Synthesis of the Carbohydrate Moiety of Glycoproteins from the Parasite Echinococcus granulosus and Their Antigenicity against Human Sera

Abstract

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the carbohydrate moiety of glycoprotein from Echinococcus granulosus have been accomplished. Trisaccharide Galβ1-3Galβ1-3GalNAcα1-R (A), tetrasaccharide Galα1-4Galβ1-3Galβ1-3GalNAcα1-R (B), and pentasaccharide Galα1-4Galβ1-3Galβ1-3Galβ1-3GalNAcα1-R (C), (R = biotinylated probe) were synthesized by stepwise condensation and/or block synthesis by the use of 5-(methoxycarbonyl)pentyl 2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside as a common glycosyl acceptor. The synthesis of the tetrasaccharide and the pentasaccharide was improved from the viewpoint of reducing the number of synthetic steps and increasing the total yield by changing from stepwise condensation to block synthesis. Moreover, hexasaccharide E, which contains the oligosaccharide sequence which occurs in E. granulosus, was synthesized from trisaccharide D. We examined the antigenicity of these five oligosaccharides by an enzyme-linked immunosorbent assay (ELISA). Although compounds of C–E did not exhibit antigenicity against cystic echinococcosis (CE) patient sera, compounds B, D, and E showed good serodiagnostic potential for alveolar echinococcosis (AE).

1. Introduction

In the course of our studies on natural oligosaccharides from invertebrate animal species, we are interested in the unique glycosphingolipids (GSLs) and glycoproteins (GPs) found in various Protostomia phyla and we have synthesized these oligosaccharides in order to elucidate biological functions [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18]. In particular, our interests have been focused on the unique oligosaccharide structures of GSLs and GPs found in several parasites including Echinococcus multilocularis [2,7,8,11,12,13,15], Schistosoma mansoni [5,10], Ascaris suum [1,17], and Toxocara canis [6]. Among them, GSLs and GPs from E. multilocularis have attracted our special attention. 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. In 1992, Persat et al. reported [19] 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. Hülsmeier et al. reported [20] in 2002 that Em2, an antigen extracted from E. multilocularis, is a mucin-type glycoprotein. Based on this information, we synthesized four glycosphingolipids including Galβ1→6Galβ1-Cer as a core structure [15] and five carbohydrate structures of glycoproteins including a reducing terminal Galβ1→3GalNAcα1—which is a core 1 of mucin-type O-glycans of E. multilocularis [8,11], and examined antigenicity of the pure compounds by ELISA for their serodiagnostic potential [7,12,13].

More recently, Díaz et al. reported [21] that the extracellular matrix of a related cestode Echinococcus granulosus contains a novel mucin-type O-glycan capping motif consisting of Galpα1-4Gal linkages at the non-reducing end (Figure 1). E. granulosus is a parasitic cestode causing cystic echinococcosis (CE) in intermediate hosts like humans. The adult worm lives in the small intestine of a carnivore (definitive host), and the intermediate larval stage can infect a wide range of mammal species—including humans—that acquire the infection through accidental ingestion of eggs. Díaz et al. elucidated in full the major glycan of the E. granulosus laminated layer (LL) and these are conventional core 1 and 2 O-glycans modified with Galα1-4Gal that are linked to three kinds of carbohydrates (Gal, GalNAc, and GlcNAc). Based on this information, we report here on the synthesis of the biotinylated glycan portions A–E (Figure 1 and Figure 2) of the glycoprotein antigen of E. granulosus in order to elucidate the interactions between the oligosaccharides and sera, and the structure-activity relationships involved in the antigen recognition. Compound D is a synthetic intermediate derived from the process of synthesizing compound E.

2. Results and Discussion

2.1. Syntheses of the Target Oligosaccharides A, B, and C

The synthetic strategy for oligosaccharides A–C is shown in Figure 3 (NMR spectra provided in the Supplemental Data).

Suitably protected monosaccharide derivatives (4, 5, 9, and 12) were chosen as building blocks. The 5-(Methoxycarbonyl)pentyl group was chosen as the temporary protecting group for all the target compounds to ensure future conjugation with biotin for the ELISA assay as previously shown by us [8]. The synthetic routes for target compounds A–C are outlined in Scheme 1, Scheme 2, Scheme 3, Scheme 4, Scheme 5 and Scheme 6. Initially, disaccharide acceptor 8 was prepared in a way that serves as a common acceptor for the syntheses of compounds A, B, and C. Disaccharide 8 was prepared from thioglycoside donor 3, which was obtained from phenyl-1-thio-β-d-galactopyranoside (1) by regioselective 2-naphthylbenzylation of the in situ prepared stannylidene derivative with 2-naphthylbenzyl bromide (NapBr) and tetrabutylammonium bromide followed by benzoylation (Scheme 1). The glycosylation of 3 with 5 [8] in the presence of N-iodosuccinimide (NIS)/trifluoromethanesulfonic acid (TfOH) [22] and AW-300 molecular sieves (MS AW-300) in CH₂Cl₂ afforded desired disaccharide 6 in 71% yield. The nature of the new β-glycosidic linkage was determined by the vicinal coupling constant of the anomeric proton (H-1 of Gal, δ = 5.11 ppm, J = 7.9 Hz). Condensation of 5 with the 3-O-chloroacetyl (ClAc) donor 4, which was prepared by selective removal of the 3′-O-NAP group in 3 with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) followed by chloroacetylation afforded desired disaccharide (7) in higher yield (89%). Oxidative removal of the 3′-O-NAP group in 6 with DDQ gave disaccharide acceptor 8 in 63% yield and removal of the 3′-O-ClAc group in 7 with thiourea produced the same acceptor 8 in 84% yield. Comparing the protecting groups at the 3’-position, the ClAc group consistently gave higher yields than the NAP group in both, glycosylation and deprotection steps in the synthesis of 8. The glycosylation of disaccharide acceptor 8 with thioglycosyl donor 9 in the presence of NIS/TfOH and MS AW-300 in CH₂Cl₂ afforded desired trisaccharide (10) in 78% yield. The nature of the new glycosidic linkage was determined as β by the vicinal coupling constant of the anomeric proton (H-1 of Gal, δ = 4.69 ppm, J = 8.1 Hz). Selective removal of the 4″-O-ClAc protecting group from 10 was achieved by thiourea to produce trisaccharide acceptor 11 in 85% yield, which was the direct precursor of compound A and used directly for the next glycosylation step of compound B. The Glycosylation of 11 with 12 [23] using NIS/TfOH and MS AW-300 in CH₂Cl₂ produced desired tetrasaccharide (13) in 85% yield. The newly formed α-glycosidic linkage (H-1 of Galc, δ = 4.86 ppm, J = 2.6 Hz) was confirmed by ¹H NMR spectroscopy.

Global deprotection of the precursors for A and B was performed by a combination of protection/deprotection steps. Initially, we attempted the simultaneous reduction of the azido group and removal of the benzyl protecting groups in 13 by catalytic hydrogenolysis with 10% Pd/C. However, since this conversion was not successful, we studied stepwise conversion. Selective hydrogenolysis using 10% Pd/C of the azido group in the presence of ammonia followed by hydrogenolytic cleavage of the benzyl groups over 10% Pd/C in acetic acid and exposure to Ac₂O in pyridine resulted in the O- and N-acylation. However, the benzylidene acetal was not removed under these conditions. Thus, the benzylidene acetal was removed with TsOH followed by O-acetylation with Ac₂O in pyridine to afford 14 in 51% yield after five steps. After deacetylation of 14 under Zemplén conditions, 5-(methoxycarbonyl)pentyl glycoside 15 was converted into the ethylenediamine monoamide by exposure to ethylenediamine and conjugated to biotin using our previously established methodology [8] to afford tetrasaccharide-biotin conjugate A in 67% yield after column chromatographic purification on Sephadex LH-20 (Scheme 2).

Tetrasaccharide B was synthesized in a multi-step sequence as outlined in Scheme 3. At first, selective removal of the DTBS group in 13 was achieved with HF/pyridine and the benzylidene acetal was removed by acidic hydrolysis followed by acetylation with acetic anhydride in pyridine to afford 16 in 65% yield. Then, the azido-group was converted to an acetamido moiety by reduction with PPh₃ followed by N-acylation with Ac₂O in pyridine and debenzylation by catalytic hydrogenolysis using Pearlman’s catalyst followed by O-acetylation to produce protected tetrasaccharide 17 in 65% yield. The deacetylation of 17 under Zemplén conditions yielded unprotected tetrasaccharide 18 in 60% yield. Compound 18 was then used for ligation to biotin to provide target tetrasaccharide B after column chromatographic purification on Sephadex LH-20 (Scheme 3).

Originally, it was planned to prepare pentasaccharide C by elongation from previously prepared disaccharide acceptor 8 and thioglycoside donor 4. However, preparation of the intermediate trisaccharide 19 by glycosylation of thioglycoside donor 4 with disaccharide acceptor 8 by NIS/TfOH-promoted activation was not successful. Similarly, the glycosylation of donor 20 [5] as well as donor 22 [24] with disaccharide acceptor 8 was also not successful in the presence of NIS and TfOH. The latter reaction afforded undesired α-glycosylated trisaccharide 24 in 67% yield (Scheme 4). These results suggest that 4 and 20 are unreactive (mismatched) donors to react with disaccharide acceptor 8. On the other hand, donor 22 displays superior reactivity but exclusively produces undesired α-anomer because of the absence of a C-2 acyl neighboring group participation.

The failure to generate trisaccharide intermediates 19, 21, and 23 forced us to modify our original synthetic strategy as outlined in Figure 4. Compound 20 was selected as a new glycosyl donor of the di- and trisaccharides and the elongation of the carbohydrate chain was repeated by glycosylation and dechloroacetylation as shown in Scheme 5 and Scheme 6. The glycosylation of the acceptor 5 with 20 in the presence of NIS/TfOH provided disaccharide 25 in 76% yield. The disaccharide acceptor 26 was obtained in 76% yield from 25 after treatment with thiourea, which was used directly for the next glycosylation step. Trisaccharide derivative 27 was obtained by glycosylation of glycosyl donor 20 with 26. The deprotection of the ClAc group in 27 was performed as described for compound 26 to provide trisaccharide acceptor 28 (Scheme 5).

