Synthesis and Antigenicity against Human Sera of a Biotin-Labeled Oligosaccharide Portion of a Glycosphingolipid from the Parasite Echinococcus multilocularis

Abstract

Synthesis of a biotinylated analog of the carbohydrate portion of a glycosphingolipid from the parasite Echinococcus multilocularis has been achieved. We synthesized β-D-Galp-(1→6)-β-D-Galp-(1→6)-[α-L-Fucp-(1→3)]-β-D-Galp-(1→R: biotin probe) (1) and compared the antigenicity by an enzyme linked immunosorbent assay (ELISA) with biotinylated trisaccharide α-D-Galp-(1→4)-β-D-Galp-(1→3)-α-D-Galp-(1→R: biotin probe) (F), which has been shown to have significant antigenicity. Both of the oligosaccharides reacted with sera of alveolar echinococcosis (AE) patients, but showed different reactivity. Among the 60 sera of AE patients, more sera reacted with the linear sequence Galα1→4Galβ1→3GalNAcα1→R of oligosaccharide (F) than for branched compound 1. Some sera showed high specificity to one of the compound, indicating that the antibodies in the sera of AE patients differ in their specificity to recognize carbohydrate sequences of glycosphingolipids. Our results demonstrate that both of the biotinylated oligosaccharides 1 and F have good serodiagnostic potential and are complementary to detect infections caused by the parasite Echinococcus multilocularis.

In our continuing studies to elucidate the biological function of carbohydrate moieties of glycosphingolipids (GSLs) and glycoproteins (GPs) from invertebrates, we have synthesized oligosaccharides found in various Protostomia phyla.^1–16) Our interests have been focused on the unique saccharide chain structure of GSLs and GPs found in several parasites, Echinococcus multilocularis,^5,6,8,12,16) Schistosoma mansoni,^3,7) Ascaris suum^13,14) and Toxocara canis.⁴⁾ Among them, we are strongly interested in the structures of GSLs and GPs from E. multilocularis. E. multilocularis is a parasite, which belongs to the class Cestoda of the phylum Platyhelminthes and causes alveolar echinococcosis (AE), a severe parasitic zoonosis that can be fatal without appropriate treatment. Therefore, a simple and reliable diagnosis method is important. Diagnosis of AE is based on immunological response, detected by enzyme-linked immunosorbent assay (ELISA) for primary screening and by the Western blotting method for secondary confirmation tests, of the serum of patients to the crude antigen extracted from E. multilocularis cysts.^17–19) However, no perfect antigen exists for the serological detection of AE. Persat et al. reported^20,21) that sera from AE patients recognized a neutral glycosphingolipid fraction from E. multilocularis and determined the structures of some of the glycosphingolipids isolated from this fraction. Furthermore, Hülsmeier et al. reported²²⁾ that Em2, an antigen extracted from E. multilocularis, is a mucin-type glycoprotein. Based on these information, we synthesized four glycosphingolipids¹²⁾ and five carbohydrate structures of glycoproteins^6,8) of E. multilocularis, and examined antigenicity of the pure compounds by ELISA for their serodiagnostic potential.^5,6,23) Among the synthesized compounds (A–I), a glycosphingolipid, Galβ1→6(Fucα1→3)Galβ1→6Galβ1-Cer (D), and a biotinylated carbohydrate, Galα1→4Galβ1→3GalNAcα1-biotin-probe (F), showed good serodiagnostic potential for AE (Fig. 1). However, the potential of the two compounds cannot be compared directly because of the difference of the reducing end structure, a ceramide and a biotin probe. In this paper, we describe the synthesis of the biotinylated analog of D (1) and compare the antigenicity against sera of AE patients with compound F.

Fig. 1. Structures of the Compounds A–I, Derivatives of the Oligosaccharides from the Parasite Echinococcus multilocularis, Synthesized in the Previous Report

Results and Discussion

Chemistry

The synthetic strategy for oligosaccharide 1 is shown in Chart 1. Suitably protected monosaccharide derivatives (2–5) were chosen as building blocks. As a temporary protecting group of the reducing end for the target compound, 5-(methoxycarbonyl)pentyl group was chosen to ensure future conjugation to biotin for ELISA. The synthetic route for the target compound 1 is shown in Charts 2–4. Initially, reducing end monosaccharide acceptor 2 was prepared from commercially available 1,2,3,4,6-pentaacetyl-β-D-galactose in a five-step process (Chart 2). At first, coupling of 1,2,3,4,6-pentaacetyl-β-D-galactose with methyl 6-hydroxyhexanoate in the presence of SnCl₄ in dry CH₂Cl₂ for 16 h at room temperature gave compound 6 (47%). Compound 6 was converted to the glycosyl acceptor 2 by a series of reactions involving deacetylation, protection of primary alcohol with tert-butyldiphenylsilyl (TBDPS) chloride and benzoylation (7) (57%, 3 steps), followed by removal of the TBDPS group (94%). Acceptor 3 was prepared from known 2-(trimethylsilyl)ethyl 3-O-(2-naphthylmethyl)-β-D-galactopyranoside (8)²⁴⁾ by the following five-step procedure. Protection of primary alcohol with tert-butyldimethylsilyl (TBDMS) chloride, followed by benzoylation gave compound 9 (79%, 2 steps). Selective removal of the 3-O-naphthylmethyl (Nap) group from 9 by 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) followed by chloroacetylation produced compound 10 (86%, 2 steps), and finally deprotection of the TBDMS group in 10 under an acidic condition by TsOH afforded the acceptor 3 in 81% yield (Chart 3).

