.5 Hz, J_5,6b = 2.0 Hz, J_6a,6b = 12.4 Hz, H-6a, 6b), 3.60 (1H, dd, H-3), 3.37 (1H, br s, H-5), 1.17 and 1.10 (18H, each s, Si[(CH₃)₃]₂. ¹³C-NMR (125 MHz, CDCl₃) δ: 165.4, 135.3, 134.3, 133.0, 132.9, 132.1, 130.1, 129.9, 128.7, 128.4, 128.2, 127.7, 127.6, 127.5, 126.4, 126.1, 125.9, 125.7, 87.6 (C-1), 79.1 (C-3), 75.1 (C-5), 70.1 (Nap methylene), 69.7 (C-4), 69.4 (C-2), 67.3 (C-6), 27.61 and 27.58 [2× (CH₃)₃], 23.4, 20.7. HR-ESI-MS: Calcd for C₃₈H₄₄O₆SSiNa: m/z 679.2526. Found: m/z 679.2531 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2-O-Benzoyl-3-O-2-napthylmethyl-4,6-O-di-tert-butylsilylene-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (12)

To a solution of 9 (0.59 g, 1.12 mmol) and 11 (0.94 g, 1.43 mmol) in dry CH₂Cl₂ (10 mL) was added powdered MS-AW300 (1.0 g), and the mixture was stirred under Ar atmosphere for 2 h at room temperature, then cooled to −40°C. NIS (0.45 g, 2.0 mmol) and TfOH (44.9 µL, 0.46 mmol) were added to the mixture, which was stirred for 1 h at −50°C, 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 silica gel column chromatography (5 : 1 toluene–EtOAc) to give 12 (0.96 g, 0.90 mmol, 80%). [α]_D²⁴ +151.2 (c = 1.8, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 7.94–7.01 (21H, m, Ph), 5.50 (1H, d, J_1′,2′ = 3.7 Hz, H-1′), 5.37 (1H, d, J_2′,3′ = 10.3 Hz, H-2′), 5.18 (1H, d, J_1,2 = 8.3 Hz, H-1), 5.13 (1H, s, PhCH), 4.78–4.70 (2H, m, napthylmethylene), 4.72 (1H, d, J_2,3 = 11.3 Hz, H-2), 4.64 (1H, d, J_3,4 = 3.1 Hz, H-3), 4.24 (1H, d, J_6a,6b = 11.8 Hz, H-6a), 4.23 (1H, br, H-4′), 4.14 (1H, d, H-4), 3.93 (1H, d, H-6b), 3.83 (1H, dd, J_2′,3′ = 2.6 Hz, H-3′), 3.82–3.79 (1H, m, –OCH₂–), 3.59 (3H, s, CH₃), 3.55–3.51 (2H, m, H-6′), 3.47 (1H, s, H-5), 3.38–3.34 (1H, m, –OCH₂–), 3.47 (1H, s, H-5′), 2.35–0.88 (26H, m, –OCH₂CH₂CH₂CH₂CH₂–, t-bu × 2). ¹³C-NMR (125 MHz, CDCl₃) δ: 73.9, 173.9, 168.6, 167.5, 137.8, 137.5, 135.9, 134.3, 133.2, 133.0, 132.9, 131.40, 131.36, 129.6, 129.5, 129.0, 128.4, 128.3, 128.2, 128.0, 127.9, 127.8, 127.6, 126.0, 125.9, 125.7, 125.5, 125.3, 123.5, 123.0, 100.2, 98.2 (C-1), 95.9 (C-1′), 75.2(C-3′), 73.7 (C-3), 72.4 (C-4), 71.4, 71.0 (C-4′), 70.3 (C-2′), 69.0 (C-6), 68.8, 68.0 (C-5′), 66.48 (C-5), 66.46 (C-6′), 52.1 (C-2), 51.3, 33.7, 28.8, 27.6, 27.5, 27.3, 27.2, 25.3, 24.4, 23.3, 21.4, 20.7. HR-ESI-MS: Calcd for C₆₀H₆₉NO₁₅SiNa: m/z 1094.4334. Found: m/z 1094.4342 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2-O-Benzoyl-4,6-O-di-tert-butylsilylene-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (13)

A solution of 12 (414 mg, 0.39 mmol) in CH₂Cl₂–H₂O (19 : 1, 5 mL) was treated with DDQ (134 mg, 0.60 mmol) at room temperature and then was stirred for 15 h. After concentration, the residue was added to the water, extracted with CHCl₃, and the organic layer was proceeded as usual. The product was purified by silica gel column chromatography (5 : 1 toluene–EtOAc) as eluent to give 13 (315 mg, 89%). [α]_D²⁴ +23.5 (c = 2.4, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 7.95–7.12 (21H, m, Ph), 5.35 (1H, d, J_1′,2′ = 3.8 Hz, H-1′), 5.27 (1H, s, PhCH), 5.20–5.16 (1H, m, H-2′), 5.17 (1H, d, J_1,2 = 8.2 Hz, H-1), 4.77 (1H, d, J_2,3 = 11.2 Hz, H-2), 4.66 (1H, d, J_3,4 = 3.3 Hz, H-3), 4.28 (1H, d, J_6a,6b = 12.1 Hz, H-6a), 4.22 (1H, d, H-4), 4.20 (1H, d, J_3′,4′ = 2.3 Hz, H-4′), 3.98 (1H, d, H-6b), 3.83 (1H, br, H-3′), 3.84–3.80 (1H, m, –OCH₂–), 3.71 (1H, dd, J_6′a,6′b = 12.1 Hz, J_5′,6′a = 2.0 Hz, H-6′a), 3.60 (3H, s, CH₃), 3.60–5.57 (1H, m, H-6b′), 3.51 (1H, s, H-5), 3.40–3.35 (1H, m, –OCH₂–), 3.40 (1H, s, H-5′), 2.35–0.88 (26H, m, –OCH₂CH₂CH₂CH₂CH₂–, t-bu × 2). ¹³C-NMR (125 MHz, CDCl₃) δ: 173.9, 168.7, 167.4, 166.8, 137.4, 134.5, 134.4, 133.1, 131.5, 131.4, 129.8, 129.3, 128.5, 128.3, 127.9, 126.0, 123.7, 123.1, 100.2, 98.2 (C-1), 95.8 (C-1′), 73.6 (C-3), 73.2 (C-4′), 71.4 (C-4), 71.4 (C-2′), 69.1 (C-6), 68.8, 68.0 (C-3′), 67.8 (C-5′), 66.5 (C-5), 66.2 (C-6′), 52.1 (C-2), 51.4, 33.7, 28.8, 27.5, 27.4, 27.3, 27.2, 25.3, 24.4, 23.2, 20.6. HR-ESI-MS: Calcd for C₄₉H₆₁NO₁₅SiNa: m/z 931.3810. Found: m/z 931.3852 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2-O-Benzoyl-3-O-2-napthylmethyl-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (14)