The coupling of acceptor 28 to thioglycoside donor 9 afforded protected tetrasaccharide 29 in 53% yield. Selective removal of the 4-O-ClAc group in 29 by thiourea afforded tetrasaccharide acceptor 30 in 97% yield. In order to prepare the Galα1-4Gal-sequence with high α-stereoselectivity, we selected 4,6-O-di-tert-butylsilylene (DTBS)-protected galactose donor 12. Previous studies have indicated that DTBS-protected galactose donors induce high α-selectivity in glycosylation reactions [23]. NIS/TfOH-promoted activation of thioglycoside donor 12 and coupling to tetrasaccharide acceptor 30 generated protected pentasaccharide 31 in 68% yield (Scheme 6).

Although we were able to achieve the synthesis of the desired protected target pentasaccharide using the stepwise elongation approach, the amount obtained was not sufficient to undergo global deprotection. As a result, we investigated a block synthesis approach in which a non-reducing terminal disaccharide derivative was synthesized in advance and condensed with the reducing end terminal di- and tri-saccharide derivatives to synthesize the tetra- and the penta-saccharides (Figure 5).

Disaccharide donor 36 was obtained by using the synthetic strategy outlined in Scheme 7. At first, the glycosylation of the known monosaccharide acceptor 32 [25] with monosaccharide donor 33 (commercially available) afforded benzylgroup-protected disaccharide 34 in 79% yield. Removal of the benzyl protecting groups of 34 by catalytic hydrogenation over 10% Pd-C in THF-MeOH followed by O-acetylation produced protected disaccharide 35. Selective removal of the 2-(trimethylsilyl)ethyl (TMS-ethyl) group in 35 with TFA, followed by exposure of resulted hemiacetal to CCl₃CN and 1,8-diazabicyclo [5,4,0]-7-undecene (DBU) afforded corresponding α-trichloroacetimidate donor 36 in 84% yield (Scheme 7).

The glycosylation of acceptor 26 with donor 36 in the presence of TMSOTf and MS AW-300 in CH₂Cl₂ afforded desired tetrasaccharide 37 in 34% yield. The nature of the new β-glycosidic linkage was determined by the vicinal coupling constant of the anomeric proton (H-1 of Galb, δ = 5.01 ppm, J = 8.1 Hz). The deprotection and biotinylation of 37 proceeded in excellent yield to give target tetrasaccharide B (Scheme 8).

Pentasaccharide C was obtained in moderate yields from trisaccharide acceptor 28 and disaccharide donor 36 in a similar manner (Scheme 9). Deprotection and biotinylation of pentasaccharide 40 were performed as described for compound B to provide target trisaccharide C in an excellent 85% yield (Scheme 9).

2.2. Syntheses of the Target Oligosaccharides D and E

We next devised a synthetic strategy for trisaccharide D and hexasaccharide E as shown in Figure 6 (NMR spectra provided in the Supplemental Data). Trisaccharide D constitutes the partial structure of hexasaccharide E. Trisaccharide 44 served as starting material for the preparation of D. Trisaccharide 45 was prepared by condensation of 2,6-dimethyl-thiophenyl-trisaccharide donor 44 [11] with 5-(methoxycarbonyl)pentyl alcohol in the presence of NIS/TfOH and MS AW-300 in CH₂Cl₂ in 89% yield. The reduction and N-acetylation of the Troc groups of 45 with Zn-Cu THF/AcOH/Ac₂O followed by debenzylation with catalytic hydrogenolysis over 10% Pd/C in MeOH and acetylation afforded 46. Deacylation of 46 followed by biotinylation of 47 was performed as described above to provide target trisaccharide D (Scheme 10).

Compound E is a hexasaccharide which combines two trisaccharide components: Galα1-4Galβ1-4GlcNAc and Galβ1-3Galβ1-3GalNAc. The former component can be conveniently installed using synthetic intermediate 44 while the latter component 48 was obtained from the regioselective reductive ring-opening of the benzylidene acetal in compound 10 as a glycosyl acceptor (Scheme 11). The glycosylation of 44 with 48 in the presence of NIS/TfOH and MS AW-300 in CH₂Cl₂ afforded desired disaccharide (49) in 94% yield. The new β-glycosidic linkage was confirmed by ¹H NMR using the coupling constant of H-1 of GlcN (δ 4.62 J_1,2 7.0 Hz) as a diagnostic tool as well as from the ¹³C-NMR value for C-1 of GlcN (δ 100.9). The removal of the Troc-protecting group of 49 was achieved with Zn in an Ac₂O and AcOH mixture to produce protected N-acylated hexasaccharide 50. The removal of benzyl protecting groups in 50 was initially attempted by hydrogenolysis using Pd-C. However, this reaction failed and resulted in side reactions involving the ClAc protecting group leading to an intractable mixture of products. In contrast, significantly improved yields were obtained by deacylation under Zemplén conditions followed by hydrogenolytic cleavage of the benzyl protecting group to produce the desired hexasaccharide 51. Biotinylation was performed as the usual method to provide target hexasaccharide E in 79% yield.

2.3. Antigenicity of Oligosaccharides by ELISA

The reactivity of the five oligosaccharides A–E (NMR spectra provided in the Supplemental Data). to alveolar echinococcosis (AE) patient sera and cystic echinococcosis (CE) was examined using microplates coated with streptavidin. Contrary to expectations, the antibody response of the CE patient group was not significantly different from that of the normal healthy (NH) group (Figure 7).

Although A–E display oligosaccharide structures specific to E. granulosus, only compounds A and B showed a modest effect of antigenicity to CE patient serum while no effect was observed with saccharides C–E. This suggests that the presence of the terminal Galα1-4Gal sequence in oligosaccharides B–E of E. granulosus may suppress a host immune response or the cell surface oligosaccharides on E. granulosus may be associated with lower host specificity than E. multirocularis [26]. Interestingly, we previously reported that the trisaccharide sequence Galα1-4Galβ1-3GalNAc found on the surface of E. multilocularis showed the strongest antigenic response to the AE group among a series of oligosaccharides [8]. Rather unexpected is our finding that oligosaccharides B, D, and E that occur on E. granulosus display strong antigenicity to AE patient sera.

3. Conclusions

We have developed an efficient synthetic strategy for the preparation of five oligosaccharide-biotin conjugates A–E which display carbohydrate structures that occur on the surface of E. granulosus. The oligosaccharide-biotin conjugates were prepared to study the antigenicity of the compounds to detect antibodies in patient sera infected with E. granulosus the cause of CE. Surprisingly, none of the oligosaccharide structures C–E was able to detect antibodies in sera from patients suffering from CE using our ELISA assay while only a modest response was seen with compounds A and B. However, glycoconjugates B, D, and E showed good serodiagnostic potential to recognize antibodies in AE patients. Although the oligosaccharide sequence of compound E has not been reported in E. multilocularis, it showed strong antigenicity to the serum of AE patients. Overall, our results suggest that oligosaccharide-based structures on the cell surface of E. granulosus may serve as a diagnostic tool to detect AE. The reasons for this phenomenon are currently not understood. Possible explanations for this observation are: (i) E. granulosus induces a suppressed host immune response when compared to E. multilocularis; (ii) the presence of Galα1-4Gal terminal capping linkage in E. granulosus reduces a host immune response and (iii) oligosaccharide structures present in E. granulosus may also be present in E. multilocularis. Overall, our investigation encourages future studies in the development of carbohydrate-based antigens as serodiagnostic tools to detect parasitic infections. In particular, our findings that oligosaccharide-biotin probes D and E can differentiate between sera from AE and CE patients warrant further studies toward the development of serodiagnostic tools to detect parasite-specific infections in humans.

4. Experimental

4.1. General Procedures

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 (Palo Alto, CA, USA). Me₄Si and acetone were used as internal standards for CDCl₃ and D₂O, respectively. ESI-HRMS was recorded on a JEOL MS T-100 mass spectrometer (Tokyo, Japan). MALDI-TOFMS was recorded on an AB SCIEX Voyager RP mass spectrometer (Framingham, MA, USA). TLC was performed on Silica Gel 60 F254 (E. Merck, Darmstadt, Germany) with detection by the quenching of UV fluorescence and by charring with 10% H₂SO₄. Column chromatography was carried out on Silica Gel 60. 5-(Methoxycarbonyl)pentyl 2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (5) [8], phenyl-2-O-benzoyl-3,6-di-O-benzyl-4-O-chloroacetyl-1-thio-β-d-galactopyranoside (9), phenyl 2,3-di-O-benzyl-4,6-O-di-tert-butylsilylene-1-thio-β-d-galactopyranoside (12) [23], phenyl 4,6-di-O-benzylidene-2-O-benzoyl-3-O-chloroacetyl-1-thio-β-d-galactopyranoside (20) [5], phenyl 2,4,6-tri-O-benzyl-3-O-chloroacetyl-1-thio-β-d-galactopyranoside (22) [25] 2-(trimethylsilyl)ethyl 2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranoside (32) [26], 2,6-dimethylphenyl 4,6-di-O-acetyl-2,3-di-O-benzyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl-(1→4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-1-thio-β-d-glucopyranoside (44) [11] were prepared as reported. Phenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-d-galactopyranoside (33) was purchased from Tokyo Chemical Industry Co., Ltd. (TCI), (Tokyo, Japan).