Chart 1. Synthetic Strategy of Compound 1

Chart 2. Preparation of Monosaccharide Acceptor 2

Chart 3. Preparation of Monosaccharide Acceptor 3

Synthesis of the oligosaccharide 1 was initiated by coupling of glycosyl donor 4²⁵⁾ to galactose-based acceptor 3 in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf)²⁶⁾ to produce disaccharide 13 in 90% yield. The β-glycosidic linkage in 13 was confirmed by ¹H-NMR spectroscopy. The anomeric proton of the nonreducing end galactose moiety appeared as a doublet with a homonuclear coupling constant of 7.8 Hz. Selective removal of 2-(trimethylsilyl)ethyl (TMS-ethyl) group in 13 with trifluoroacetic acid (TFA) in CH₂Cl₂, followed by treatment with CCl₃CN in the presence of 1,8-diazabicyclo[5,4,0]-7-undecene (DBU)²⁷⁾ provided α-trichloroacetimidate 14. Glycosylation of disaccharide donor 14 with acceptor 2 in the presence of TMSOTf and MS4Å in CH₂Cl₂ produced desired trisaccharide 15 in 90% yield. Removal of the 3-O-chloroacetyl group in 15 was achieved with thiourea yielding trisaccharide acceptor 16 in 93% yield. Glycosylation of the acceptor 16 with the donor 5²⁸⁾ in the presence of N-iodosuccinimide (NIS), trifluoromethanesulfonic acid²⁹⁾ and MS 4 Å in CH₂Cl₂ afforded desired disaccharide 17 in 99% yield, as evidenced by ¹H-NMR spectroscopy (H-1 of Fuc δ 5.06 ppm, J=3.6 Hz). Deprotection of the benzyl group in 17 under neutral condition by hydrogenation over 10% Pd–C, and removal of the benzoyl groups under the Zemplén condition, followed by purification by column chromatography on Sephadex LH-20, furnished desired precursor 18. Conversion of 18 into its ethylenediamine monoamide³⁰⁾ by exposure to ethylenediamine followed by conjugation with N-hydroxy succinimide ester of biotin (biotin-NHS) afforded the target biotinylated tetrasaccharide 1 after column chromatographic purification on Sephadex LH-20 (Chart 4). The structure and purity of 1 was demonstrated by its ¹H-NMR and high resolution-electrospray ionization (HR-ESI)-MS data.

Char 4. Synthesis of Tetrasaccharide 1

Antigenicity of the Oligosaccharides

Reactivity of the oligosaccharides 1 and F to sera of AE patients was examined by ELISA using microplates coated with streptavidin (Fig. 2). As reported previously,⁶⁾ compound F showed strong response to the AE group. On the contrary, although some of the patients’ sera reacted strongly and the average of absorbance values of AE group (n=60) was significantly higher than that of the NH group (n=60) (p<0.001, Student’s t-test), the average reactivity of compound 1 was not strong compared with that of F. However, comparison of the reactivity of individual serum against 1 and F revealed that each serum showed different reactivity to the oligosaccharides (Fig. 3). Most of the sera which reacted strongly (absorbance >0.5) with F showed weak reactivity (absorbance <0.5) with 1. On the contrary, the sera which reacted strongly (absorbance >0.5) with 1 showed less reactivity to F except for 2 sera. Ten of the sera reacted to F more than 10 fold (maximum 25 fold) compared with 1 and one serum showed specificity to 1 (12 fold compared with F). These results indicate that the specificity of the antibodies in the sera is not uniform, and the two oligosaccharides are complementary as the antigen for serological diagnosis of AE. The average reactivity of F was stronger than that of 1, suggesting that the linear trisaccharide structure of F is important as a core structure for the antigenicity.⁶⁾ However, further studies will be necessary to elucidate structure–antigenicity relationship of oligosaccharides found in E. multilocularis.

Fig. 2. ELISA Reaction of the Oligosaccharides 1 and F with Human Sera

AE: alveolar echinococcosis patient group; NH: normal healthy group 1–3: compound 1 with AE sera; 4–6: compound F with AE sera; 7–9: compound 1 with NH sera; 10–12: compound F with NH sera.

Fig. 3. Correlation of the Reactivity of Each Serum to Oligosaccharides 1 and F

Conclusion

We prepared oligosaccharide-biotin conjugate 1 and compared the antigenicity with the previously synthesized oligosaccharide derivative F by ELISA for their serodiagnostic potential. Sixty sera of AE patients showed different reactivity to the two oligosaccharides, which indicated that the antibodies in the sera of AE patients recognize a different glycosphingolipid sequences. As the antigenicity of the two oligosaccharides was complementary, a mixture of the two compounds will show superior serodiagnostic potential for diagnosis of AE than a single compound.