A solution of 12 (2.33 g, 2.15 mmol) and acetic acid (0.65 µL, 10.8 µmol) in tetrahydrofuran (THF) (20 mL) was treated with 1 M TBAF in THF (6.46 µL, 6.46 mmol) at room temperature and then was stirred for 15 h. After concentration, the residue was added to the water, extracted with EtOAc, and the organic layer was proceeded as usual. The product was purified by silica gel column chromatography using 3 : 1 toluene–acetone as eluent to give 14 (1.47 g, 73%): [α]_D²⁴ +155.3 (c = 1.8, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 7.95–7.12 (21H, m, Ph), 5.35 (1H, d, J_1′,2′ = 3.9 Hz, H-1′), 5.30 (1H, d, J_2′,3′ = 9.6 Hz, H-2′), 5.16 (1H, d, J_1,2 = 8.3 Hz, H-1), 5.14 (1H, s, PhCH), 4.74 (1H, d, J_2,3 = 11.1 Hz, H-2), 4.69, 4.66 (2H, each d, napthylmethylene), 4.67 (1H, dd, J_3,4 = 2.9 Hz,), 4.19 (1H, d, J_6a,6b = 11.8 Hz, H-6a), 4.15 (1H, d, J_3,4 = 2.6 Hz, H-4), 3.89–3.87 (3H, m, H-6b, H-3′, H-5′), 3.81–3.76 (1H, m, –OCH₂–), 3.57 (3H, s, CH₃), 3.42–3.11 (5H, m, H-4′, H-5′, H-6, –OCH₂–), 3.11 (1H, br, 6′-OH), 2.57 (1H, br, 4′-OH), 2.0–1.88 (2H, m, –CH₂COOMe), 1.42–1.28 (4H, m, –OCH₂CH₂CH₂CH₂–), 1.07–1.00 (2H, m, –OCH₂CH₂CH₂–). ¹³C-NMR (125 MHz, CDCl₃) δ: 173.8, 168.9, 167.5, 166.3, 137.4, 134.9, 134.2, 133.1, 133.0, 132.8, 131.4, 131.3, 129.5, 129.2, 128.9, 128.3, 128.2, 128.1, 128.1, 127.9, 127.7, 127.5, 126.2, 126.0, 125.9, 125.8, 125.3, 125.2, 123.3, 123.2, 100.0, 98.1 (C-1), 95.2 (C-1′), 74.9 (C-3′), 73.3 (C-3), 72.5, 72.0 (C-4), 70.5 (C-2′), 69.6 (C-4′), 68.9 (C-6), 68.8, 68.5 (C-5′), 66.3 (C-5), 62.2 (C-6′), 52.1 (C-2), 51.3, 33.6, 28.7, 25.2, 24.3, 21.3. HR-ESI-MS: Calcd for C₅₂H₅₃NO₁₅Na: m/z 954.3313. Found: m/z 954.3375 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-acetyl-α-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-galactopyranoside (15)

A solution of 13 (188 mg 0.20 mmol) and acetic acid (57 µL, 1.0 mmol) in THF (2 mL) was treated with 1 M TBAF in THF (0.6 µL, 0.60 mmol) at room temperature and then was stirred for 10 h. After concentration, the residue was added to the water, extracted with EtOAc, and the organic layer was proceeded as usual. The product was purified by silica gel column chromatography using 30 : 1 CHCl₃–MeOH as eluent to give the de-DTBS compound (138 mg, 87%). A solution of the residue (138 mg 0.17 mmol) in 80% AcOH (5 mL) was stirred at 70°C for 3 h, then was diluted with toluene and concentrated. The product was purified by silica gel column chromatography (8 : 1 CHCl₃–MeOH) to give the compound (95 mg, 78%). The residue was deprotected of phthaloyl and acyl group with ethylenediamine in BuOH. After concentrate on, the residue was acetylated with acetic anhydride (1.0 mL) in pyridine (1.5 mL). The product was purified by silica gel column chromatography using 3 : 1 toluene–acetone as eluent to give 15 (42 mg, 28%, 4 steps). [α]_D²⁵ +68.2 (c = 1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 6.08 (1H, d, NH), 5.42 (1H, s, H-2′), 5.34 (1H, d, J_3,4 = 2.8 Hz, H-4), 5.27–5.25 (1H, m, H-3′), 5.23 (1H, d, J_1′,2′ = 2.9 Hz, H-1′), 5.01 (1H, d, J_1,2 = 7.9 Hz, H-1), 4.57 (1H, d, J_2,3 = 10.0 Hz, H-3), 4.24–4.15 (3H, m, H-4′, 5′, 6a′), 4.10 (2H, d, J_5,6 = 6.5 Hz, H-6), 3.91–3.83 (2H, m, H-5, OCH₂CH₂), 3.67 (3H, s, OCH₃), 3.51–3.47 (2H, m, H-2, OCH₂CH₂), 2.35–2.16 (2H, m, CH₂COOCH₃), 2.14–1.96 (21H, m, COCH₃ × 7), 1.69–1.25 (6H, m, (CH₂)₃). ¹³C-NMR(125 MHz, CDCl₃) δ: 174.2, 171.0, 170.7, 170.4, 170.3, 170.13, 170.07, 169.8, 129.0, 128.2, 125.3, 99.5 (C-1), 94.2 (C-1′), 72.2, 70.6, 69.9, 67.5, 67.3, 66.8, 66.3, 65.3, 61.7, 61.1, 54.4, 51.5, 33.9, 29.7, 28.9, 25.5, 24.5, 20.78, 20.75, 20.74, 20.70, 20.6. HR-ESI-MS: Calcd for C₃₃H₄₉NO₁₉Na: m/z 786.2796. Found: m/z 786.2776 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl α-D-Galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-galactopyranoside (16)

To a solution of 15 (18.4 mg, 24.1 µmol) in MeOH (4.0 mL) was added NaOMe (5 mg) at room temperature and the mixture was stirred for 5 h, then neutralized with Amberlite IR 120 [H⁺]. The mixture was filtered off and concentrated. The product was purified by Sephadex LH-20 column chromatography in H₂O to give 16 (12.3 mg, quant.). [α]_D²⁵ +76.7 (c = 0.3, CH₃OH). ¹H-NMR (500 MHz, CD₃OD) δ: 5.00 (1H, d, J_1′,2′ = 3.3 Hz, H-1′), 4.39 (1H, d, J_1,2 = 8.5 Hz, H-1). ¹³C-NMR (125 MHz, CD₃OD) δ: 175.9, 173.7, 102.9 (C-1), 97.6 (C-1′), 78.3, 76.3, 72.8, 71.4, 71.1, 70.2, 70.0, 65.7, 62.7, 62.5, 52.1, 52.0, 34.8, 30.8, 30.3, 26.7, 25.7, 23.2. HR-ESI-MS: Calcd for C₂₁H₃₇NO₁₃Na: m/z 534.2163. Found: m/z 534.2174 [M + Na]⁺.

Biotinylateddisaccharide (1)

Compound 16 (20.4 mg, 39.8 mmol) was dissolved in neat anhydrous ethylenediamine (8 mL) and heated at 70°C for 48 h. The mixture was concentrated with toluene and the product was purified by Sephadex LH-20 column chromatography in H₂O to give an amine intermediate. The amine was dissolved in DMF (4 mL), and the pH was adjusted to 8–9 using DIPEA. Biotin-NHS (18.8 mg, 55.2 µmol) was added and the reaction stirred for 12 h at room temperature. Toluene was added to and evaporated from the residue several times. The product was purified by Sephadex LH-20 column chromatography in H₂O to give 1 (26.3 mg, 86%). [α]_D²⁵ +37.5 (c = 0.3, MeOH). ¹H-NMR (500 MHz, D₂O) δ: 5.13 (1H, d, J_1′,2′ = 3.6 Hz, H-1′), 4.53 (1H, d, J_1,2 = 8.6 Hz, H-1). ¹³C-NMR (125 MHz, D₂O) δ: 180.1, 177.74, 177.69, 174.9, 101.8 (C-1), 95.7 (C-1′), 76.1, 75.4, 71.9, 70.8, 70.0, 69.7, 68.7, 64.4, 62.7, 62.6, 61.6, 61.5, 60.88, 60.86, 55.9, 51.3, 40.3, 39.2, 37.9, 36.4, 36.2, 28.9, 28.87, 28.5, 28.3, 28.28, 26.4, 25.8, 25.6, 25.5, 25.3, 22.9. HR-ESI-MS: Calcd for C₃₂H₅₅N₅O₁₄SNa: m/z 788.3364. Found: m/z 788.3341 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-O-di-tert-butylsilylene-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (18)