• Phenyl 2,4,6-tri-O-benzoyl-3-O-(2-naphthyl)methyl-1-thio-β-d-galactopyranoside (3)

A solution of 1 (5.00 g, 18.4 mmol) and dibutyltin oxide (5.50 g, 22.1 mmol) in 150 mL of dry MeOH was stirred under reflux for 4 h. MeOH was distilled off, the stannylidene derivative was dissolved in toluene (70 mL) and Bu₄NBr (7.12 g, 22.1 mmol) and NapBr (6.1 g, 27.6 mmol) were added at room temperature. After being stirred for 15 h at 100 °C, the solution was concentrated. To a solution of this residue (2) in pyridine (4 mL) benzoyl chloride (60 mL, 82.8 mmol) was added, and the reaction mixture was stirred for 5 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 8:1 hexane-EtOAc as eluent to give 3 (8.46 g, 64% 2 steps). ${\left[\alpha \right]}_{\mathrm{D}}^{22}$ + 66.0 (c =1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.01–7.16 (m, 27H, 4×Ph, naphtyl), 5.97 (d, 1H, J_{3, 4} = 2.5 Hz, H-4), 5.55 (t, 1H, J_{1, 2} = J_{2, 3} = 9.8 Hz, H-2), 4.83 and 4.64 (each d, 2H, J_gem = 12.8 Hz, naphtylmethylene), 4.82 (d, 1H, H-1), 4.58 (dd, 1H, J_{5, 6a} = 7.4 Hz, J_{6a, 6b} = 11.6 Hz, H-6a), 4.50–4.47 (dd, 1H, J_{5, 6b} = 5.0 Hz, H-6b), 4.15 (br.t, 1H, H-5), 3.88 (dd, 1H, H-3). ¹³C-NMR (125 MHz, CDCl₃): δ 166.1, 165.8, 165.1, 134.5, 133.43, 133.41, 133.3, 133.1, 132.96, 132.93, 132.0, 130.1, 129.9, 129.8, 129.5, 129.2, 128.7, 128.5, 128.44, 128.36, 128.2, 128.0, 127.8, 127.6, 126.9, 126.0, 125.89, 125.86, 86.0 (C-1), 77.2 (C-3), 75.2 (C-5), 71.0 (naphtylmetylene), 69.4 (C-2), 66.8 (C-4), 63.1 (C-6). HR-ESIMS: calcd for C₄₄H₃₆O₈SNa: m/z 747.2029; found: m/z 747.2003 [M + Na]⁺.

• Phenyl 2,4,6-tri-O-benzoyl-3-O-chloroacetyl-1-thio-β-d-galactopyranoside (4)

A solution of 3 (8.46 g, 11.07 mmol) in CH₂Cl₂—H₂O (20:1, 84 mL) was treated with DDQ (5.31 g, 23.4 mmol) at room temperature and then was stirred for 6 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 (3:1 hexane-EtOAc) as eluent to give intermediate (6.19 g, 96%). [MALDI-TOFMS: calcd for C₃₃H₂₈O₈SNa, m/z 607.1; found, m/z 607.6 [M + Na]⁺]. To a solution of this compound (4.00 g, 6.84 mmol) in CH₂Cl₂/pyridine (15:1, 64 mL) was added chloroacetyl chloride (ClAcCl ) (817 μL, 10.3 mmol), and the reaction mixture was stirred for 1 h at 0°C. 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 4 (3.9 g, 83% 2 steps). ${\left[\alpha \right]}_{\mathrm{D}}^{22}$ + 32.9 (c =1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.03–7.24 (m, 20H, 4×Ph), 5.85 (d, 1H, J_{3, 4} = 3.2 Hz, H-4), 5.59 (d, 1H, J_{1, 2} = J_{2, 3} = 9.8 Hz, H-2), 5.45 (dd, 1H, H-3), 4.97 (d, 1H, H-1), 4.64 (dd, 1H, J_{5, 6a} = 7.0 Hz, J _{6a, 6b} = 11.4 Hz, H-6a), 4.40 (dd, 1H, J_{5, 6b} = 5.8 Hz, H-6b), 4.32 (t, 1H, H-5), 3.88 and 3.83 (each d, 1H, J_gem = 15.2 Hz, CH₂Cl). ¹³C-NMR (125 MHz, CDCl₃): δ 166.7, 166.0, 165.7, 165.1, 134.0, 133.8, 133.6, 133.4, 133.1, 133.0, 131.0, 130.0, 129.9, 129.8, 129.3, 129.1, 128.9, 128.7, 128.6, 128.5, 85.9 (C-1), 74.9 (C-5), 74.1 (C-3), 67.9 (C-4), 67.6 (C-2), 62.3 (C-6), 40.4 (CH₂Cl). HR-ESIMS: calcd for C₃₅H₂₉ClO₉SNa: m/z 683.1119; found: m/z 683.1142 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2,4,6-tri-O-benzoyl-3-O-(2-naphthyl)methyl-β-d-galacto-pyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (6)

To a solution of 5 (100 mg, 0.237 mmol) and 3 (223 mg, 0.308 mmol) in dry CH₂Cl₂ (2 mL) powdered AW300 (300 mg) was added, and the mixture was stirred under Ar atmosphere for 2 h at room temperature, then cooled to −40 °C. NIS (139 mg, 0.62 mmol) and TfOH (5.5 μL, 0.06 mmol) were added to the mixture, which was stirred for 0.5 h at –40 °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 (70:1 toluene-acetone) to give 6 (175 mg, 71%). ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ + 103.1 (c =1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.19–6.85 (m, 27H, 4×Ph, naphtylmethylene), 5.96 (d, 1H, J_{3′, 4′} = 2.4 Hz, H-4′), 5.68 (dd, 1H, J_{1′, 2′} = 8.1 Hz, J_{2′, 3′} = 9.9 Hz, H-2′), 5.46 (s, 1H, PhCH), 4.53 (d, 1H, H-1′), 4.89 (d, 1H, J_{1, 2} = 3.3 Hz, H-1), 4.87–4.80 (m, 1H, naphtylmethylene), 4.72–4.68 (m, 1H, H-6′a), 4.68—4.65 (m, 1H, naphtylmethylene), 4.45–4.43 (m, 1H, H-6′b), 4.42 (d, 1H, J_{3, 4} = 2.6 Hz, H-4), 4.16 (t, 1H, H-5′), 4.11–4.07 (m, 1H, H-6a), 4.03 (dd, 1H, J_{2, 3} = 10.9 Hz, H-3), 3.88 (dd, 1H, J_{3′ 4′} = 3.3 Hz, H-3′), 3.72–3.68 (m, 2H, H-2, H-6b), 3.66—3.59 (m, 4H, -OCH₂-_, -OCH₃), 3.43 (s, 1H, H-5), 3.41–3.38 (m, 1H, -OCH₂-), 2.30–2.26 (m, 2H, -CH₂-), 1.64–1.53 (m, 4H, 2×-CH₂-), 1.37–1.25 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 166.0, 165.2, 137.7, 134.5, 133.46, 133.43, 132.94, 132.91, 130.4, 130.13, 130.08, 129.9, 129.8, 129.7, 129.6, 129.3 129.1, 128.7, 128.6, 128.5, 128.4, 128.19, 128.16. 128.05, 127.79, 127.57, 126.8, 126.1, 126.0, 125.9, 125.8, 102.9 (C-1′), 100.6 (PhCH), 98.6 (C-1), 76.2 (C-3′), 76.1 (C-3), 75.9 (C-4), 71.4 (C-5′),71.0 (naphtylmethylene), 70.96 (C-2′), 68.95 (C-6), 68.2(OCH₂), 66.6 (C-4′), 63.0 (C-5, C-6′), 58.4 (C-2), 51.4 (OCH₃), 33.8, 28.9, 25.6, 24.6. HR-ESIMS: calcd for C₅₈H₅₇N₃O₁₅Na: m/z 1058.3687; found: m/z 1058.3667 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2,4,6-tri-O-benzoyl-3-O-chloroacetyl-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (7)

To a solution of 5 (200 mg, 0.48 mmol) and 4 (377 mg, 0.57 mmol) in dry CH₂Cl₂ (5 mL) was added powdered MS AW300 (600 mg), and the mixture was stirred under Ar atmosphere for 2 h at room temperature, then cooled to −40 °C. NIS (192 mg, 0.85 mmol) and TfOH (15 μL, 0.17 mmol) were added to the mixture, which was stirred for 0.5 h at −40 °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 (15:1 toluene-EtOAc) to give 7 (413 mg, 89%). ${\left[\alpha \right]}_{\mathrm{D}}^{22}$ + 96.3 (c =1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.13–7.15 (m, 20H, 4×Ph), 5.85 (d, 1H, J_{3′, 4′} = 2.6 Hz, H-4′), 5.73 (br.t, 1H, H-2′), 5.49 (s, 1H, PhCH), 5.46 (dd, 1H, J_{2′, 3′} = 10.5 Hz, H-3′), 5.11 (d, 1H, J_{1′, 2′} = 7.9 Hz, H-1′), 4.92 (d, 1H, J_{1, 2} = 2.8 Hz, H-1), 4.78–4.73 (m, 1H, H-6′a), 4.44 (d, 1H, J_{3, 4} = 1.8 Hz, H-4), 4.40–4.34 (m, 2H, H-5′, H-6′b), 4.14–4.12 (m, 2H, H-3, H-6a), 3.91 and 3.85 (each d, J_gem = 15.2 Hz, 2H, CH₂Cl), 3.76–3.71 (m, 2H, H-2, H-6b), 3.67–3.63 (m, 4H, -OCH₂-, -OCH₃), 3.49 (s, 1H, H-5), 3.46–3.42 (m, 1H, -OCH₂-), 2.35–2.30 (m, 2H, -CH₂-), 1.68–1.58 (m, 4H, 2×-CH₂-), 1.41–1.26 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 166.8, 165.84, 165.82, 165.0, 137.7, 133.8, 133.5, 133.3, 130.0, 129.7, 129.6, 129.3, 129.27, 129.2, 129.0, 128.98, 128.76, 128.70, 128.65, 128.5, 128.3, 128.2, 128.1, 126.1, 125.2, 102.8 (C-1′), 100.6 (PhCH), 98.5 (C-1), 76.3 (C-3), 75.8 (C-4), 73.0 (C-3′), 71.2 (C-5′), 69.2 (C-2′), 69. 0 (C-6), 68.3 (-OCH₂-), 67.7 (C-4′), 63.0 (C-5), 62.2 (C-6′), 58.4 (C-2), 51.5 (OCH₃), 40.4 (-CH₂Cl), 33.8, 29.6, 28.9, 25.6, 24.6, 21.4. HR-ESIMS: calcd for C₄₉H₅₀ClN₃O₁₆Na: m/z 994.2777; found: m/z 994.2764 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2,4,6-tri-O-benzoyl-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (8)

(I) A solution of 6 (175 mg, 0.17 mmol) in CH₂Cl₂-H₂O (19:1, 2 mL) was treated with DDQ (96 mg, 0.42 mmol) at room temperature and then was stirred for 6 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 (2.5:1 hexane-AcOEt) as eluent to give 8 (94.5 mg, 63%).