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. ESI-HR-MS was recorded on a JEOL MS T-100 mass spectrometer. 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 carried out on Silica Gel 60 (E. Merck). 2,3,4,6-Tri-O-benzoyl-α-D-galactopyranosyl trichloroacetimidate (4),²⁵⁾ 2-(trimethylsilyl)ethyl 3-O-(2-naphthylmethyl)-β-D-galactopyranoside (8),²⁴⁾ phenyl 2,3,4-tri-O-benzyl-l-thio-α-L-fucopyranoside (5)²⁹⁾ were prepared as reported. 1,2,3,4,6- Pentaacetyl-β-D-galactose was purchased from Tokyo Chemical Industry Co.

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranoside (6)

To a solution of 1,2,3,4,6-penta-O-acetyl-β-D-galactopyranose (500 mg, 1.28 mmol) in CH₂Cl₂ (5.0 mL) cooled at 0°C were added methyl 6-hydroxyhexanoate (277 mg, 1.92 mmol), and 4 Å powdered molecular sieves (250 mg) and SnCl₄ (150 µL, 1.28 mmol). The mixture was stirred for 17 h at room temperature. Et₃N (0.1 mL) was added, and the mixture was filtered, washed with cold water, dried (MgSO₄), and concentrated. The residue was purified by silica gel column chromatography using 2 : 1 hexane–EtOAc as eluent to give 6 (288 mg, 47%). [α]_D²⁵ −14.9° (c=0.1, CHCl₃). ¹H-NMR (CDCl₃) δ: 5.39 (1H, d, J_3,4=3.1 Hz, H-4), 5.20 (1H, dd, J_2,3=10.3 Hz, J_1,2=7.9 Hz, H-2), 5.02 (1H, dd, H-3), 4.46 (1H, d, H-1), 4.20–4.11 (2H, m, H-6a, b), 3.92–3.87 (2H, m, H-5, –OCH₂CH₂CH₂CH₂CH₂–), 3.67 (3H, s, –OCH₃), 3.50–3,46 (1H, m, –OCH₂CH₂CH₂CH₂CH₂–), 2.15–1.99 (12H, m, Ac×4). ¹³C-NMR (CDCl₃) δ: 173.9, 170.3, 170.2, 170.1, 169.2, 101.2 (C-1), 70.8 (C-3), 70.5 (C-5), 69.7 (–OCH₂CH₂CH₂CH₂CH₂–), 68.8 (C-2), 67.0 (C-4), 61.2 (C-6), 51.4 (CH₃), 33.8, 29.0, 25.3, 24.5, 20.61, 20.55, 20.5. HR-ESI-MS: Calcd for C₁₂H₃₂O₁₂Na: m/z 499.1791. Found: 499.1793 [M+Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4-Tri-O-benzoyl-6-O-tert-butyldiphenylsilyl-β-D-galactopyranoside (7)

To a solution of 6 (270 mg, 0.57 mmol) in MeOH (5.0 mL) was added NaOMe (10 mg) and stirred for 30 min at room temperature, then neutralized with Amberlite IR 120 [H⁺]. The mixture was filtered and concentrated to give a residue. To a solution of this residue in N,N-dimethylformamide (DMF) (2.5 mL) was added imidazole (73.5 mg 1.08 mmol) and tert-butyldiphenylsilyl-chloride (169 µL, 0.65 mmol) at 0°C, and the reaction mixture was stirred for 3 h at 0°C. The mixture was diluted with 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 toluene–acetone as eluent to give 5-(methoxycarbonyl)pentyl 6-O-tert-butyldiphenylsilyl-β-D-galactopyranoside. To a solution of this compound in pyridine (2 mL) was added benzoyl chloride (198 µL, 1.70 mmol). The mixture was stirred for 18 h at 0°C, then diluted with CHCl₃, washed with 5% HCl, aq NaHCO₃ and water, dried (MgSO₄), and concentrated. The product was purified by silica gel column chromatography using 20 : 1 toluene-acetone as eluent to give compound 7 (279 mg, 57% 3 steps); [α]_D²⁵ +80.6° (c=1.1, CHCl₃). ¹H-NMR (CDCl₃) δ: 8.04–7.09 (25H, m, 5×Ph), 6.04 (1H, d, J_3,4=3.4 Hz, H-4), 5.70 (1H, d, J_1,2=7.6 Hz, J_2,3=10.4 Hz, H-2), 5.62 (1H, dd, H-3), 4.72 (1H, d, H-1), 4.06 (1H, m, H-5), 3.91 (1H, dt, –OCH₂CH₂CH₂CH₂CH₂), 3.88–3.82 (2H, m, H-6a, b), 3.60 (3H, s, –OCH₃), 3.49 (1H, dt, –OCH₂CH₂CH₂CH₂CH₂). ¹³C-NMR (CDCl₃) δ: 173.9, 165.6, 165.4, 165.2, 135.5, 135.4, 133.2, 133.1, 132.9, 132.5, 130.0, 129.8, 129.7, 129.6, 129.53, 129.48, 129.45, 129.0, 128.5, 128.3, 128.2, 127.7, 127.6, 101.6 (C-1), 73.8 (C-5), 71.9 (C-3), 70.1 (C-2), 69.8 (–OCH₂CH₂CH₂CH₂CH₂–), 67.9 (C-4), 61.3 (C-6), 51.4 (CH₃), 33.7, 29.0, 26.6, 25.3, 24.4, 19.0. HR-ESI-MS: Calcd for C₅₀H₅₄O₁₁NaSi: m/z 881.3333. Found: 881.3324 [M+Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4-Tri-O-benzoyl-β-D-galactopyranoside (2)