To a solution of 13 (156 mg, 0.17 mmol) and 17 (249 mg, 0.51 mmol) in dry CH₂Cl₂ (3.0 mL) was added MS 4 Å (500 mg), and the mixture was stirred for 2 h at room temperature, then cooled to −20°C. TMSOTf (17.6 µL, 101 µmol) was added, and the mixture was stirred for 3 h at −40°C, then neutralized with Et₃N. The precipitates were filtrated off and washed with CHCl₃. The combined filtrate and washings were successively washed with water, dried (MgSO₄), and concentrated. The product was purified by silica gel column chromatography (5 : 1 toluene–EtOAc) to give 18 (156 mg, 74%). [α]_D²⁴ +85.9 (c = 2.5, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 7.92–7.13 (14H, m, Ph), 5.45 (1H, d, J_1′,2′ = 3.8 Hz, H-1′), 5.30 (1H, d, J_3″,4″ = 2.6 Hz, H-4″), 5.26 (1H, dd, J_2′,3′ = 10.5 Hz, H-2′), 5.18 (1H, d, J_1,2 = 8.2 Hz, H-1), 5.08 (1H, dd, J_1″,2″ = 7.9, J_2″,3″ = 10.3 Hz, H-2″), 5.06 (1H, s, PhCH), 4.84 (1H, dd, H-3″), 4.73 (1H, d, J_2,3 = 11.1 Hz, H-2), 4.68 (1H, d, H-1″), 4.65 (1H, d, J_3,4 = 3.1 Hz, H-3), 4.40 (1H, d, J_3′,4′ = 2.3 Hz, H-4′), 4.21 (1H, d, J_6a,6b = 12.0 Hz, H-6a), 4.09 (1H, d, H-4), 4.01–3.78 (6H, m, H-6b, 3′, 5″, 6″a, 6″b, –OCH₂–), 3.60 (3H, s, OCH₃), 3.52–3.46 (3H, H-5, 6′a, 6′b), 3.40–3.35 (1H, m, –OCH₂–), 3.26 (1H, s, H-5′), 2.07, 1.99, 1.89 (9H, each s, 3 × OAc), 1.97–0.94 (26H, m, –OCH₂CH₂CH₂CH₂CH₂–, t-bu × 2). ¹³C-NMR (125 MHz, CDCl₃) δ: 173.7, 170.1, 170.0, 168.9, 168.4, 167.6, 166.1, 137.4, 134.5, 134.4, 133.2, 131.41, 131.37, 129.5, 129.1, 128.5, 128.3, 127.8, 126.0, 123.4, 123.2, 101.6 (C-1″), 100.1, 98.1 (C-1 of GalN), 96.0 (C-1′), 77.2, 75.7 (C-3′), 74.1 (C-3 of GalN), 73.0 (C-4′), 72.5 (C-4 of GalN), 70.9 (C-3″), 70.3 (C-5″), 69.9 (C-2′), 68.8, 67.9 (C-5′), 66.8 (C-4″), 66.4 (C-5 of GalN), 66.4 (C-6′), 61.2 (C-6″), 52.0 (C-2 of GalN), 51.3, 33.6, 28.7, 27.3, 27.0, 25.2, 24.3, 23.1, 20.5, 20.5, 20.4, 20.4, 19.7. HR-ESI-MS: Calcd for C₆₃H₇₉NO₂₄SiNa: m/z 1284.4659. Found: m/z 1284.4698 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→3)-2,4,6-tri-O-acetyl-α-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2- deoxy-β-D-galactopyranoside (19)

A solution of 18 (80 mg, 63.4 µmol) in THF (0.6 mL) was added TBAF (0.19 mL, 0.19 mmol) and AcOH (18 µL, 0.32 mmol) at room temperature and then was stirred for 22 h. Toluene was added and co-evaporated several times. The product was purified by silica gel column chromatography (3 : 1 toluene–acetone) to give the de-DTBS-compound. A solution of this compound in 80% AcOH (1.0 mL) was stirred at 80°C for 4 h, then was diluted with toluene and concentrated. The product was purified by silica gel column chromatography (2 : 1 toluene–acetone) to give deprotection of benzylidene acetal compound. The residue was deproducted of phthaloyl group with ethylenediamine (0.5 mL) in BuOH (2 mL) at 90°C for 7 h. After concentration, the residue was acetylated with acetic anhydride (0.2 mL) in pyridine (1.0 mL) at room temperature for 14 h. After the reaction, toluene was added and co-evaporated several times. The product was purified by silica gel column chromatography (3 : 1 toluene–acetone) to give 19 (9.8 mg, 15%, 4 steps). [α]_D +33.3 (c = 0.2, CHCl₃). ¹H-NMR (400 MHz, CDCl₃) δ: 6.01 (1H, d, J = 8.2 Hz, NH), 5.37–4.93 (6H, m, H-4, 2′, 4′, 2″, 3″, 4″), 5.17 (1H, d, J_1,2 = 3.5 Hz, H-1′), 4.63 (1H, d, J_1,2 = 7.9 Hz, H-1), 4.62 (1H, d, J_1,2 = 7.9 Hz, H-1″). 3.67 (3H, s, OCH₃), 2.17, 2.16, 2.133, 2.130, 2.11, 2.05 × 2, 2.00, 1.971, 1.968 (30H, each s, 10 × OAc), ¹³C-NMR (150 MHz, CDCl₃) δ: 174.2, 171.5, 170.43 × 2, 170.37, 170.2, 170.1, 169.8, 169.7, 169.1, 100.9 (C-1″), 100.6 (C-1), 93.8 (C-1′), 73.0, 70.8, 70.7, 70.6, 70.6, 69.5, 69.3, 69.1, 68.8, 66.8, 66.7, 64.9, 62.3, 61.6, 60.7, 51.5, 33.9, 29.7, 29.3, 28.9, 25.4, 24.5, 23.2, 21.0, 20.9, 20.9, 20.7, 20.7, 20.6, 20.5, 20.5. HR-ESI-MS: Calcd for C₄₅H₆₅NO₂₇Na: m/z 1074.3642. Found: m/z 1074.3610 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl β-D-Galactopyranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-galactopyranoside (20)

To a solution of 19 (9.8 mg, 9.3 µmol) in 1,4-dioxane–MeOH (1 : 1, 1.0 mL) was added NaOMe (10 mg) at room temperature and the mixture was stirred at 50°C for 12 h, then neutralized with Amberlite IR 120 [H⁺]. The mixture was filtered off and concentrated. The product was purified by Sephadex LH-20 column chromatography in H₂O to give 20 (7.5 mg, quant.). [α]_D²⁵ +80.6 (c = 0.2, MeOH). ¹H-NMR (600 MHz, CD₃OD) δ: 5.04 (1H, d, J_1,2 = 3.9 Hz, H-1′), 4.48 (1H, d, J_1,2 = 7.7 Hz, H-1″), 4.40 (1H, d, J_1,2 = 8.5 Hz, H-1). 3.65 (3H, s, OCH₃), 1.99 (9H, s, OAc). ¹³C-NMR (150 MHz, CD₃OD) δ: 173.7, 106.6 (C-1′), 102.9 (C-1), 97.4 (C-1″), 81.8, 78.5, 76.7, 76.2, 74.6, 73.0, 72.5, 70.6, 70.2, 68.9, 65.7, 62.7, 62.5, 62.5, 52.2, 52.0, 34.8. 30.7, 30.3, 26.7, 25.7, 23.3. ESI-HR-MS: Calcd for C₂₇H₄₇NO₁₈Na, m/z 696.2691. Found: m/z 696.2725 [M + Na]⁺.