(II) A solution of 7 (720 mg, 0.74 mmol) in MeOH-Pyr. (3:1, 8 mL) was treated with thiourea (169 mg, 2.22 mmol) under reflux for 2 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 (18:1 toluene-acetone) as eluent to give 8 (558 mg, 84%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ + 44.2 (c =1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.15–7.26 (m, 20H, 4×Ph), 5.75 (d, 1H, J_{3′, 4′} = 3.1 Hz, H-4′), 5.46 (dd, 1H, J_{1′, 2′} = 7.8 Hz, J_{2′, 3′} = 10 Hz, H-2′), 5.44 (s, 1H, PhCH), 5.05 (d, 1H, H-1′), 4.95 (d, 1H, J_{1, 2} = 3.5 Hz, H-1), 4.69 (dd, 1H, J_{5′, 6′a}= 7.4 Hz, J_{6′a, 6′b} = 11.5 Hz, H-6′a), 4.44–4.41 (m, 2H, H-4, H-6′b), 4.23–4.21 (m, 1H, H-5′), 4.16–4.08 (m, 3H, H-3, H-6a, H-3′), 3.77 (dd, J_{2, 3} = 10.7 Hz, 1H, H-2), 3.71–3.63 (m, 5H, H-6b, -OCH_2, CH₃), 3.48–3.45 (m, 1H, -OCH_2,-), 3.43 (s, 1H, H-5), 3.00 (d, 1H, OH), 2.34–2.31 (m, 2H, -CH₂-), 1.69–1.59 (m, 4H, 2×-CH₂-), 1.41–1.26 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 167.3, 166.2, 166.0, 137.7, 133.6, 133.45, 133.43, 130.1, 129.9, 129.7, 129.6, 129.4, 129.1, 128.8, 128.7, 128.5, 128.3, 128.1, 126.1, 102.6 (C-1′), 100.7 (PhCH), 98.6 (C-1), 76.3 (C-3), 76.0 (C-4), 73.7 (C-2′), 72.2 (C-3′), 71.6 (C-5′), 70.5 (C-4′), 69.0 (C-6), 68.3 (OCH₂), 63.0 (C-5), 62.8 (C-6′), 58.6 (C-2), 51.5 (OCH₃), 33.9, 29.6, 29.0, 25.6, 24.7. HR-ESIMS: calcd for C₄₇H₄₉N₃O₁₅Na: m/z 918.3061; found: m/z 918.3045 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-3,6-di-O-benzyl-4-O-chloroacetyl-β-d-galacto-pyranosyl-(1→3)-2,4,6-tri-O-benzoyl-β-d-galactopyranosyl-(1→3)-2-azido–4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (10)

Compound 10 was prepared from 8 (648 mg, 0.72 mmol) and 9 (554 mg, 0.87 mmol) as described for preparation of 7. The product was purified by silica gel column chromatography (30:1 toluene-acetone) to give 10 (802 mg, 78%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ + 53.3 (c =1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.10–6.96 (m, 35H, 7×Ph), 5.74 (d, 1H, J_{3′, 4′} = 3.6 Hz, H-4′), 5.57 (dd, 1H, J_{1′, 2′} = 8.1 Hz, J_{2′, 3′} = 9.9 Hz, H-2′), 5.52 (d, 1H, J_{3′, 4′} = 2.8 Hz, H-4″), 5.34 (s, 1H, PhCH), 5.11 (dd, 1H, J_{1′, 2′} = 7.9 Hz, J_{2′, 3′} = 10.2 Hz, H-2″), 4.89 (d, 1H, H-1′), 4.83 (d, 1H, J_{1, 2} = 3.3 Hz, H-1), 4.69 (d, 1H, H-1″), 4.58–4.13 (m, 7H, H-4, H-5′, H-6′a, b, H-3″, 2×PhCH₂), 4.02 (br. dd, 2H, H-6″a, b), 3.93 (dd, 1H, J_{2, 3} = 11.0 Hz, J_{3, 4} = 3.0 Hz, H-3), 3.87–3.29 (m, 13H, H-2, H-3, H-5, H-6a, b, H-3′, -CH₂Cl, -OCH₂-, -OCH₃), 2.34–2.31 (m, 2H, -CH₂-), 1.69–1.59 (m, 4H, -CH₂-), 1.41–1.26 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 166.8, 166.3, 166.0, 164.5, 164.4, 137.6, 137.4, 136.8, 133.3, 133.1, 132.6, 132.5, 130.2, 129.81, 129.77, 129.63, 129.60, 129.4, 128.57, 128.50, 128.47, 128.2, 128.1, 128.03, 127.98, 127.95, 127.88, 127.6, 126.0, 102.8 (C-1 of Gal a), 101.1 (C-1 of Gal b), 100.4, 98.6 (C-1 of GalN), 77.2, 75.8, 75.7, 75.6, 73.7, 72.0, 71.9, 71.3, 70.8, 70.7, 70.5, 68.8, 68.2, 67.6, 67.1, 63.4, 63.0, 58.3, 51.5, 40.5, 33.9, 29.7, 28.9, 25.6, 24.6. HR-ESIMS: calcd for C₇₆H₇₆ClN₃O₂₂Na: m/z 1440.4507; found: m/z 1440.4543 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl-(1→3)-2,4,6-tri-O-benzoyl-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galacto-pyranoside (11)

Compound 11 was prepared from 10 (77.5 mg, 54.6 μmol) as described for preparation of 8, yielding 62.4 mg (85%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ + 43.3 (c =1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.07–6.99 (m, 35H, 7×Ph), 5.79 (d, 1H, J_{3′, 4′} = 3.6 Hz, H-4′), 5.58 (dd, 1H, J_{1′, 2′} = 8.0 Hz, J_{2′, 3′} = 10.0 Hz, H-2′), 5.52 (d, 1H, J_{3′, 4′} = 2.8 Hz, H-4“), 5.35 (s, 1H, PhCH), 5.21 (dd, 1H, J_{1′, 2′} = 7.8 Hz, J_{2′, 3′} = 9.6 Hz, H-2″), 4.89 (d, 1H, H-1′), 4.84 (d, 1H, J_{1, 2} = 3.5 Hz, H-1), 4.66 (d, 1H, H-1″), 4.60–3.94 (m, 10H, H-3, H-4, H-5′, H-6′a, b, H-3″, H-6″a, b, 2×PhCH₂), 3.77–3.32 (m, 11H, H-2, H-3, H-5, H-6a, b, H-3′, -OCH₂, OCH₃), 2.34–2.31 (m, 2H, -CH₂-), 1.69–1.59 (m, 4H, -2×CH₂-), 1.41–1.26 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 166.1, 165.9, 164.7, 164.4, 138.1, 137.6, 137.0, 133.3, 133.1, 132.7, 132.4, 130.2, 129.8, 129.70, 129.67, 129.60,129.56, 128.56, 128.50, 128.48, 128.40, 128.3, 128.1, 127.9, 127.79, 127.75, 127.6, 126.0, 102.8 (C-1 of Gal a), 101.1 (C-1 of Gal b), 100.4, 98.6 (C-1 of GalN), 78.0, 76.0, 75.8, 75.6, 73.7, 72.0, 71.5, 71.05, 70.95, 70.6, 68.88, 68.85, 68.2, 65.9, 63.2, 63.0, 58.3, 51.5, 33.9, 29.0, 25.6, 24.6. HR-ESIMS: calcd for C₇₄H₇₅N₃O₂₁Na: m/z 1364.4791; found: m/z 1364.4755 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2,3-di-O-benzyl-4,6-O-di-tert-butylsilylene-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl-(1→3)–2,4,6-tri-O-benzoyl-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene–2-deoxy-α-d-galactopyranoside (13)

Compound 13 was prepared from 11 (60 mg, 44.7 μmol) and 12 (39.8 mg, 67.1 μmol) as described for preparation of 6. The product was purified by silica gel column chromatography (10:1 toluene-ethyl acetate) to give 13 (69.7 mg, 85%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ + 71.2 (c =1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.02–6.95 (m, 45H, 9×Ph), 5.81 (d, 1H, J_{3′, 4′} = 3.3 Hz, H-4′), 5.61 (dd, 1H, J_{1′, 2′} = 7.9 Hz, J_{2′, 3′} = 9.7 Hz, H-2′), 5.33 (s, 1H, PhCH), 5.26 (dd, 1H, J_{1′, 2′} = 7.9 Hz, J_{2′, 3′} = 10.2 Hz, H-2″), 4.88 (d, 1H, J_{1, 2} = 6.7 Hz, H-1 of Gal a), 4.86 (d, 1H, J_{1, 2} = 2.6 Hz, H-1 of Gal c), 4.84 (d, 1H, J_{1, 2} = 3.0 Hz, H-1 of GalN), 4.67 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal b), 0.97 and 0.95 (each s, 18H, 2×C(CH₃)₃. ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 166.0, 164.7, 164.5, 139.22, 139.19, 138.2, 137.6, 137.4, 133.2, 132.9, 132.7, 132.3, 130.1, 129.8, 129.6, 129.5, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.78, 127.73, 127.67, 127.3, 127.2, 127.1, 126.0, 102.8 (C-1 of Gal a), 101.1 (C-1 of Gal b), 100.5 (C-1 of Gal c), 100.4, 98.6 (C-1 of GalN), 78.6, 78.1, 75.8, 75.7, 75.6, 74.5, 74.1, 73.4, 73.1, 72.1, 71.6, 71.4, 71.1, 70.9, 70.7, 70.5, 68.8, 68.2, 67.7, 67.0, 63.3, 63.0, 58.3, 51.5, 33.8, 29.7, 29.0, 27.6, 27.4, 25.6, 24.6, 23.2, 20.7. HR-ESIMS: calcd for C₁₀₂H₁₁₃N₃O₂₆SiNa: m/z 1846.7279; found: m/z 1846.7245 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 3,4,6-tri-O-acetyl-2-O-benzoyl-β-d-galactopyranosyl-(1→3)-2,4,6-tri-O-benzoyl-β-d-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-galactopyranoside (14)