A solution of 7 (279 mg 0.33 mmol) and acetic acid (92 µL, 1.63 mmol) in tetrahydrofuran (THF) (1.6 mL) was added 1 M tetrabutylammonium fluoride (TBAF) in THF (640 µL, 0.65 mmol) at 0°C and then stirred for 18 h. After concentration of the mixture, the residue was dissolved in water, extracted with CHCl₃, washed with aq NaHCO₃ and water, dried (MgSO₄), and concentrated. The product was purified by silica gel column chromatography using 27 : 7 toluene–EtOAc as eluent to give 2 (192 mg, 94%): [α]_D²⁵ +112.6° (c=0.2, CHCl₃). ¹H-NMR (CDCl₃) δ: 8.11–7.18 (15H, m, 3×Ph), 5.85–5.81 (2H, m, H-2, 4), 5.61 (1H, dd, J_2,3=3.0 Hz, J_3,4=10.0 Hz, H-3), 4.80 (1H, d, J_1,2=8.0 Hz), 4.05–4.02 (1H, m, H-5), 4.00–3.94 (1H, m, H-6a), 3.84 (1H, m, –OCH₂CH₂CH₂CH₂CH₂–), 3.67 (1H, m, –OCH₂CH₂CH₂CH₂CH₂–), 3.69–3.54 (4H, m, H-6b, –OCH₃). ¹³C-NMR (CDCl₃) δ: 174.0, 166.8, 165.5, 165.3, 133.8, 133.3, 133.2, 130.1, 129.8, 129.7, 129.6, 129.3, 129.0, 128.72, 128.70, 128.6, 128.4, 128.3, 128.2, 101.7 (C-1), 74.0 (C-5), 71.8 (C-3), 70.1 (C-2), 70.0 (C-6), 69.0 (C-4), 60.6 (–OCH₂CH₂CH₂CH₂CH₂–), 51.4 (CH₃), 33.7, 29.0, 25.3, 24.4. HR-ESI-MS: Calcd for C₃₄H₃₆O₁₁Na: m/z 643.2155. Found: 643.2151 [M+Na]⁺.

2-(Trimethylsilyl)ethyl 2,4-Di-O-benzoyl-6-O-tert-butyldiphenylsilyl-3-O-naphthylmethyl-β-D-galactopyranoside (9)

To a solution of 8 (605 mg, 1.44 mmol) in DMF (6.5 mL) was added imidazole (198 mg, 2.88 mmol) and tert-butyldimethylsilyl chloride (260 mg, 1.73 mmol) at 0°C, and the reaction mixture was stirred for 3 h at 0°C. The mixture was diluted with CHCl₃, washed with 5% HCl, aq NaHCO₃ and water, dried (MgSO₄), and concentrated. To a solution of the residue (628 mg) in pyridine (8 mL) was added benzoyl chloride (0.41 mL, 3.5 mmol). The mixture was stirred for 15 h at 0°C, then diluted with 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 hexane–EtOAc as eluent to give compound 9 (845 mg, 79%); [α]_D²⁵ +82.0° (c=0.6, CHCl₃). ¹H-NMR (CDCl₃) δ: 8.12–7.14 (17H, m, Ph), 5.87 (1H, d, J_3,4=3.3 Hz, H-4), 5.44 (1H, dd, J_1,2=8.0 Hz, J_2,3=10.0 Hz, H-2), 4.80 and 4.63 (2H, each d, naphthylmethylene), 4.48 (1H, d, H-1), 3.93 (1H, dt, –OCH₂CH₂–), 3.77–3.66 (4H, m, H-3, 5, 6a, b), 3.45 (1H, dt, –OCH₂CH₂–), 0.88–0.74 (11H, m, –OCH₂CH_2–, Si(CH₃)₃), –0.16 (15H, m, TBDMS). ¹³C-NMR (CDCl₃) δ: 165.8, 165.2, 135.0, 133.1, 133.0, 132.9, 130.2, 130.0, 129.9, 129.8, 128.4, 128.2, 128.1, 127.8, 127.6, 126.8, 126.1, 125.9, 125.8, 101.0 (C-1), 76.3 (C-3), 74.2 (C-5), 71.5 (naphthylmethylene), 70.8 (C-2), 67.3 (–OCH₂CH₂), 66.3 (C-4), 61.3 (C-6), 25.8, 18.2, 17.9, –1.6, –5.58, –5.62. HR-ESI-MS: Calcd for C₄₂H₅₄NaO₈Si₂: m/z 765.3255. Found: 765.3291 [M+Na]⁺.