Biotinylatedtrisaccharide (2)

Compound 2 was prepared from 20 (7.5 mg, 11.1 µmol) as described for preparation of 1. The product was purified by Sephadex LH-20 column chromatography (H₂O) to give 2 (4.3 mg, 42%). [α]_D²⁵ +41.2 (c = 0.1, H₂O). ¹H-NMR (500 MHz, D₂O) δ: 5.15 (1H, d, J_1′,2′ = 3.6 Hz, H-1′), 4.59 (1H, d, J_1″,2″ = 7.7 Hz, H-1″), 4.53 (1H, d, J_1,2 = 8.6 Hz, H-1), 2.04 (3H, s, NAc). ¹³C-NMR (125 MHz, D₂O) δ: 179.4, 177.13, 177.09, 174.2, 104.5 (C-1″), 101.2 (C-1), 95.0 (C-1′), 79.2, 75.5, 75.1, 74.8, 72.5, 71.1, 71.0, 70.2, 69.0, 68.6, 67.1, 63.8, 62.1, 61.0, 60.8, 60.3, 55.4, 50.7, 39.7, 38.8, 38.7, 38.6, 35.8, 35.6, 30.2, 30.1, 30.0, 29.9, 28.3, 27.9, 27.7, 25.2, 25.0, 24.7, 22.3, 20.1. HR-ESI-MS: Calcd for C₃₈H₆₅N₅O₁₉SNa: m/z 950.3892. Found: m/z 950.3901 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranosyl-(1→6)-2-O-benzoyl-3-O-2-napthylmethyl-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (22)

Compound 22 was prepared from 21 (278 mg, 0.40 mmol) and 14 (205 mg, 0.22 mmol) as described for preparation of 12. The product was purified by silica gel column chromatography (3 : 1 toluene–EtOAc) to give 22 (292 mg, 86%). [α]_D²⁴ +48.2 (c = 1.5, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 8.18–6.97 (41H, m, Ph), 6.06 (1H, d, J_3″,4″ = 2.8 Hz, H-4″), 5.82 (1H, dd, J_1″,2″ = 7.9 Hz, J_2″,3″ = 10.3 Hz, H-2″), 5.65 (1H, dd, H-3″), 5.29 (1H, d, J_1′,2′ = 3.7 Hz, H-1′), 5.26 (1H, dd, J_2′,3′ = 10.0 Hz, H-2′), 5.21 (1H, d, J_1,2 = 8.4 Hz, H-1), 5.15 (1H, s, PhCH), 4.80 (1H, d, H-1″), 4.78–4.70 (2H, m, H-2, 6″a), 4.56 (1H, d, J_2,3 = 11.1 Hz, J_3,4 = 3.3 Hz, H-3), 4.49–4.43 (2H, m, H-5″, 6″b), 4.26–4.18 (2H, m, H-6a, napthylmethylene), 3.97 (1H, d, H-4), 3.94–3.80 (4H, m, H-4, 6a, 6b, –OCH₂–), 3.75 (1H, br s, H-4′), 3.64–3.60 (5H, H-3′, 6′b, –OCH₃), 3.44–3.40 (4H, m, H-5, 5′, –OCH₂–), 2.47 (1H, br s, OH), 2.03–1.10 (8H, m, –OCH₂CH₂CH₂CH₂CH₂–). ¹³C-NMR (125 MHz, CDCl₃) δ: 173.9, 168.7, 167.7, 166.1, 166.0, 165.6, 165.5, 165.1, 137.3, 134.8, 134.3, 134.2, 133.6, 133.4, 133.3, 133.1, 133.0, 132.8, 131.62, 131.59, 130.1, 129.8, 129.7, 129.6, 129.5, 129.3, 129.1, 128.8, 128.62, 128.61, 128.5, 128.35, 128.31, 128.0, 127.9, 127.8, 127.6, 126.2, 126.0, 125.9, 125.3, 123.6, 123.3, 101.3 (C-1″), 100.4, 98.2 (C-1 of GalN), 94.5 (C-1′), 75.4 (C-3′), 72.6 (C-3 of GalN), 72.0, 71.9 (C-3″), 71.8 (C-4 of GalN), 71.3 (C-5″), 70.1 (C-2′), 69.9 (C-2″), 69.1 (C-6 of GalN), 69.0 (–OCH₂–), 68.1 (C-4″), 68.0 (C-5′), 67.2 (C-6′), 66.3 (C-5 of GalN), 66.0 (C-4′), 61.9 (C-6″), 52.5 (C-2 of GalNAc), 51.3 (–OCH₃), 33.8, 29.0, 25.4, 24.5. ESI-HR-MS: Calcd for C₈₆H₇₉NO₂₄Na, m/z 1532.4890. Found: m/z 1532.4848 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,4-di-O-benzoyl-3-O-2-napthylmethyl-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (23)

To a solution of 22 (210 mg, 0.14 mmol) in Pyr. (2.5 mL) was added DMAP (50 mg) and benzoyl chloride (48 µL, 0.42 mmol) at 0°C, and the reaction mixture was stirred for 23 h at 45°C. Toluene was added and evaporated, then the residue was dissolved in CHCl₃, washed with 5% HCl, aq NaHCO₃ and water, dried (MgSO₄), and concentrated. The product was purified by silica gel column chromatography using 15 : 1 toluene–acetone as eluent to give 23 (213 mg, 94%). ¹H-NMR (500 MHz, CDCl₃) δ: 8.25–6.97 (46H, m, Ph), 5.94 (1H, d, J_3″,4″ = 3.0 Hz, H-4″), 5.78 (1H, dd, J_1″,2″ = 8.0, J_2″,3″ = 10.1 Hz, H-2″), 5.62 (1H, br s, H-4′), 5.58 (1H, dd, H-3″), 5.46 (1H, d, J_1′,2′ = 3.7 Hz, H-1′), 5.27 (1H, dd, J_2′,3′ = 10.4 Hz, H-2′), 5.22 (1H, d, J_1,2 = 8.3 Hz, H-1), 5.07 (1H, s, PhCH), 4.73 (1H, J_2,3 = 11.0 Hz, H-2), 4.61 (1H, d, J_1,2 = 8.0 Hz, H-1″), 4.58–4.56 (2H, m, H-3, napthylmethylene), 4.30–4.21 (4H, m, H-4, H-6a, 6″a, napthylmethylene), 4.09 (1H, dd, J_5,6″b = 6.8, J_6″a,6″b = 10.6 Hz, H-6″b), 4.00 (1H, d, H-5″), 3.91–3.78 (5H, m, H-3′, 5′, 6′a, 6b, –OCH₂–), 3.60 (3H, s, OCH₃), 3.44–3.38 (2H, m, H-5, –OCH₂–), 3.02 (1H, dd, H-6′b), 2.36–1.01 (8H, m, –OCH₂CH₂CH₂CH₂CH₂–). ¹³C-NMR (125 MHz, CDCl₃) δ: 173.9, 168.6, 167.5, 166.2, 165.9, 165.6, 165.6, 165.1, 165.0, 153.6, 140.7, 137.2, 135.2, 137.4, 134.2, 133.7, 133.2, 133.1, 133.0, 131.55, 131.45, 130.4, 130.3, 130.2, 129.81, 129.76, 129.6, 129.4, 129.1, 128.8, 128.6, 128.6, 128.5, 128.5, 128.3, 128.0, 127.8, 127.7, 127.5, 126.2, 125.8, 125.7, 125.8, 123.3, 120.7, 107.7, 100.4 (C-1″), 100.3, 98.2 (C-1), 95.0 (C-1′), 73.6 (C-3′), 73.3 (C-3), 72.0 (C-5″), 71.9 (C-3″), 71.5 (CH₂), 71.0 (C-4), 70.5 (C-2′), 70.0 (C-2″), 69.1 (C-6), 68.9 (–OCH₂–), 67.9 (C-4″), 67.8 (C-5′), 67.0 (C-4′), 66.2 (C-5), 66.0 (C-6′), 61.4 (C-6″), 52.3 (C-2), 51.4 (–OCH₃), 33.8, 28.9, 25.4, 24.5. ESI-HR-MS: Calcd for C₉₃H₈₃NO₂₅Na, m/z 1613.5254. Found: m/z 1613.5287 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,4-di-O-benzoyl-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (24)