To a solution of 11 (137 mg, 0.10 mmol) in MeOH—THF—NH₃aq. (3:1:0.1, 4.1 mL) Pd/C (150 mg) was added. The mixture was stirred for 80 min at room temperature under H₂ atmosphere. After completion of the reaction, the mixture was filtered through Celite. The filtrate was concentrated with toluene. To a solution of this compound in AcOH (2.0 mL) was hydrogenolysed in the presence of Pd/C (150 mg) for 18 h at room temperature. The mixture was filtered and concentrated, and the residue was acetylated with acetic anhydride (3.0 mL) in pyridine (5.0 mL). After concentration, the residue underwent de-benzylidenation with TsOH (30 mg) in CHCl₃-MeOH (2:1, 6 mL) for 15 h at room temperature, and was then neutralized with Et₃N. After concentration, the residue was acetylated with acetic anhydride (1.0 mL) in pyridine (1.5 mL) for 5 h at room temperature. After the reaction, toluene was added and co-evaporated several times. The product was purified by silica gel column chromatography (5:1 CHCl₃-MeOH) to give 14 (68 mg, 51%, 5 steps). ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ + 69.2 (c=1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.10–7.17 (m, 20H, 4×Ph), 5.83 (d, 1H, J_{3′, 4′} = 3.1 Hz, H-4′), 5.49–5.46 (m 2H, H-4, 2′), 5.38 (d, 1H, J = 8.3 Hz, NH), 5.27 (d, 1H, J_{3″, 4″} = 2.4 Hz, H-4″), 5.23 (dd, 1H, J_{1′, 2′} = 7.8 Hz, J_{2′, 3′} = 10.5 Hz, H-2″), 4.77 (d, 1H, J_{1″, 2″} = 7.6 Hz, H-1″), 4.82 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of Gal N), 4.81 (d, 1H, J_{1, 2} = 8.1 Hz, H-1of Gal’). 4.50–3.23 (m 14H, H-2, 3, 5, 6a, 6b, 2′, 5′, 6′a, 6′b, 5″, 6″a, 6″b, -OCH₂-), 3.67 (s, 3H, OMe), 2.26–2.23 (m, 2H, -CH₂-), 2.05, 2.002, 1.999, 1.989 (each s, 12H, 4×Ac), 1.74–1.72 (m, 4H, -CH₂-), 1.43–1.21 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 170.4, 170.3, 170.2, 170.0, 169.9, 169.7, 169.2, 165.7, 164.3, 133.2, 133.13, 133.08, 132.7, 130.1, 129.7, 129.5, 129.44, 129.37, 129.1, 128.9, 128.42, 128.37, 128.35, 128.0, 101.0 (C-1″), 100.8 (C-1′), 97.1 (C-1), 76.7, 74.7, 72.1, 71.9, 70.7, 70.5, 70.1, 69.7, 69.2, 68.6, 67.7, 67.1, 63.0, 62.7, 60.9, 51.5, 48.9, 33.7, 28.5, 25.6, 24.64, 22.3, 20.7, 20.63, 20.59, 20.4, 20.3. HR-ESIMS: calcd for C₆₅H₇₃NO₂₇Na: m/z 1322.4268; found: m/z 1322.4249 [M + Na]⁺.

• Biotinylated trisaccharide (A)

To a solution of 14 (68 mg, 52.3 μmol) in MeOH (1.0 mL) NaOMe (30 mg) was added and the mixture was stirred at 40 °C for 2 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 15 (38 mg, quant.) [MALDI-TOFMS: calcd for C₂₇H₄₇NO₁₈Na, m/z 696.3; found, m/z 696.7 [M + Na]⁺]. The residue (26 mg, 37 μmol) was dissolved in anhydrous ethylenediamine (5 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 (4.0 mL), and the pH was adjusted to 8–9 using DIPEA. Biotine-NHS (15.2 mg, 45.0 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 A (23.0 mg, 67%). ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ + 82.4 (c=0.4, CHCl₃). ¹H-NMR (500 MHz, D₂O): δ 4.70 (d, 1H, J_{1, 2} = 4.0 Hz, H-1), 4.42 (d, 1H, J_{1, 2} = 7.0 Hz, H-1″), 4.34 (d, 1H, J_{1, 2} = 7.5 Hz, H-1′). ¹³C-NMR (125 MHz, CDCl₃): δ 176.8, 176.6, 174.0, 164.9, 104.0 (C-1″), 103.9 (C-1′), 96.6 (C-1), 81.6, 77.2, 74.6, 74.2, 72.1, 70.6, 70.1, 69.4, 68.2, 68.0, 67.4, 61.6, 60.8, 60.5, 59.8, 54.9, 48.3, 39.3, 38.2, 38.1, 35.4, 35.1, 27.8, 27.5, 27.3, 24.70, 24.69, 24.5, 21.6, 20.7, 20.63, 20.59, 20.4, 20.3. HR-ESIMS: calcd for C₃₈H₆₅N₅O₁₉SNa: m/z 950.3892.; found: m/z 950.3984 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2,3-di-O-benzyl-4,6-di-O-acetyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl-(1→3)-2,4,6-tri-O-benzoyl-β-d-galactopyranosyl-(1→3)-2-azido-4,6-di-O-acetyl-2-deoxy-α-d-galactopyranoside (16)

A solution of 13 (217 mg, 0.12 mmol) in Pyr. (2.5 mL) was added HF—Pyr. (1.0 mL) at 0 °C and then was stirred for 4 h. The reaction mixture was added to water, extracted with ethyl acetate, and the organic layer was washed with saturated aqueous NaHCO₃ and water, dried (MgSO₄), and concentrated. A solution of the residue in 80% AcOH (4 mL) was stirred at 70 °C for 5 h, then was diluted with toluene and concentrated. The residue was treated with Ac₂O (1.1 mL) in pyridine (2 mL). The reaction mixture was poured into ice-water and extracted with CHCl₃. The extract was washed sequentially with 5% HCl, saturated aqueous NaHCO₃ and water, dried (MgSO₄), and concentrated. The product was purified by silica gel column chromatography (10:1 toluene-acetone) to give 16 (136 mg, 65%, 3 steps). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +66.8 (c = 0.50, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.06–6.94 (m, 40H, 8×Ph), 5.84 (d, 1H, J_3,4 = 3.1 Hz, H-4 of Gal b), 5.55–5.49 (m 4H, H-4 of GalN, H-2, 4 of Gal a, H-4 of Gal c), 5.30 (dd, 1H, J_1,2 = 7.5 Hz, J_2,3 = 10.0 Hz, H-2 of Gal b), 4.97 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of Gal c), 4.81 (d, 1H, J_{1, 2} = 7.0 Hz, H-1 of Gal a), 4.80 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of GalN), 4.67 (d, 1H, J_{1, 2} = 7.0 Hz, H-1 of Gal b), 3.65 (s, 3H, OMe), 2.36–2.31 (m, 2H, -CH₂-), 2.02×2, 1.98, 1.85 (each s, 12H, 4×Ac), 1.66–1.54 (m, 4H, -CH₂-), 1.35–1.25 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 173.9, 170.5, 169.5, 166.1, 165.6, 164.5, 138.9, 138.3, 137.2, 133.0, 132.8, 130.0, 129.9, 129.7, 129.5, 128.4, 128.3, 128.2, 128.2, 128.1, 127.9, 127.9, 127.7, 127.6, 127.5, 127.3, 101.9 (C-1 of Gal a), 101.3 (C-1 of Gal b), 100.5 (C-1 of Gal c), 97.9 (C-1 of GalN), 76.4, 75.8, 74.6, 73.9, 73.7, 73.0, 72.0, 71.9, 71.6, 71.5, 69.8, 68.1, 67.8, 67.2, 67.0, 62.8, 61.5, 59.1, 51.5, 33.9, 29.7, 28.9, 25.6, 24.5, 20.8, 20.7, 20.6. HR-ESIMS: calcd for C₉₅H₁₀₁N₃O₃₀Na: m/z 1786.6368.; found: m/z 1786.6384 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl-(1→4)-3,6-di-O-acetyl-2-O-benzoyl-β-d-galactopyranosyl-(1→3)-2,4,6-tri-O-benzoyl-β-d-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-galactopyranoside (17)

To a solution of 16 (300 mg, 0.17 mmol) in THF—H₂O (6:1, 7.0 mL) was added PPh₃ (50 mg, 0.19 mmol). The mixture was stirred under reflux for 3.5 h after completion of the reaction, the reaction mixture was added to water, extracted with ethyl acetate, and the organic layer was washed with saturated aqueous NaHCO₃ and water, dried (MgSO₄), and concentrated. The residue was acetylated with acetic anhydride (4.0 mL) in pyridine (6.0 mL). After the reaction was quenched with MeOH, the residue was diluted with toluene and concentrated. The product was purified by silica gel column chromatography (10:1 toluene-acetone) to give acetamide compound. This compound in THF-MeOH (1:1, 2.0 mL) was hydrogenolysed in the presence of Pd(OH)₂/C (75 mg) for 19 h at room temperature, and the mixture was filtered and concentrated. The residue was acetylated with acetic anhydride (1.0 mL) in pyridine (1.0 mL). After the reaction was quenched with MeOH, toluene was added and co-evaporated several times. The product was purified by silica gel column chromatography (10:1 toluene-acetone) to give 17 (175 mg, 65%, 4 steps). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +91.6 (c = 0.59, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.14–7.16 (m, 20H, 4×Ph), 5.90 (d, 1H, J_3,4 = 3.1 Hz, H-4 of Gal a), 5.51–5.13 (m 8H, H-4 of GalN, H-2 of Gal a, H-2,3 of Gal b, H-2,3,4 of Gal c, NH), 4.99 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of Gal c), 4.86 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of GalN), 4.80 (d, 1H, J_{1, 2} = 7.2 Hz, H-1 of Gal a), 4.79 (d, 1H, J_{1, 2} = 7.8 Hz, H-1 of Gal b), 3.67 (s, 3H, OMe), 2.36–2.25 (m, 2H, -CH₂-), 2.18, 2.11, 2.06, 2.04, 2.02, 2.00 × 2, 1.94, 1.91 (each s, 27H, 9×Ac), 1.68–1.56 (m, 4H, -CH₂-), 1.35–1.25 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 175.6, 174.0, 170.6, 170.5, 170.4, 170.07, 169.92, 169.8, 169.3, 166.2, 165.3, 164.5, 164.1, 137.9, 133.1, 133.0, 132.7, 131.0, 130.2, 129.8, 129.7, 129.5, 129.3, 129.2, 129.01, 128.96, 128.4, 128.2, 128.1, 125.3, 101.0 (C-1 of Gal a), 100.6 (C-1 of Gal b), 98.6 (C-1 of Gal c), 97.1 (C-1 of GalN), 75.6, 74.4, 72.10, 71.95, 69.6, 69.3, 68.6, 68.4, 68.1, 67.8, 67.4, 67.2, 67.1, 63.0, 62.7, 62.1, 61.6, 61.1, 51.5, 49.0, 42.8, 33.8, 29.7, 29.3, 28.8, 28.7, 25.7, 25.6, 25.3, 24.4, 22.4, 21.4, 20.82, 20.75, 20.71, 20.61, 20.56. HR-ESIMS: calcd for C₇₇H₈₉NO₃₅Na: m/z 1610.5113.; found: m/z 1610.5152 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl α-d-galactopyranosyl-(1→4)-β-d-galactopyranosyl-(1→3)-β-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-α-d-galactopyranoside (18)