2-(Trimethylsilyl)ethyl 2,4-Di-O-benzoyl-6-O-tert-butyldiphenylsilyl-3-O-chloroacethyl-β-D-galactopyranoside (10)

A solution of 9 (208 mg, 0.28 mmol) in CH₂Cl₂–H₂O (19 : 1, 2.0 mL) was added DDQ (127 mg, 0.56 mmol) at room temperature and then stirred for 17 h. The precipitates were filtrated off and washed with CHCl₃. After concentration, the residue was dissolved in water, extracted with CHCl₃, washed with saturated aqueous NaHCO₃, dried (MgSO₄), and concentrated. The product was purified by silica gel column chromatography (6 : 1 hexane–AcOEt) as eluent to give 6-hydroxyl compound (148 mg). To a solution of this compound in CH₂Cl₂ (2.4 mL) was added ClCH₂COCl (39 µL, 0.50 mmol) and pyridine (0.4 mL). The reaction mixture was stirred for 45 min. at 0°C, then diluted with 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 hexane–EtOAc as eluent to give compound 10 (164 mg, 86% 2 steps). [α]_D²⁵ +58.1° (c=0.5, CHCl₃). ¹H-NMR (CDCl₃) δ: 8.06–7.21 (10H, m, 2×Ph), 5.71 (1H, d, J_3,4=3.4 Hz, H-4), 5.48 (1H, dd, J_1,2=7.9 Hz, J_2,3=10.6 Hz, H-2), 5.33 (1H, dd, H-3), 4.65 (1H, d, H-1), 3.96 (1H, dt, –OCH₂CH₂–), 3.87 (1H, br t, H-5), 3.85 and 3.78 (2H, each d, J=14.7 Hz, –OCH₂Cl), 3.77–3.65 (2H, m, H-6a, b), 3.52 (1H, dt, –OCH₂CH₂–), 0.76–0.74 (2H, m, –OCH₂CH₂–), −0.10 and −0.16 (15H, m, –OSi(CH₃)₂, –C(CH₃)₃), –0.14 (9H, m, Si(CH₃)₃). ¹³C-NMR (CDCl₃) δ: 206.9, 166.8, 165.8, 165.0, 133.4, 133.2, 129.9, 129.7, 128.5, 128.4, 100.9 (C-1), 73.6 (C-5), 73.3 (C-3), 69.7 (C-2), 67.6 (–OCH₂CH₂–), 67.5 (C-4), 60.6 (C-6), 40.5 (ClCH₂CO–), 30.8, 25.6, 18.0 (–OCH₂CH₂–), −1.6, −5.7, −5.8. HR-ESI-MS: Calcd for C₃₃H₄₇ClO₉NaSi₂: m/z 701.2345. Found: 701.2398 [M+Na]⁺.

2-(Trimethylsilyl)ethyl 2,4-Di-O-benzoyl-3-O-chloroacetyl-β-D-galactopyranoside (3)

A solution of 10 (113 mg, 0.17 mmol) in CH₂Cl₂–MeOH (2 : 1, 1.8 mL) was added TsOH (9.5 mg, 0.51 mmol) at room temperature and then stirred for 14 h. Et₃N was added and concentrated. The product was purified by silica gel column chromatography (3 : 1 hexane–ethyl acetate) to give 3 (78 mg, 81%). [α]_D²⁵ +98.5° (c=0.4, CHCl₃). ¹H-NMR (CDCl₃) δ: 8.07–7.20 (10H, m, 2×Ph), 5.66 (1H, d, J_3,4=3.4 Hz, H-4), 5.58 (1H, dd, J_1,2=7.8 Hz, J_2,3=10.2 Hz, H-2), 5.38 (1H, dd, H-3), 4.70 (1H, d, H-1), 3.98 (1H, dt, –OCH₂CH₂–), 3.91 (1H, t, J_5,6a=J_5,6b=6.6 Hz, H-5), 3.85–3.76 (3H, m, H-6a, –OCH₂Cl), 3.62–3.53 (2H, m, H-6b, –OCH₂CH₂–), 2.54 (1H, br s, OH), 0.91–0.79 (2H, m, –OCH₂CH₂–), −0.14 (9H, m, Si(CH₃)₃). ¹³C-NMR (CDCl₃) δ: 166.8, 166.7, 165.2, 133.9, 133.4, 130.1, 129.7, 129.3, 128.6, 128.5, 128.4, 100.9 (C-1), 73.7 (C-5), 73.0 (C-3), 69.7 (C-2), 68.4 (–OCH₂CH₂–), 67.9 (C-4), 60.6 (C-6), 40.4 (ClCH₂CO–), 18.0 (–OCH₂CH₂–), –1.6. HR-ESI-MS: Calcd for C₂₇H₃₃ClO₉NaSi: m/z 587.1480. Found: 587.1464 [M+Na]⁺.

2-(Trimethylsilyl)ethyl 2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,4-di-O-benzoyl-3-O-chloroacetyl-β-D-galactopyranoside (13)