Compound 24 was prepared from 23 (213 mg, 0.13 mmol) as described for preparation of 13. The product was purified by silica gel column chromatography (12 : 1 toluene–acetone) to give 24 (162 mg, 83%). [α]_D²⁴=+38.8 (c = 1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 8.20–6.98 (39H, m, Ph), 5.95 (1H, d, J_3″,4″ = 3.2 Hz, H-4″), 5.77 (1H, dd, J_1″,2″ = 8.1, J_2″,3″ = 10.0 Hz, H-2″), 5.60 (1H, dd, H-3″), 5.42 (1H, d, J_1′,2′ = 3.5 Hz, H-1′), 5.31 (1H, br s, H-4′), 5.24 (1H, d, J_1,2 = 8.4 Hz, H-1), 5.19 (1H, s, PhCH), 5.16 (1H, dd, J_2′,3′ = 10.3 Hz, H-2′), 4.78 (1H, J_2,3 = 11.0 Hz, H-2), 4.67 (1H, d, J_1,2 = 7.6 Hz, H-1″), 4.66 (1H, dd, H-3), 4.37–4.25 (3H, m, H-5, H-6a, 6″a), 4.14–4.11 (2H, m, H-4, H-6″b), 3.99 (1H, dd, H-3′), 3.92–3.82 (4H, m, 5′, 6′a, 6′b, –OCH₂–), 3.60 (3H, s, OCH₃), 3.50 (1H, br s, H-5), 3.41 (1H, dd, –OCH₂–), 3.19 (1H, dd, H-6′b), 2.35–1.09 (8H, m, –OCH₂CH₂CH₂CH₂CH₂–). ¹³C-NMR (125 MHz, CDCl₃) δ: 173.9, 168.8, 167.5, 166.7, 166.1, 170.0, 165.6, 165.5, 164.9, 137.9, 137.2, 134.4, 134.6, 133.6, 133.4, 133.3, 133.2, 131.6, 130.2, 130.1, 129.9, 129.8, 129.8, 129.6, 129.6, 129.4, 129.3, 129.1, 128.8, 128.6, 128.5, 128.5, 128.4, 128.3, 128.2, 127.8, 125.9, 125.3, 123.6, 123.5, 100.6 (C-1′), 100.4 (PhCH), 98.2 (C-1), 94.0 (C-1″), 72.3 (C-3), 71.8 (C-3″), 71.6 (C-4), 71.3 (C-2′), 71.1 (C-5″), 70.4 (C-4′), 69.9 (C-2″), 69.2 (C-6), 69.0 (–OCH₂–), 68.0 (C-4″), 67.7 (C-5′), 67.0 (C-3′), 66.3 (C-5, 6′), 61.4 (C-6″), 52.3 (C-2), 51.4 (–OCH₃), 47.1, 33.8, 29.0, 25.4, 24.5. ESI-HR-MS: Calcd for C₈₂H₇₅NO₂₅Na, m/z 1473.4628. Found: m/z 1473.4672 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-acetyl-α-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-galactopyranoside (25)

A solution of 24 (90.4 mg 61.3 µmol) in 80% AcOH (2 mL) was stirred at 80°C for 4 h, then was diluted with toluene and concentrated. The residue was deproducted of phthaloyl group with ethylenediamine (1.5 mL) in BuOH (6.0 mL). After concentration, the residue was acetylated with acetic anhydride (0.4 mL) in pyridine (2.0 mL) at 45°C for 20 h. After the reaction, toluene was added and co-evaporated several times. The product was purified by silica gel column chromatography (2 : 1 toluene–acetone) to give 25 (30.2 mg, 47%, 3 steps). [α]_D²⁵ +35.0 (c = 1.0, CHCl₃); ¹H-NMR (500 MHz, CDCl₃) δ: 6.09 (1H, d, J = 9.6 Hz, NH), 5.41–5.39 (2H, m, H-4′, 4″), 5.30 (1H, d, J_3,4 = 2.9 Hz, H-4), 5.21 (1H, dd, J_2′,3′ = 6.6, J_3′,4′ = 2.9 Hz, H-3′), 5.12–5.09 (3H, m, H-2′, 2″, 3″), 5.07 (1H, d, J_1′,2′ = 3.0 Hz, H-1′), 4.72 (1H, d, J_1,2 = 8.2 Hz, H-1), 4.43 (1H, d, J_1,2 = 7.3 Hz, H-1″), 4.40–4.27 (2H, m, H-2, H-6a), 3.66 (3H, s, OCH₃), 2.16, 2.15, 2.12, 2.09, 2.06 × 2, 2.05 × 2, 2.02 (27H, each s, 9 × OAc), 2.23–1.34 (8H, m, –OCH₂CH₂CH₂CH₂CH₂–). ¹³C-NMR (125 MHz, CDCl₃) δ: 174.0, 170.6, 170.5, 170.5, 170.3, 170.2, 170.1, 169.9, 169.9, 169.6, 101.6 (C-1), 101.3 (C-1″), 94.3 (C-1′), 74.4, 70.7, 70.6, 70.1, 69.1, 68.8, 68.5, 67.4, 67.4, 66.9, 66.8, 65.1, 62.1, 60.9, 51.5, 33.9, 29.7, 29.2, 25.4, 24.6, 23.3, 21.2, 20.8, 20.8, 20.6, 20.5, 20.5. ESI-HR-MS: Calcd for C₄₅H₆₅NO₂₇Na, m/z 1074.3642. Found: m/z 1074.3625 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl β-D-Galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-galactopyranoside (26)

To a solution of 25 (30.2 mg, 28.1 µmol) in 1,4-dioxane–MeOH (1 : 1, 1.0 mL) was added NaOMe (20 mg) at room temperature and the mixture was stirred at 50°C for 15 h, then neutralized with Amberlite IR 120 [H⁺]. The mixture was filtered off and concentrated. The product was purified by Sephadex LH-20 column chromatography in H₂O to give 26 (15.0 mg, 79%.). [α]_D²⁵ +36.6 (c = 0.5, MeOH–H₂O = 1 : 1). ¹H-NMR (500 MHz, CD₃OD) δ: 4.98 (1H, d, J_1,2 = 3.7 Hz, H-1′), 4.57 (1H, br s, H-1), 4.41 (1H, d, J_1,2 = 7.6 Hz, H-1″), 3.65 (3H, s, OCH₃), 2.02 (3H, s, NAc), 1.63–0.86 (8H, m, –OCH₂CH₂CH₂CH₂CH₂–). ¹³C-NMR (125 MHz, CD₃OD) δ: 176.8, 174.9, 106.0 (C-1′), 103.8 (C-1), 98.5 (C-1″), 79.0, 77.5, 77.0, 73.8, 73.2, 72.2, 71.9, 71.2, 71.0, 70.9, 70.4, 66.7, 63.3, 52.9, 50.5, 35.7, 31.6, 31.2, 27.5, 26.6, 24.6, 24.4. ESI-HR-MS: Calcd for C₂₇H₄₇NO₁₈Na, m/z 696.2691. Found: m/z 696.2675 [M + Na]⁺.