To a solution of 17 (23 mg, 14.5 mmol) in MeOH (2 mL) was added NaOMe (20 mg) at room temperature and the mixture was stirred at 40 °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 MeOH to give 18 (7.3 mg, 60%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ + 68.2 (c = 0.18, MeOH). ¹H-NMR (500 MHz, CH₃OH): δ 4.97 (d, 1H, J_{1, 2} = 3.7 Hz, H-1 of Gal c), 4.84 (d, 1H, J_{1, 2} = 3.7 Hz, H-1 of GalN), 4.55 (d, 1H, J_{1, 2} = 7.3 Hz, H-1 of Gal a), 4.49 (d, 1H, J_{1, 2} = 7.6 Hz, H-1 of Gal b), 3.66 (s, 3H, OMe), 2.36–2.25 (m, 2H, -CH₂-), 1.97 (s, 3H, Ac), 1.67–1.60 (m, 4H, -CH₂-), 1.47–1.38 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CH₃OH): δ 176.0, 173.9, 106.4 (C-1of Gal b), 105.8 (C-1 of Gal a), 102.6 (C-1 of Gal c), 98.9 (C-1 of GalN), 84.8, 79.6, 79.1, 76.2, 74.4, 73.0, 72.8, 72.1, 71.6, 71.4, 71.1, 70.6, 70.0, 69.6, 68.8, 62.8, 62.7, 62.5, 61.5, 52.0, 50.4, 49.9, 49.5, 49.3, 48.7, 48.5, 34.7, 33.1, 30.8, 30.5, 26.8, 25.8, 23.7, 22.8, 14.4, 1.5. HR-ESIMS: calcd for C₃₃H₅₇NO₂₃Na: m/z 858.3219.; found: m/z 858.3225 [M + Na]⁺.

• Biotinylated tetrasaccharide (B)

Compound 18 (7.3 mg, 8.7 μmol) was dissolved in neat anhydrous ethylenediamine (1.5 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 (2 mL), and the pH was adjusted to 8–9 using DIPEA. Biotin-NHS (4.0 mg 11.5 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 B (3.9 mg, 41% 2 steps). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ + 56.9 (c = 0.1, H₂O). ¹H-NMR (600 MHz, D₂O): δ 4.84 (br.s, 1H, H-1 of Gal c), 4.77 (br.s, 1H, H-1 of GalN), 4.57 (d, 1H, J_{1, 2} = 7.4 Hz, H-1 of Gal a), 4.40 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal b). ¹³C-NMR (150 MHz, D₂O): δ 215.0, 176.8, 176.6, 174.0, 164.9, 104.0 (C-1 of Gal a,b), 99.9 (C-1 of Gal c), 96.6 (C-1 of GalN), 74.7, 71.7, 70.6, 69.5, 68.5, 68.2, 61.6, 60.1, 59.8, 48.3, 39.3, 38.1, 35.1, 29.8, 27.5, 27.3, 24.7. HRFABMS: calcd for C₄₄H₇₅N₅O₂₄SNa, m/z 1112.4420; found, m/z 1114.4495 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2,4,6-tri-O-benzyl-3-O-chloroacetyl-α-d-galactopyranosyl-(1→3)-2,4,6-tri-O-benzoyl-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (24)

Compound 24 was prepared from 8 (100 mg, 0.11 mmol) and 22 (83 mg, 0.13 mmol) as described for preparation of 7. The product was purified by silica gel column chromatography (10:1 toluene-ethyl acetate) to give 24 (103 mg, 67%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +61.8 (c = 1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.11–7.04 (m, 35H, 7×Ph), 5.89 (d, 1H, J_3,4 = 3.1 Hz, H-4′), 5.76 (br. t, 1H, H-2′), 5.44 (s, 1H, PhCH), 5.28 (d, 1H, J_{1″, 2″} = 3.5 Hz, H-1″), 4.93(d, 1H, J_{1′, 2′} = 8.0 Hz, H-1′), 4.92 (d, 1H, J_{1, 2} = 3.5 Hz, H-1), 3.65 (s, 3H, OMe), 2.35–2.31 (m, 2H, -CH₂-), 1.69–1.59 (m, 4H, 2×-CH₂-), 1.42–1.26 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 166.2, 165.98, 165.97, 164.8, 138.2, 138.0, 137.8, 137.7, 133.5, 133.4, 133.0, 130.1, 129.97, 129.72, 129.60, 129.56, 129.1, 128.7, 128.6, 128.5, 128.43, 128.40, 128.3, 128.2, 128.1, 128.13, 127.98 127.86, 127.86, 127.81, 127.6, 127.4, 126.1, 102.9 (C-1′), 100.6, 98.7 (C-1), 93.9 (C-1″), 75.9, 75.3, 75.0, 74.9, 73.9, 73.3, 73.2, 72.7, 72.3, 71.6, 69.0, 68.5, 68.3, 65.9, 63.0, 62.8, 58.4, 51.5, 40.4, 33.9, 29.1, 25.6, 24.6. ESI-HRMS: calcd for C₇₆H₇₈ClN₃O₂₁Na, 1426.4714 m/z; found, 1426.4867 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (25)

Compound 25 was prepared from 5 (500 mg, 1.18 mmol) and 20 (964 mg, 1.78 mmol) as described for preparation of 7. The product was purified by silica gel column chromatography (10:1 toluene-ethyl acetate) to give 25 (770 mg, 76%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ + 56.3 (c = 0.5, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.02–7.22 (m, 15H, 3×Ph), 5.74 (dd, 1H J_2′,3′ = 10.5, J_1′,2′ = 8.0 Hz, H-2′), 5.55 and 5.54 (each s, 2H, 2×PhCH), 5.22 (dd, 1H, J_2,3 = 10.5 Hz, J_3,4 = 3.5, H-3′), 5.08 (d, 1H, J_1′,2′ = 8.0 Hz, H-1′), 4.94 (d, 1H, J_{1, 2} = 3.5 Hz, H-1), 4.50 and 4.49 (each d, 2H, J_3,4 = 3.0Hz, J_{3′, 4′} = 3.5Hz, H-4, 4′), 4.40–3.93 (m, 7H, H-3, 6a, 6b, 6′a, 6′b, -CH₂Cl), 3.81–3.69 (m, 3H, H-5, 5′, -OCH₂-), 3.66 (s, 3H, OMe), 3.52–3.46 (m, 1H, -OCH₂-), 2.33–2.30 (m, 2H, -CH₂-), 1.67–1.61 (m, 4H, 2×-CH₂-), 1.42–1.36 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 167.3, 165.0, 137.6, 137.4, 133.2, 129.8, 129.5, 129.2, 128.6, 128.4, 128.3, 128.0, 126.4, 126.1, 101.4 (C-1′), 101.1 (PhCH), 100.5 (PhCH), 98.7 (C-1), 75.8, 73.9 (C-3′), 73.6, 73.1, 69.1, 69.0, 68.9, 68.3, 66.3, 58.8, 51.5, 40.7, 33.9, 29.1, 25.7, 24.6. ESI-HRMS: calcd for C₄₂H₄₆ClN₃O₁₄Na, 874.2566 m/z; found, 874.2646 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (26)

A solution of 25 (100 mg, 0.12 mmol) in MeOH-Pyr. (3:1, 2 mL) was treated with thiourea (27 mg, 0.35 mmol) under reflux for 2 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 (20:1 toluene-acetone) as eluent to give 26 (69 mg, 76%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +46.3 (c = 0.3, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.08–7.23 (m, 15H, 3×Ph), 5.54 and 5.51 (each s, 2H, 2×PhCH), 5.42 (dd, 1H J_2′,3′ = 10.0 Hz, J_1′,2′ = 8.0 Hz, H-2′), 4.96 (d, 1H, J_1′,2′ = 8.0 Hz, H-1′), 4.95 (d, 1H, J_{1, 2} = 2.5 Hz, H-1), 4.40–3.93 (m, 6H, H-4, 6a, 6b, 4′, 6′a, 6′b), 3.85 (dt, 1H, H-3), 3.71–3.66 (m, 3H, H-5, 5′, -OCH₂-), 3.65 (s, 3H, OMe), 3.50–3.44 (m, 1H, -OCH₂-), 2.73 (d, 1H, OH), 2.34–2.29 (m, 2H, -CH₂-), 1.69–1.60 (m, 4H, 2×-CH₂-), 1.42–1.36 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.1, 166.4, 137.7, 137.6, 133.1, 130.0 129.9, 129.3, 128.6, 128.3, 128.3, 128.0, 126.5, 126.1, 101.4, 101.4 (C-1′), 100.6, 98.7 (C-1), 76.0, 75.5, 73.3, 72.9, 71.9, 69.1, 69.0, 68.3, 66.6, 63.1, 58.8, 51.5, 33.9, 29.1, 25.7, 24.7 ESI-HRMS: calcd for C₄₀H₄₅N₃O₁₃Na, 798.2850 m/z; found, 798.2912 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O- benzylidene-2-deoxy-α-d-galactopyranoside (27)