To a solution of 3 (165 mg, 0.29 mmol) and 4 (342 mg, 0.74 mmol) in dry CH₂Cl₂ (3 mL) was added MS 4 Å (500 mg), and the mixture was stirred for 2 h at room temperature, then cooled to 0°C. TMSOTf (16.0 µL, 89.2 µmol) was added, and the mixture was stirred for 1 h at 0°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 hexane–EtOAc) to give 13 (300 mg, 90%). [α]_D²⁵ +53.0° (c=0.3, CHCl₃). ¹H-NMR (CDCl₃) δ: 8.13–7.16 (30H, m, 6×Ph), 5.93 (1H, d, J_3′,4′=3.4 Hz, H-4′), 5.80 (1H, d, J_3,4=3.4 Hz, H-4), 5.76 (1H, dd, J_1′,2′=7.9 Hz, J_2′,3′=10.4 Hz, H-2′), 5.59–5.53 (2H, m, H-2, 3′), 5.34 (1H, dd, J_2,3=3.3 Hz, J_3,4=10.3 Hz, H-3), 4.88 (1H, d, J_1′,2′=7.8 Hz, H-1′), 4.64 (1H, d, J_1,2=8.2 Hz, H-1), 4.30–4.09 (5H, m, H-5, 5′, 6a, 6′a, 6′b), 3.95–3.81 (4H, m, H-6b, ClCH₂CO–, –OCH₂CH₂–), 3.46 (1H, dt, –OCH₂CH₂–), 0.86–0.69 (2H, m, –OCH₂CH₂–), –0.11 (9H, m, Si(CH₃)₃). ¹³C-NMR (CDCl₃) δ: 166.6, 165.7, 165.6, 165.44, 165.38, 165.0, 133.5, 133.22, 133.18, 130.0, 129.9, 129.70, 129.67, 129.6, 129.3, 129.24, 129.22, 128.94, 128.91, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 100.8 (C-1′), 100.7 (C-1), 76.7, 72.9 (C-3), 72.3 (C-5), 71,6 (C-5′), 71.1 (C-3′), 69.7 (C-2), 69.5 (C-2′), 67.9 (C-4), 67.8 (C-4′), 67.5 (–OCH₂CH₂–), 66.7 (C-6), 61.5 (C-6′), 40.4 (ClCH₂CO–), 17.7, −1.5. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-MS: Calcd for C₆₁H₅₉ClO₁₈NaSi: m/z 1165.3057. Found: 1165 [M+Na]⁺.

2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,4-di-O-benzoyl-3-O-chloroacetyl-α-D-galactopyranosyl Trichloroacetimidate (14)

To a solution of 13 (653 mg, 0.57 mmol) in CH₂Cl₂ (2.5 mL) cooled to 0°C was added CF₃CO₂H (5.0 mL), and the mixture was stirred for 30 min at room temperature and concentrated. EtOAc–toluene (1 : 2) were added and the mixture was concentrated to give the reducing sugar. To a solution of the residue in CH₂Cl₂ (5.0 mL) cooled at 0°C were added DBU (25 µL, 171 µL) and CCl₃CN (575 µL, 5.71 mmol). The mixture was stirred for 2 h at 0°C. The mixture was concentrated and the residue was purified by silica gel column chromatography using 2 : 1 hexane–EtOAc as eluent to give 14 (680 mg, quant.). [α]_D²⁵ +75.0° (c=0.8, CHCl₃). ¹H-NMR (CDCl₃) δ: 8.36 (1H, s, NH), 8.10–7.20 (30H, m, 6×Ph), 6.73 (1H, d, J_1.2=3.5 Hz, H-1), 5.96 (1H, d, J_3′,4′=3.7 Hz, H-4′), 5.90 (1H, d, J_3,4=3.7 Hz, H-4), 5.84 (1H, dd, J_2,3=3.3 Hz, J_3,4=10.7 Hz, H-3), 5.74–5.67 (2H, m, H-2, H-2′), 5.54 (1H, dd, J_2′,3′=3.5 Hz, J_3′,4′=10.3 Hz, H-3′), 4.87 (1H, d, J_1′,2′=7.9 Hz, H-1′), 4.61 (1H, t, J_5,6a=J_5,6b=6.4 Hz, H-5), 4.26–4.09 (4H, m, H-5′, 6a, 6′a, 6′b), 3.98–3.85 (3H, m, H-6b, ClCH₂CO–). ¹³C-NMR (CDCl₃) δ: 166.5, 165.8, 165.5, 165.4, 165.0, 160.4, 133.7, 133.6, 133.23, 133.17, 130.1, 129.9, 129.80, 129.75, 129.72, 129.68, 129.32, 129.30, 129.24, 128.94, 128.89, 128.7, 128.6, 128.53, 128.48, 128.4, 128.3, 128.2, 100.6 (C-1′), 93.4 (C-1), 90.5 (–CCl₃), 76.7, 71.7 (C-3′), 71.2 (C-5′), 70.7 (C-5), 69.6 (C-2′), 69.5 (C-3), 68.2 (C-4), 67.8 (C-4′), 67.6 (C-2), 66.6 (C-6), 61.5 (C-6′), 40.5 (ClCH₂CO–).

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,4-di-O-benzoyl-3-O-chloroacetyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranoside (15)