Biotinylated Trisaccharide (3)

Compound 3 was prepared from 26 (14.3 mg, 21.2 µmol) as described for preparation of 1, yielding 8.4 mg (43%). [α]_D²⁵ +28.6 (c = 0.15, H₂O). ¹H-NMR (600 MHz, D₂O) δ: 5.12 (1H, d, J_1,2 = 3.6 Hz, H-1′), 4.53 (1H, d, J_1,2 = 8.5 Hz, H-1), 4.48 (1H, d, J_1,2 = 7.7 Hz, H-1″), 2.07 (3H, s, NAc).¹³C-NMR (150 MHz, D₂O) δ: 216.1, 177.8, 177.7, 175.1, 103.8 (C-1′), 101.8 (C-1), 96.0 (C-1″), 76.4, 75.8, 75.4, 73.5, 71.5, 70.9, 70.8, 69.8, 69.7, 69.3, 69.0, 68.6, 64.6, 62.8, 61.7, 61.6, 60.9, 56.0, 51.4, 40.4, 39.3, 39.2, 36.5, 36.2, 29.0, 28.6, 28.4, 25.8, 25.7, 25.4, 23.1, 20.7. ESI-HR-MS: Calcd for C₃₈H₆₅N₅O₁₉SNa, m/z 950.3892. Found: m/z 950.3918 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranosyl-(1→6)-[2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→3)]-2,4-di-O-benzoyl-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (27)

Compound 27 was prepared from 24 (118 mg, 81.6 µmol) and 17 (119 mg, 242 µmol) as described for preparation of 18. The product was purified by silica gel column chromatography (3 : 1 toluene–ethyl acetate) to give 27 (92.7 mg, 63%). [α]_D²⁴ +16.7 (c = 1.1, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 8.31–6.84 (39H, m, Ph), 6.03 (1H, d, J_3,4 = 2.5 Hz, H-4 of Gal c), 5.93 (1H, dd, J_1,2 = 8.0, J_2,3 = 10.5 Hz, H-2 of Gal c), 5.57 (1H, dd, H-3 of Gal c), 5.43 (1H, d, J_1,2 = 8.5 Hz, H-1 of GalN), 5.38 (1H, dd, 5.31 J_1,2 = 3.8, J_2,3 = 10.3 Hz, H-2 of Gal a), 5.29 (1H, d, H-1 of Gal a), 5.27(1H, d, J_3,4 = 2.9 Hz, H-4 of Gal b), 5.13 (1H, s, PhCH), 4.76 (1H, d, J_{1, 2} = 7.9 Hz, H-1 of Gal c), 4.22 (1H, d, J_1,2 = 8.1 Hz, H-1 of Gal b), 3.61 (3H, s, OCH₃), 2.35, 2.04, 1.91, 1.83 (12H, each s, 4 × OAc), 2.01–1.05 (8H, m, –OCH₂CH₂CH₂CH₂CH₂–). ¹³C-NMR (125 MHz, CDCl₃) δ: 173.9, 170.23, 170.20, 168.3, 167.8, 166.1, 165.9, 165.7, 165.6, 165.2, 153.6, 137.8, 137.1, 134.4, 133.5, 133.3, 133.2, 131.7, 130.3, 130.2, 130.0, 129.84, 129.82, 129.5, 129.4, 129.2, 129.0, 128.9, 128.6, 128.5, 128.53, 128.47, 128.4, 128.3, 128.21, 128.18, 127.7, 127.6, 125.8, 125.3, 123.8, 1203.2, 101.1 (C-1 of Gal c), 100.8 (C-1 of Gal b), 100.5, 98.1 (C-1 of GalN), 92.0 (C-1 of Gal a), 77.3, 77.0, 76.8, 73.8, 72.2, 71.1, 70.8, 70.6, 70.2, 70.2, 69.8, 69.2, 69.1, 68.6, 68.5, 68.1, 66.3, 66.1, 61.8, 60.4, 52.5, 51.4, 34.3, 33.8, 29.1, 25.5, 24,5, 22.8, 21.4, 20.7, 20.7, 20.4, 19.6. ESI-HR-MS: Calcd for C₉₆H₉₃NO₃₄Na, m/z 1826.5477. Found: m/z 1826.5492 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→6)-[2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→3)]-2,4-di-O-acetyl-α-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-galactopyranoside (28)

Compound 28 was prepared from 27 (60.7 mg 33.7 µmol) as described for preparation of 25. The product was purified by silica gel column chromatography (2 : 1 toluene–acetone) to give 28 (15.0 mg, 33%, 3 steps). [α]_D²⁵ +33.3 (c = 0.2, CHCl₃). ¹H-NMR (400 MHz, CDCl₃) δ: 6.15 (1H, d, J = 10.0 Hz, NH), 5.07 (1H, d, J_1,2 = 8.6 Hz, H-1 of GalN), 5.02 (1H, d, J_1,2 = 3.7 Hz, H-1 of Gal a), 4.66 (1H, d, J_1,2 = 8.4 Hz, H-1 of Gal c), 4.58 (1H, d, J_1,2 = 8.0 Hz, H-1 of Gal b), 3.66 (3H, s, OCH₃). ¹³C-NMR (150 MHz, CDCl₃) δ: 174.0, 170.8, 170.6, 170.5, 170.4, 170.3, 170.3, 170.1, 170.1, 169.9, 169.8, 169.1, 101.9 (C-1 of Gal c), 101.3 (C-1 of Gal b), 100.8 (C-1 of GalN), 94.0 (C-1 of Gal a), 72.0, 70.7, 70.7, 70.7, 70.6, 70.5, 69.9, 69.2, 68.9, 68.7, 68.5, 68.3, 66.8, 66.8, 65.0, 62.1, 60.8, 60.8, 51.5, 50.1, 33.9, 29.7, 29.2, 25.4, 24.6, 23.2, 21.3, 21.0, 20.9, 20.8, 20.7, 20.6, 20.5, 20.4. ESI-HR-MS: Calcd for C₅₇H₈₁NO₃₅Na, m/z 1362.4487. Found: m/z 1362.4455 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl β-D-Galactopyranosyl-(1→6)-[β-D-galactopyranosyl-(1→3)]-α-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-galactopyranoside (29)

Compound 29 was prepared from 28 (15.0 mg, 11.1 µmol) as described for preparation of 20, yielding 7.0 mg (78%). [α]_D²⁵ +29.9 (c = 0.25, MeOH). ¹H-NMR (600 MHz, CD₃OD) δ: 5.03 (1H, d, J_1,2 = 4.2 Hz, H-1 of Gal a), 4.59 (1H, br s, H-1 of GalN), 4.47 (1H, d, J_1,2 = 7.4 Hz, H-1 of Gal c), 4.41 (1H, d, J_1,2 = 7.7 Hz, H-1 of Gal b), 3.65 (3H, s, OCH₃), 2.03 (3H, s, NAc). ¹³C-NMR (150 MHz, CD₃OD) δ: 171.1, 103.6 (C-1 of Gal c), 102.2 (C-1 of Gal b), 100.0 (C-1 of GalN), 94.3 (C-1 of Gal a), 78.6, 75.1, 73.7, 73.6, 73.1, 72.2, 71.6, 70.0, 70.0, 69.1, 67.8, 67.4, 67.2, 67.1, 66.7, 65.9, 62.8, 59.5, 59.4, 49.1, 49.0, 31.8, 27.7, 27.3, 23.7, 22.7, 20.6. ESI-HR-MS: Calcd for C₃₃H₅₇NO₂₃Na, m/z 858.3219. Found: m/z 858.3235 [M + Na]⁺.

Biotinylated Tetrasaccharide (4)

Compound 4 was prepared from 29 (15.0 mg, 11.1 µmol) as described for preparation of 2, yielding 8.2 mg (89%). [α]_D²⁵ +17.8 (c = 0.2, D₂O). ¹H-NMR (600 MHz, D₂O) δ: 5.16 (1H, d, J_1,2 = 3.8 Hz, H-1 of Gal a), 4.59 (1H, d, J_1,2 = 7.7 Hz, H-1 of GalN), 4.54 (1H, d, J_1,2 = 8.5 Hz, H-1 of Gal c), 4.48 (1H, d, J_1,2 = 7.7 Hz, H-1 of Gal b), 2.02 (3H, s, NAc). ¹³C-NMR (150 MHz, D₂O) δ: 177.8, 177.7, 175.1, 105.2 (C-1 of Gal c), 103.7 (C-1 of Gal b), 101.8 (C-1 of Gal N), 95.9 (C-1 of Gal a), 79.7, 76.3, 75.8, 75.7, 75.4, 73.5, 73.2, 71.7, 71.5, 70.8, 70.6, 69.7, 69.3, 69.2, 68.9, 67.7, 64.6, 62.7, 61.7, 61.6, 60.9, 56.0, 51.4, 40.4, 39.8, 39.2, 36.5, 36.2, 29.0, 28.5, 28.4, 25.8, 25.7, 25.5, 25.4, 23.1. ESI-HR-MS: Calcd for C₄₄H₇₅N₅O₂₄SNa, m/z 1112.4420. Found, m/z 1112.4464 [M + Na]⁺.