Compound 27 was prepared from 26 (300 mg, 0.39 mol) and 20 (417 mg, 0.77 mol) as described for preparation of 7. The product was purified by silica gel column chromatography (10:1 toluene-ethyl acetate) to give 27 (204 mg, 44%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ + 81.3 (c = 1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 7.86–7.16 (m, 25H, 5×Ph), 5.65–5.58 (m, 2H, H-2 of Gal a,b), 5.48, 5.47 and 5.46 (each s, 3H, 3×PhCH), 5.01 (d, 1H, J_1,2 = 8.0 Hz, H-1 of Gal b), 4.99 (d, 1H, J_{3, 4} = 3.0 Hz, H-3 of Gal b), 4.98 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal a), 4.91 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of GalN), 3.66 (s, 3H, OMe), 3.50–3.44 (m, 1H, -OCH₂-), 2.31–2.28 (m, 2H, -CH₂-), 1.66–1.55 (m, 4H, 2×-CH₂-), 1.40–1.35 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 167.2, 137.6, 137.3, 132.8, 129.7, 129.6, 129.2, 128.6, 128.4, 128.3, 128.2, 128.0, 127.8, 126.3, 126.2, 126.1, 101.4 (C-1 of Gal a), 101.0, 100.7 (C-1 of Gal b), 100.3, 100.1, 98.8 (C-1 of GalN), 75.8, 75.7, 75.4, 73.8, 73.0, 72.3, 69.0, 68.8, 68.2, 66.9, 66.3, 63.2, 58.7, 51.5, 40.6, 29.1, 25.6, 24.6. ESI-HRMS: calcd for C₆₂H₆₄ClN₃O₂₀Na, 1228.3669 m/z; found, 1228.3792 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (28)

Compound 28 was prepared from 27 (200 mg, 0.17 mmol) as described for preparation of 11. The product was purified by silica gel column chromatography (5:1 toluene-ethyl acetate) to give 28 (160 mg, 85%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +120.4 (c = 0.5, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 7.96–7.17 (m, 25H, 5×Ph), 5.61 (dd, 1H, J_{1, 2} = 8.0 Hz, J_{2, 3} = 10.0 Hz, H-2 of Gal b), 5.50, 5.48 and 5.42 (each s, 3H, 3×PhCH), 5.31 (dd, 1H, J_{1, 2a}= 8.1 Hz, J_{2, 3} = 9.8 Hz, H-2 of Gal a), 4.98 (d, 1H, J_{1, 2} = 8.5 Hz, H-1 of Gal a), 4.91 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal b), 4.91 (d, 1H, J_{1, 2} = 4.0 Hz, H-1 of GalN), 3.65 (s, 3H, OMe), 3.50–3.44 (m, 1H, -OCH₂-), 2.31–2.28 (m, 2H, -CH₂-), 1.65–1.57 (m, 4H, 2×-CH₂-), 1.40–1.35 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.1, 166.5, 164.8, 137.7, 137.7, 137.4, 132.9, 132.8, 129.8, 129.7, 129.5, 129.3, 128.7, 128.4, 128.3, 128.2, 127.8, 126.4, 126.3, 126.1, 101.5 (C-1 of Gal a), 101.3, 100.8, 100.3, 99.9 (C-1 of Gal b), 98.8 (C-1 of GalN), 77.3, 75.9, 75.7, 75.5, 75.5, 75.1, 72.5, 72.5, 71.9, 70.9, 69.0, 68.9, 68.8, 68.2, 66.8, 66.7, 63.16, 58.7, 51.5, 33.9, 29.1, 25.6, 24.6. ESI-HRMS: calcd for C₆₀H₆₃N₃O₁₉Na, 1152.3953 m/z; found, 1152.4071 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-3,6-di-O-benzyl-4-O-chloroacetyl-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (29)

Compound 29 was prepared from 28 (123 mg, 0.11 mmol) and 9 (83 mg, 0.13 mmol) as described for preparation of 7. The product was purified by silica gel column chromatography (10:1 toluene-ethyl acetate) to give 29 (94 mg, 53%). ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ + 55.3 (c = 0.5, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 7.92–6.94 (m, 40H, 8×Ph), 5.57 (d, 1H, J_3,4 = 3.0Hz, H-4 of Gal c), 5.49 (dd, 1H, J_{1, 2} = 8.0 Hz, J_{2, 3} = 10.0 Hz, H-2 of Gal c), 5.43 (dd, 1H, J_{1, 2} = 8.0 Hz, J_{2, 3} = 10.0 Hz, H-2 of Gal b), 5.45, 5.32 and 5.28 (each s, 3H, 3×PhCH), 5.26 (dd, 1H, J_{1, 2} = 8.5 Hz, J_{2, 3} = 9.8 Hz, H-2 of Gal a), 4.92 (d, 1H, J_{1, 2} = 7.5 Hz, H-1 of Gal a), 4.90 (d, 1H, J_{1, 2} = 3.0 Hz, H-1 of GalN), 4.86 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal b), 4.74 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal c), 3.65 (s, 3H, OMe), 2.32–2.28 (m, 2H, -CH₂-), 1.65–1.58 (m, 4H, 2×-CH₂-), 1.39–1.35 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.1, 166.9, 165.0, 164.7, 164.7, 137.8, 137.7, 137.6, 132.8, 132.6, 132.5, 130.3, 129.7, 129.6, 129.5, 129.4, 129.0, 128.8, 128.6, 128.4, 128.4, 128.2, 128.1, 128.1, 128.1, 128.0, 128.0, 127.9, 127.8, 127.8, 127.7, 126.2, 126.2, 126.2, 126.1, 126.1, 101.5 (C-1 of Gal a), 100.6 (C-1 of Gal c), 100.5, 100.4, 99.7 (C-1 of Gal b), 98.8 (C-1 of GalN), 76.2, 75.7, 75.4, 73.7, 73.6, 72.6, 71.7, 71.0, 70.8, 70.6, 69.9, 69.0, 68.8, 68.1, 67.8, 67.0, 66.8, 63.1, 58.6, 51.5, 40.9, 33.9, 29.7, 29.5, 29.0, 25.6, 24.6. ESI-HRMS: calcd for C₈₉H₉₀ClN₃O₂₆Na, 1675.5477 m/z; found, 1675.5602 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (30)

Compound 30 was prepared from 29 (80 mg, 48 mmol) as described for preparation of 11. The product was purified by silica gel column chromatography (4:1 toluene-EtOAc) to give 30 (74 mg, 97%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +64.1 (c = 0.5, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 7.86–6.97 (m, 40H, 8×Ph), 5.47–5.32 (m, 3H, H-2 of Gal a, b and c), 5.44, 5.38 and 5.27 (each s, 3H, 3×PhCH), 4.96 (d, 1H, J_{1, 2} = 7.5 Hz, H-1 of Gal a), 4.92 (d, 1H, J_{1, 2} = 6.5 Hz, H-1 of Gal b), 4.86 (d, 1H, J_{1, 2} = 2.5 Hz, H-1 of GalN), 4.65 (d, 1H, J_{1, 2} = 7.0 Hz, H-1 of Gal c), 3.64 (s, 3H, OMe), 2.35–2.30 (m, 2H, -CH₂-), 1.65–1.56 (m, 4H, 2×-CH₂-), 1.39–1.34 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.1, 165.1, 164.8, 164.7, 137.9, 137.8, 137.8, 137.7, 136.9, 132.7, 132.5, 132.4, 130.1, 129.7, 129.6, 129.5, 129.0, 128.6, 128.5, 128.4, 128.4, 128.3, 128.2, 128.1, 128.0, 128.0, 127.9, 127.9, 127.8, 127.8, 127.6, 127.5, 126.3, 126.3, 126.2, 125.3, 101.6(C-1 of Gal a), 100.9 (C-1 of Gal c), 100.6, 100.5, 100.4, 99.8 (C-1 of Gal b), 98.8 (C-1 of GalN), 75.7, 75.5, 75.3, 73.6, 73.2, 71.2, 70.8, 70.7, 70.2, 69.0, 68.9, 68.7, 66.7, 66.7, 65.8, 63.1, 58.5, 51.5, 33.9, 29.7, 29.0, 25.6, 24.6, 21.4. ESI-HRMS: calcd for C₈₇H₈₉N₃O₂₅Na, 1598.5683 m/z; found, 1598.5839 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2,3-di-O-benzyl-4,6-O-di-tert-butylsilylene-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (31)

Compound 31 was prepared from 30 (64 mg, 41 mmol) and 12 (36 mg, 61 mmol) as described for preparation of 7. The product was purified by silica gel column chromatography (5:1 toluene-ethyl acetate) to give 31 (57 mg, 68%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ + 124.5 (c = 1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 7.94–6.96 (m, 50H, 10×Ph), 5.50–5.40 (m, 3H, H-2 of Gal a, b and c), 5.45, 5.32 and 5.25 (each s, 3H, 3×PhCH), 4.94 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal a), 4.92 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of GalN), 4.92 (d, 1H, J_{1, 2} = 7.5 Hz, H-1 of Gal b), 4.74 (d, 1H, J_{1, 2} = 4.0 Hz, H-1 of Gal d), 4.63 (d, 1H, J_{1, 2} = 7.5 Hz, H-1 of Gal c), 3.65 (s, 3H, OMe), 2.35–2.31 (m, 2H, -CH₂-), 1.67–1.56 (m, 4H, 2×-CH₂-), 1.37–1.34 (m, 2H, -CH₂-), 0.96 and 0.92 (each s, 18H, 2×(CH₃)₃). ¹³C-NMR (125 MHz, CDCl₃): δ 174.1, 165.1, 164.8, 164.7, 137.9, 137.85, 137.80, 137.7, 132.7, 132.5, 132.5, 130.2, 129.68, 129.62, 129.5, 129.0, 128.6, 128.5, 128.41, 128.36, 128.29, 128.23. 128.1, 128.02, 127.97, 127.93, 127.90, 127.84, 127.78, 127.6, 127.5, 126.31, 126.2, 125.3, 101.6 (C-1 of Gal a), 100.87, 100.6 (C-1 of Gal c), 100.5, 100.3 (C-1 of Gal b), 99.8 (C-1 of Gal d), 98.8 (C-1 of GalN), 78.7, 77.8, 75.9, 75.7, 75.5, 75.2, 74.4, 73.6, 73.6, 73.4, 73.2, 72.3, 71.2, 70.8, 70.6, 70.5, 70.1, 70.0, 69.0, 68.8, 68.5, 68.2, 67.6, 67.5, 67.1, 66.8, 63.1, 58.6, 51.5, 33.9, 29.0, 27.6, 27.4, 27.3, 27.3, 25.6, 24.6, 23.3, 21.5, 20.7. ESI-HRMS: calcd for C₁₁₅H₁₂₇N₃O₃₀SiNa, 2080.8171 m/z; found, 2080.8264 m/z [M + Na]⁺.