Compound 15 was prepared from 14 (441 mg, 0.37 mmol) and 2 (192 mg, 0.31 mmol) as described for preparation of 13. The product was purified by silica gel column chromatography (3 : 2 hexane–EtOAc) to give 15 (460 mg, 90%). [α]_D²⁵ +76.2° (c=1.1, CHCl₃). ¹H-NMR (CDCl₃) δ: 8.11–7.21 (45H, m, 9×Ph), 5.87–5.85 (2H, m, H-1 of Gal c, Gal a), 5.79 (1H, d, J_3,4=3.3 Hz, H-4 of Gal b), 5.69–5.62 (2H, m, H-2 of Gal c, Gal a), 5.54–5.48 (3H, m, H-2 of Gal b, H-3 of Gal c, Gal a), 5.34 (1H, dd, J_2,3=10.5 Hz, H-3 of Gal b), 4.68 (1H, J_1,2=8.1 Hz, H-1 of Gal c), 4.66 (1H, J_1,2=7.9 Hz, H-1 of Gal b), 4.61 (1H, d, J_1,2=7.9 Hz, H-1 of Gal a). ¹³C-NMR (CDCl₃) δ: 173.9, 166.6, 165.7, 165.5, 165.4, 165.3, 165.2, 165.0, 164.9, 133.6, 133.3, 133.2, 133.1, 130.1, 130.03, 129.99, 129.74, 129.70, 129.6, 129.4, 129.31, 129.26, 129.24, 129.17, 129.1, 128.94, 128.9, 128.7, 128.64, 128.56, 128.49, 128.47, 128.44, 128.38, 128.3, 128.22, 128.20, 101.4 (C-1 of Gal a), 101.0 (C-1 of Gal b), 100.4 (C-1 of Gal c), 76.7, 72.8, 72.7, 71.9, 71.62, 71.57, 71.0, 69.83, 69.77, 69.6, 69.5, 68.6, 67.8, 67.7, 67.3, 65.5, 61.3, 51.4, 40.5, 33.6, 28.9, 25.3, 24.4. MALDI-TOF-MS: Calcd for C₉₀H₈₁ClO₂₈Na: m/z 1667.4501. Found: 1667 [M+Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,4-di-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranoside (16)

To a solution of 15 (410 mg, 0.25 mmol) in EtOH–pyridine (6 : 1, 5.0 mL) was added thiourea (95 mg, 1.25 mmol), and the mixture was stirred for 2 h at 80°C. The mixture was diluted with CHCl₃, washed with saturated aqueous NaHCO₃ and water, dried (MgSO₄), and concentrated. The product was purified by silica gel column chromatography (5 : 1 toluene–EtOAc) to give 16 (364 mg, 93%). [α]_D²⁵ +69.6° (c=1.3, CHCl₃). ¹H-NMR (CDCl₃) δ: 8.12–7.21 (45H, m, 9×Ph), 5.90–5.84 (2H, m, H-4 of Gal a, Gal c), 5.71–5.65 (3H, m, H-4 of Gal b, H-2 of Gal a, Gal c), 5.56–5.49 (2H, m, H-3 of Gal a, Gal c), 5.31 (1H, dd, J_1,2=7.6 Hz, J_2,3=9.9 Hz, H-2 of Gal b), 4.67 (1H, d, J_1,2=7.9 Hz, H-1 of Gal c), 4.63 (1H, d, J_1,2=7.9 Hz, H-1 of Gal a), 4.62 (1H, d, J_1,2=7.9 Hz, H-1 of Gal b), 3.97–3.92 (1H, m, H-3 of Gal b). ¹³C-NMR (CDCl₃) δ: 173.9, 166.4, 166.3, 165.8, 165.5, 165.4, 165.3, 165.2, 165.0, 133.6, 133.4, 133.3, 133.2, 133.14, 133.11, 130.1, 130.0, 129.8, 129.73, 129.71, 129.68, 129.61, 129.59, 129.4, 129.29, 129.27, 129.2, 129.0, 128.9, 129.7, 128.6, 128.5, 128.4, 128.3, 128.23, 128.17, 101.4 (C-1 of Gal a), 100.8 (C-1 of Gal c), 100.7 (C-1 of Gal b), 73.3, 72.8, 72.3, 71.6, 71.5, 71.1, 70.3, 69.9, 69.8, 69.6, 68.6, 67.9, 67.7, 66.6, 61.5, 51.4, 33.6, 28.9, 25.3, 24.4. HR-ESI-MS: Calcd for C₈₈H₈₀O₂₇Na: m/z 1591.4785. Found: 1591.4896 [M+Na]⁺.

5-(Methoxycarbonyl)pentyl 2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranosyl-(1→6)-[2,3,4-tri-O-benzyl-α-L-fucopyranosyl-(1→3)]-2,4-di-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-galactopyranoside (17)

To a solution of 16 (314 mg, 0.20 mmol) and 5 (210 mg, 0.40 mmol) in dry CH₂Cl₂ (3.0 mL) was added powdered MS 4 Å (300 mg), and the mixture was stirred under Ar atmosphere for 2 h at room temperature, then cooled to −60°C. NIS (90 mg, 400 µmol) and TfOH (3.5 µL, 40 µmol) were added to the mixture and the mixture was stirred for 30 min. 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 saturated aqueous Na₂S₂O₃ and water, dried (MgSO₄), and concentrated. The product was purified by silica gel column chromatography (14 : 1 toluene–EtOAc) to give 17 (393 mg, 99%). [α]_D²⁵ +58.1° (c=2.0, CHCl₃). ¹H-NMR (CDCl₃) δ: 5.06 (1H, d, J_1,2=3.6 Hz, H-1 of Fuc), 4.73 (1H, d, J_1,2=7.7 Hz, H-1 of Gal c), 4.65–4.53 (2H, m, H-1 of Gal a, Gal b). ¹³C-NMR (CDCl₃) δ: 101.4 (C-1 of Gal a), 101.3 (C-1 of Gal b), 100.8 (C-1 of Gal c), 98.6 (C1 of Fuc). MALDI-TOF-MS: Calcd for C₁₁₅H₁₀₈O₃₁Na: m/z 2007.6772. Found: 2007 [M+Na]⁺.