Phenyl 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-thio-β-D-galactopyranoside (32)

Compound 32 was prepared from 30 (408 mg, 0.70 µmol) and 31 (205 mg, 0.35 µmol) as described for preparation of 18. The product was purified by silica gel column chromatography (3 : 1 toluene–EtOAc) to give 32 (310 mg, 88%). [α]_D²⁴ +104.4 (c = 1.8, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 7.64–7.18 (24H, m, Ph), 5.76–5.72 (2H, m, H-4, 4′), 5.57 (1H, t, J_2′,3′ = J_3′,4′ = 9.5 Hz, H-3′), 5.48 (1H, d, J_1′,2′ = 8.5 Hz, H-1′), 5.45 (1H, dd, J_2′,3′ = 10 Hz, J_3,4 = 3.0 Hz, H-3), 5.15 (1H, t, J_1,2 = J_2,3 = 9.0 Hz, H-2), 4.85 (1H, d, H-1), 4.35 (1H, dd, H-2′), 4.21–4.10 (3H, m, H-6′a, H-6′b, H-5), 3.91 (1H, dd, J_6a,6b = 11.0 Hz, H-6a), 3.82–3.77 (2H, m, H-6b, H-5′), 2.04 (9H, each s, 3 × OAc). ¹³C-NMR (125 MHz, CDCl₃) δ: 170.6, 170.1, 169.4, 165.3, 165.1, 165.0, 134.1, 134.0, 133.4, 133.2, 133.1, 131.4, 130.9, 129.8, 129.7, 129.7, 129.3, 128.9, 128.8, 128.7, 128.5, 128.3, 128.3, 128.2, 123.7, 98.2 (C-1′), 85.0 (C-1), 76.5 (C-5), 72.9 (C-3), 72.0 (C-5′), 70.8 (C-3′), 68.8 (C-4′), 68.6 (C-4) 68.5 (C-6), 67.7 (C-2), 61.7 (C-6′), 54.4 (C-2′), 20.7, 20.6, 20.4. ESI-HR-MS: Calcd for C₅₃H₄₇NO₁₇SNa, m/z 1024.2462. Found: m/z 1024.2455 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2-O-benzoyl-3-O-2-napthylmethyl-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (33)

Compound 33 was prepared from 32 (201 mg, 0.20 mmol) and 14 (124 mg, 0.13 mmol) as described for preparation of 12. The product was purified by silica gel column chromatography (3 : 1 toluene–EtOAc) to give 33 (178 mg, 73%). [α]_D²⁴ +128.1 (c = 0.8, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 8.08–6.94 (40H, m, Ph), 5.82–5.14 (7H, m, H-2a, 2c, 3c, 3 of GlcN, 4c, 4 of GlcN, PhCH), 5.46 (1H, d, J_1,2 = 8.4 Hz, H-1 of GlcN), 5.36 (1H, d, J_1,2 = 3.8 Hz, H-1 of Gal a), 5.24 (1H, d, J_1,2 = 8.4 Hz, H-1 of GalN), 4.78 (1H, dd, H-2 of GalN), 4.63 (1H, dd, H-3 of GalN), 4.56 (1H, d, J_1,2 = 7.9 Hz, H-1 of Gal b) 3.61 (3H, s, OMe), 2.09, 2.03, 1.86 (9H, each s, 3 × OAc), 2.08–1.12 (8H, m, –OCH₂CH₂CH₂CH₂CH₂–). ¹³C-NMR (125 MHz, CDCl₃) δ: 177.5, 173.9, 170.7, 170.1, 169.5, 168.9, 167.6, 166.3, 165.5, 165.1, 147.4, 134.9, 134.9, 134.5, 134.4, 134.2, 133.6, 133.32, 133.3, 133.1, 133.0, 132.8, 131.7, 131.6, 131.2, 130.0, 129.9, 129.8, 129.6, 129.4, 129.4, 129.0, 128.8, 128.6, 128.5, 128.4, 128.3, 127.9, 127.7, 127.6, 126.0, 125.9, 125.8, 125.3, 123.8, 123,4, 100.8 (C-1 of Gal c), 100.2, 98.2 (C-1 of GalN), 98.2 (C-1 of GlcN), 94.0 (C-1 of Gal a), 75.6, 72.9, 72.1, 71.8, 71.7, 71.6, 70.7, 70.1, 69.9, 69.2, 69.0, 68.8, 68.7, 68.4 (C-6 of Gal a), 67.8, 66.4, 66.3, 65.5, 61.8, 54.5, 52.4, 51.4, 33.8, 29.5, 29.0, 25.4, 24.5, 20.8, 20.3, 20.2. ESI-HR-MS: Calcd for C₉₉H₉₄N₂O₃₂Na, m/z 1845.5687. Found: m/z 1845.5631 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,4-di-O-benzoyl-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (34)

To a solution of 33 (159 mg, 87.2 µmol) in Pyr. (5.0 mL) was added benzoyl chloride (50.2 µL, 0.43 mmol) at 0°C, and the reaction mixture was stirred for 14 h at 25°C. Toluene was added and evaporated. A mixture of the residue and DDQ (58.4 mg, 0.26 mmol) in CH₂Cl₂–H₂O (19 : 1, 2 mL) was stirred for 15 h at room temperature. After completion of the reaction, the mixture was filtered and the filtrate was diluted with water and extracted with CHCl₃. The extract was washed with water, dried (MgSO₄), and concentrated. The product was purified by silica gel column chromatography using 5 : 1 toluene–EtOAc as eluent to give 34 (88 mg, 56%). ¹H-NMR (500 MHz, CDCl₃) δ: 8.06–6.96 (38H, m, Ph), 5.77–5.10 (8H, m, H-2a, 2c, 3c, 3 of GlcN, 4a, 4c, 4 of GlcN, PhCH), 5.30 (d, 1H, J_1,2 = 8.5 Hz, H-1 of GlcN), 5.24 (d, 1H, J_1,2 = 8.3 Hz, H-1 of Gal GalN), 5.10 (d, 1H, J_1,2 = 3.0 Hz, H-1 of Gal a), 4.80 (1H, dd, H-2 of GalN), 4.67 (1H, dd, H-3 of GalN), 4.53 (d, 1H, J_1,2 = 7.3 Hz, H-1 of Gal c), 3.60 (3H, s, OMe), 2.06, 2.02, 1.86 (9H, each s, 3 × OAc), 2.14–1.10 (8H, m, –OCH₂CH₂CH₂CH₂CH₂–). ¹³C-NMR (125 MHz, CDCl₃) δ: 177.2, 172.5, 172.1, 171.4, 170.7, 168.8, 168.1, 167.5, 167.1, 166.9, 166.7, 166.1, 170.0, 165.6, 165.5, 164.9, 147.1, 137.2, 134.2, 134.6, 133.5, 133.1, 133.0, 133.2, 131.6, 130.2, 129.7, 129.8, 129.6, 129.3, 129.1, 129.3, 129.1, 128.7, 128.6, 128.5, 128.5, 128.4, 128.2, 128.2, 127.9, 125.9, 125.3, 123.2, 123.0, 100.8 (C-1 of Gal c), 100.2, 98.2 (C-1 of Gal a), 98.2 (C-1 of GlcN), 94.0 (C-1 of Gal GlcN), 75.5, 72.9, 72.2, 71.4,, 71.6, 70.9, 70.0, 69.9, 69.5, 69.0, 68.8, 68.5, 68.4, 68.0 67.6, 66.3, 66.3, 65.5, 61.8, 54.5, 52.4, 51.4, 33.8, 29.7, 29.0, 25.3, 24.5, 20.9, 20.3, 20.0. ESI-HR-MS: Calcd for C₉₅H₉₀N₂O₃₃Na, m/z 1809.5324. Found: m/z 1809.5367 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranosyl-(1→6)-[2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→3)]-2,4-di-O-benzoyl-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (35)