5-(Methoxycarbonyl)pentyl β-D-Galactopyranosyl-(1→6)-[α-L-fucopyranosyl-(1→3)]-β-D-galactopyranosyl-(1→6)-β-D-galactopyranoside (18)

Compound 17 (150 mg, 75.5 μmol) in 1 : 1 THF–MeOH (6.0 mL) was hydrogenolysed in the presence of Pd/C (150 mg) for 2 h at room temperature. The mixture was filtered and concentrated. To a solution of the residue in 1 : 1 1,4-dioxane–MeOH (4.0 mL) was added NaOMe (50 mg) at room temperature and the mixture was stirred at 40°C for 16 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 MeOH to give 18 (28 mg, 48%). [α]_D²⁵ −58.1° (c=0.4, MeOH). ¹H-NMR (CD₃OD) δ: 5.12 (1H, d, J_1,2=3.9 Hz, H-1 of Fuc), 4.40 (1H, d, J_1,2=7.6 Hz, H-1 of Gal c), 4.31 (1H, d, J_1,2=7.5 Hz, H-1 of Gal b), 4.22 (1H, d, J_1,2=7.3 Hz, H-1 of Gal a). ¹³C-NMR (CDCl₃) δ: 176.9, 106.2, 105.9, 105.7, 103.6, 77.6, 76.0, 75.9, 75.8, 75.7, 75.6, 74.5, 73.4, 73.3, 72.6, 72.5, 71.4, 71.1, 70.83, 70.78, 70.6, 70.5, 69.0, 63,4, 52.9, 50.7, 50.4, 49.4, 35.6, 31.3, 27.5, 26.6, 17.7. HR-ESI-MS: Calcd for C₃₁H₅₄O₂₂Na: m/z 801.3004. Found: 801.3030 [M+Na]⁺.

Biotinylated Tetrasaccharide (1)

Compound 18 (28 mg, 36.0 µmol) was dissolved in neat anhydrous ethylenediamine (3 mL) and heated at 70°C for 48 h. The mixture was concentrated by azeotropic distillation with toluene and the product was purified by Sephadex LH-20 column chromatography in H₂O to give an amine intermediate (29.0 mg quant.). The amine was dissolved in DMF (5.7 mL), and the pH was adjusted to 8–9 using N,N-diisopropylethylamine (DIPEA). Biotine–NHS (30.0 mg, 87.9 µmol) was added and the mixture was stirred for 18 h at room temperature. The mixture was concentrated by azeotropic distillation with toluene several times. The product was purified by Sephadex LH-20 column chromatography in H₂O to give 1 (29 mg, 78%). [α]_D²⁵ −21.5° (c=0.4, H₂O). ¹H-NMR (D₂O) δ: 5.16 (1H, d, J_1,2=4.1 Hz, H-1 of Fuc), 4.53 (1H, d, J_1,2=7.6 Hz, H-1 of Gal a), 4.45 (1 H, d, J_1,2=7.6 Hz, H-1 of Gal c), 4.40 (1H, d, J_1,2=7.9 Hz, H-1 of Gal b). ¹³C-NMR (D₂O) δ: 104.0 (C-1 of Gal b), 103.7 (C-1 of Gal a), 103.4 (C-1 of Gal c), 101.6 (C1 of Fuc). HR-ESI-MS: Calcd for C₄₂H₇₂N₄O₂₃SNa: m/z 1055.4206. Found: 1055.4276 [M+Na]⁺.

Serum Samples

Serum samples of 60 patients, who were confirmed to have AE_, and those of 60 healthy individuals, which are kept in Hokkaido Institute of Public Health, were used for ELISA assay under approval of the institute.

ELISA Protocol

ELISA was performed using as previously described^31,32) with some modifications. The oligosaccharides in H₂O (13 pmol per well) were placed in the wells of flat-bottomed microplates (Streptavidin C96, No. 236001; Nunc, Roskilde, Denmark) coated with streptavidin and incubated for 1 h at 37°C. After removal of the solution, the wells were washed with 0.05% Tween-PBS (250 µL per well). Serum samples diluted 1 : 250 with 0.05% Tween-PBS (200 µL per well) were then added to the wells and incubated overnight at 4°C. After removal of the serum and washing with 0.05% Tween-PBS, 200 µL of anti-human immunoglobulin G (IgG)/horseradish peroxidase (HRP) (P0214; DakoCytomation, Denmark; 1 : 1000 in 0.05% Tween-PBS) was added, and the microplate was incubated for 1 h at 37°C. After washing of the wells, bound antibodies were detected by the addition of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) peroxidase substrate solution (KPL, Gaithersburg, MD, U.S.A, 200 µL per well). After incubation period of 8 min at 37°C, the reaction was stopped by the addition of 1% sodium dodecyl sulfate (SDS), and the absorbance (A) values were read at 405 nm on a microplate reader (Model 680; BIORAD, Hercules, California, U.S.A.).

Data Analysis

The significant difference between the two groups was analyzed by the Student’s t-test.

Acknowledgments

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 (MEXT) of Japan, and 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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