Compound 35 was prepared from 34 (88 mg, 49.2 µmol) and 17 (70.4 mg, 143 µmol) as described for preparation of 18. The product was purified by silica gel column chromatography (3 : 1 toluene–EtOAc) to give 35 (47 mg, 45%). [α]_D²⁴ +149.5 (c = 0.4, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ: 5.44 (1H, d, J_1,2 = 8.4 Hz, H-1 of GlcN), 5.35 (1H, d, J_1,2 = 3.8 Hz, H-1 of Gal a), 5.22 (1H, d, J_1,2 = 7.8 Hz, H-1 of Gla c), 5.10 (1H, d, J_1,2 = 8.4 Hz, H-1 of GalN), 4.55 (1H, d, J_1,2 = 7.9 Hz, H-1 of Gal b). ¹³C-NMR (125 MHz, CDCl₃) δ: 177.2, 172.5, 171.1, 170.2, 168.8, 168.1, 167.1, 166.7, 165.6, 165.4, 164.9, 147.3, 137.1, 134.6, 134.4, 133.8, 133.1, 133.0, 131.6, 130.2, 129.7, 129.6, 129.2, 129.0, 128.7, 128.6, 128.5, 128.5, 128.4, 128.2, 128.2, 127.9, 125.9, 125.3, 123.6, 123.0, 100.7 (C-1 of Gal b), 100.8 (C-1 of Gal c) 100.2, 98.8 (C-1 of GalN), 98.0 (C-1 of GlcN), 93.6 (C-1 of Gal a), 75.5, 74.1, 72.9, 72.1, 72.0, 71.52, 71.48, 71.6, 70.9, 70.0, 69.9, 69.5, 69.14, 69.05, 69.0, 68.8, 68.5, 68.4, 68.0 67.6, 66.2, 66.3, 65.6, 61.8, 54.4, 52.4, 51.1, 33.8, 29.7, 29.0, 25.3, 24.5, 20.9, 20.3, 20.0. ESI-HR-MS: Calcd for C₁₀₉H₁₀₈N₂O₄₂Na, m/z 2139.6274. Found: m/z 2139.6298 [M + Na]⁺.

5-(Methoxycarbonyl)pentyl 2-Acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-β-D-galactopyranosyl-(1→6)-[β-D-galactopyranosyl-(1→3)]-α-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-galactopyranoside (36)

A solution of 35 (40.5 mg, 19.1 µmol) in 80% AcOH (1.3 mL) was stirred at 80°C for 3 h, then was diluted with toluene and concentrated. The residue was deproducted of phthaloyl group with ethylenediamine (0.5 mL) in BuOH (2.0 mL). After concentration, the residue was acetylated with acetic anhydride (0.4 mL) in pyridine (2.0 mL) at 45°C for 13 h. After the reaction, toluene was added and co-evaporated several times. The product was purified by silica gel column chromatography (1 : 1 toluene–acetone) . To a solution of the residue in 1,4-dioxane–MeOH (1 : 1, 1.0 mL) was added NaOMe (20 mg) at room temperature and the mixture was stirred at 40°C for 15 h, then neutralized with Amberlite IR 120 [H⁺]. The mixture was filtered off and concentrated. The product was purified by Sephadex LH-20 column chromatography in H₂O to give 36 (6.8 mg, 34% 4 steps). [α]_D²⁵ +22.1 (c = 0.2, D₂O). ¹H-NMR (600 MHz, D₂O) δ: 5.15 (1H, d, J_1,2 = 3.8 Hz, H-1 of Gal a), 4.59 (1H, d, J_1,2 = 7.7 Hz, H-1 of Gal c), 4.56 (1H, d, J_1,2 = 8.5 Hz, H-1 of GlcN), 4.53 (1H, d, J_1,2 = 8.8 Hz, H-1 of GalN), 4.47 (1H, d, J_1,2 = 8.0 Hz, H-1 of Gal b), 3.70 (3H, s, OMe), 2.07, 2.05 (6H, each s, 2 × NAc), 2.41–1.31 (8H, m, –OCH₂CH₂CH₂CH₂CH₂–). ¹³C-NMR (150 MHz, D₂O) δ: 178.2, 175.2, 175.1, 105.2 (C-1 of Gal c), 103.7 (C-1 of Gal b), 102.0 (C-1 of GalN), 101.9 (C-1 of GlcN), 95.9 (C-1 of Gal a), 79.6, 76.5, 76.3, 75.7, 75.4, 74.4, 74.2, 73.3, 73.2, 71.7, 71.5, 70.8, 70.6, 70.5, 69.6, 69.3, 69.3, 69.2, 68.8, 67.7, 64.6, 61.6, 61.4, 56.1, 52.8, 51.4, 39.7, 34.3, 30.9, 28.9, 25.4, 24.6, 23.1, 22.9. ESI-HR-MS: Calcd for C₄₁H₇₀N₂O₂₈Na, m/z 1061.4013. Found: m/z 1061.4064 [M + Na]⁺.

Biotinylated Compound (5)

Compound 5 was prepared from 36 (7.5 mg, 11.1 µmol) as described for preparation of 1. The product was purified by Sephadex LH-20 column chromatography (H₂O) to give 5 (4.3 mg, 86%). [α]_D²⁵ +28.4 (c = 0.1, D₂O). ¹H-NMR (500 MHz, D₂O) δ: 5.15 (1H, d, J_1,2 = 4.1 Hz, H-1 of Gal a), 4.58 (1H, d, J_1,2 = 8.0 Hz, H-1 of GalN), 4.55 (1H, d, J_1,2 = 8.5 Hz, H-1 of Gal c), 4.53 (1H, d, J_1,2 = 8.5 Hz, H-1 of GlcN). 4.46 (1H, d, J_1,2 = 7.7 Hz, H-1 of Gal b), 2.06, 2.04 (6H, each s, 2 × NAc). ¹³C-NMR (125 MHz, D₂O) δ: 215.4. 177.1, 177.08, 174.5, 174.4, 165.4, 104.5 (C-1 of Gal c), 103.0 (C-1 of Gal b), 101.4 (C-1 of GalN), 101.2 (C-1 of GlcN), 95.3 (C-1 of Gal a), 79.0, 75.9, 75.7, 75.1, 74.7, 73.7, 73.6, 72.7, 72.5, 71.1, 70.9, 70.2, 69.95, 69.91, 68.9, 68.69, 68.67, 68.6, 68.5, 68.1, 67.1, 64.0, 62.1, 61.0, 60.7, 60.3, 55.5, 55.4, 50.7, 39.7, 39.2, 38.7, 38.68, 38.60, 35.8, 35.6, 30.2, 28.4, 28.0, 27.9, 27.7, 25.2, 25.0, 24.9, 24.8, 22.5, 22.3, 20.1. HR-ESI-MS: Calcd for C₅₂H₈₈N₆O₂₉SNa: m/z 1315.5214. Found: m/z 1315.5237 [M + Na]⁺.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research (No. 25460131) and by Ministry of Education, Culture, Sports, Science and Technology (MEXT)-supported program for the strategic research foundation at private universities (centers of excellence for research) in “molecular nanotechnology for green innovation,” FY 2012–2016.

Conflict of Interest

The authors declare no conflict of interest.

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