• 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-benzyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranoside (34)

To a solution of 32 (145 mg, 0.26 mmol) and 33 (195 mg, 0.31 mmol) in dry CH₂Cl₂—toluene (1:1, 1.6 mL) powdered AW300 (330 mg) was added, and the mixture was stirred under Ar atmosphere at room temperature for 2 h, then cooled to –10 °C. NIS (139 mg, 0.62 mmol) and TfOH (2.7 μL, 31 μmol) were added to the mixture, which was stirred at −10 °C for 10min., 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 n-hexane-EtOAc) to give 34 (222 mg, 79%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +57.6 (c 1.2, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.03–7.05 (35H, m, 7×Ph), 5.52 (1H, dd, J_{1, 2} = 8.0 Hz, J_{2, 3} = 7.9 Hz, H-2), 5.03 (1H, d, J_{1′, 2′} = 3.4 Hz, H-1′), 4.96–4.89 (2H, m, 2×PhCH₂), 4.80 (2H, s, PhCH₂), 4.72–4.66 (2H, m, 2×PhCH₂),4.58–4.53 (2H, m, H-5, PhCH₂), 4.48 (1H, d, H-1), 4.34 (1H, d, J_gem = 12.9 Hz, PhCH₂), 4.27–4.12 (8H, m, H-4, H-2′, H-3′, H-4′, 4×PhCH₂), 4.06 (1H, dd, J_{5, 6a} = 1.2 Hz, J_{6a, 6b} = 10.9 Hz, H-6a), 4.04–3.94 (1H, m, CH₂), 3.59–3.49 (5H, m, H-3, H-6a’, H-6b’, H-5′, CH₂), 3.22 (1H, dd, J_{5, 6b} = 3.9 Hz, H-6b), 0.91–0.81 (2H, m, 2×CH₂), −0.10 (9H, s, TMS). ¹³C-NMR (125 MHz, CDCl₃): δ 165.1, 139.0, 138.9, 138.7, 138.4, 138.0, 137.8, 132.8, 130.4, 129.8 × 2, 128.3 × 2, 128.22 × 2, 128.17 × 2, 128.12 × 2, 128.09 × 2, 128.06 × 2, 128.04 × 2, 128.03 × 2, 127.9 × 2, 127.67, 127.64 × 2, 127.63 × 2, 127.60, 127.5, 127.4 × 2, 127.33, 127.30, 127.26, 127.22 × 2, 101.03 (C-1), 100.96 (C-1′), 79.3 (PhCH₂), 78.6 (C-3), 76.4 (C-3′), 74.9 (PhCH₂), 74.7 (C-4), 73.9 (PhCH₂), 73.8 (C-5′), 73.7 (PhCH₂), 73.0 (C-4′), 72.8 (C-2′), 72.3 (PhCH₂), 71.3 (C-2), 71.0 (PhCH₂), 68.9 (C-5), 67.7 × 2 (C-6, C-6′), 66.9 (CH₂), 17.9 (CH₂), −1.5 × 3 (TMS). ESI-HRMS: calcd for C₆₆H₇₄O₁₂SiNa, 1109.4847 m/z; found, 1109.4930 m/z [M + Na]⁺.

• 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-acetyl-β-d-galactopyranoside (35)

To a solution of 34 (2.47 g, 2.27 mmol) in THF—MeOH (1:1, 10.0 mL) was hydrogenolysed under hydrogen in the presence of Pd(OH)₂/C (1.05 g) for 20 h at room temperature. The mixture was filtered and concentrated, and the residue was acetylated with acetic anhydride (23 mL) in pyridine (15 mL) at 50 °C for 4 h. After the reaction was quenched with MeOH (20 mL) at 0 °C, toluene was added and co-evaporated several times. The product was purified by silica gel column chromatography (2:1 n-hexane-EtOAc) to give 35 (1.55 g, 85%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +73.4 (c 1.2, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 7.99–7.18 (5H, m, Ph), 5.61 (1H, d, J_{3′, 4′} = 2.4 Hz, H-4′), 5.47–5.42 (2H, m, H-2, H-3′), 5.22 (1H, dd, J_{1′, 2′} = 3.5 Hz, H-2′), 5.05 (1H, d, H-1′), 5.02 (1H, dd, J_{2, 3} = 10.8 Hz, H-3), 4.65 (1H, d, J_{1, 2} = 7.7 Hz, H-1), 4.64–4.62 (1H, m, H-5), 4.51 (1H, dd, J_{5′, 6a’} = 6.6 Hz, J_{6a’, 6b’} = 11.1 Hz, H-6a’), 4.21–4.14 (3H, m, H-6a, H-6b, H-6b’), 4.11 (1H, d, J_{3, 4} = 2.3 Hz, H-4), 4.03–3.97 (1H, m, CH₂), 3.86 (1H, t, J_{5′, 6b’} = 6.6 Hz, H-5′), 3.60–3.55 (1H, m, CH₂), 2.14, 2.12, 2.09, 2.05, 2.00 and 1.96 (18H, each s, 6×Ac), 0.96–0.86 (2H, m, CH₂), −0.07 (9H, s, TMS). ¹³C-NMR (125 MHz, CDCl₃): δ 170.7, 170.6, 170.5 × 2, 170.1, 169.7, 164.8, 133.1, 129.7 × 2, 129.5, 128.4 × 2, 100.7 (C-1), 99.2 (C-1′), 77.0 (C-4), 72.7 (C-3), 71.8 (C-5′), 69.3 (C-2), 68.6 (C-2′), 67.9 (C-4′), 67.42 (CH₂), 67.36 (C-3′), 67.1 (C-5), 61.9, (C-6′), 60.7 (C-6), 20.8, 20.74, 20.69, 20.68, 20.6 × 2, 17.8 (CH₂), −1.5 × 3 (TMS). ESI-HRMS: calcd for C₃₆H₅₀O₁₈SiNa, 821.2664 m/z; found, 821.2721 m/z [M + Na]⁺.

• 2,3,4,6-Tetra-O-acetyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-acetyl-α-d-galactopyranosyl trichloroacetimidate (36)

To a solution of 35 (0.92 g, 1.15 mmol) in CH₂Cl₂ (8.5 mL), cooled to 0 °C was added CF₃CO₂H (8.5 mL), and the mixture was stirred at room temperature for 0.5 h and concentrated. EtOAc and toluene (1:2) were added and evaporated to give the reducing sugar. To a solution of the residue in CH₂Cl₂ (10.0 mL) cooled at 0 °C DBU (160 μL, 1.05 mmol) and CCl₃CN (1.3 mL, 1.29 mmol) were added. The reaction mixture was stirred at room temperature for 0.5 h. After completion of the reaction, the mixture was concentrated. The residue was purified by silica gel column chromatography (3:2 n-hexane-EtOAc) to give 36 (0.81 g, 84%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +110.0 (c 1.2, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.61(1H, s, NH), 7.97—7.47 (5H, m, Ph), 6.74 (1H, d, J_{1, 2} = 3.6 Hz, H-1), 5.68 (1H, dd, J_{2, 3} = 11.0 Hz, H-2), 5.60 (1H, d, J_{3′, 4′} = 2.2 Hz, H-4′), 5.57 (1H, dd, J_{3, 4} = 2.5 Hz, H-3), 5.42 (1H, dd, J_{2′, 3′} = 11.0 Hz, H-3′), 5.27 (1H, dd, J_{1′, 2′} = 3.7 Hz H-2′), 5.08 (1H, d, H-1′), 4.63 (1H, t, J_{5, 6a} = J_{5, 6b} = 6.8 Hz, H-5), 4.42–4.35 (2H, m, H-5′, H-6a’), 4.32 (1H, d, H-4), 4.18–4.09 (3H, m, H-6a, H-6b, H-6b’), 2.15, 2.14, 2.05, 2.03, 2.01 and 2.00 (each s, 18H, 6×Ac). ¹³C-NMR (125 MHz, CDCl₃): δ 170.68, 170.63, 170.4×2, 170.3, 170.0, 165.4, 160.6, 133.7, 129.9×2, 129.0, 128.7×2, 99.0 (C-1′), 93.8 (C-1), 90.9, 76.7 (C-4), 70.9 (C-5′), 69.4 (C-3′), 68.3 (C-2′), 68.0 (C-3), 67.5×2 (C-2, C-5), 67.3 (C-4′), 61.9 (C-6′), 61.0 (C-6), 21.0, 20.9, 20.85, 20.78×2, 20.7. ESI-HRMS: calcd for C₃₃H₃₈Cl₃NO₁₈Na, 864.1052 m/z; found, 864.1087 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-acetyl-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (37)

A solution of 26 (154 mg, 0.20 mmol) and 36 (211 mg, 0.25 mmol) containing activated MS-AW300 (550 mg) in dry CH₂Cl₂ (1.5 mL) was stirred under an atmosphere of argon at room temperature for 18 h, then cooled to −40 °C. TMSOTf (2.5 μL, 13.8 μmol) was added, and the mixture was stirred for 3 h at room temperature, then 36 (74 mg, 0.088 mmol) and TMSOTf (2.5 μL, 14 μmol) were added, and the mixture was stirred at −20 °C for 3 h. After the reaction, they were neutralized with Et₃N. The solids were filtrated off and washed with CHCl₃. The combined filtrate and washings were successively washed with brine, dried (MgSO₄), and concentrated. The product was purified by flash silica gel column chromatography using 4:1 toluene-acetone as eluent to give 37 (99 mg, 34%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +109.0 (c 1.1, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.08–7.19 (20H, m, 4×Ph), δ 5.63–7.19 (20H, m, 4×Ph), 5.51 (1H, s, PhCH), 5.49 (1H, s, PhCH), 5.02 (1H, d, J_{1, 2} = 3.3 Hz, H-1 of Gal c), 5.01 (1H, d, J_{1, 2} = 8.1 Hz, H-1 of Gal b), 4.96 (1H, d, J_{1, 2} = 7.5 Hz, H-1 of Gal a), 4.92 (1H, d, J_{1, 2} = 3.5 Hz, H-1 of GalN), 3.67 (3H, s, OMe), 2.29 (2H, t, -CH₂-), 2.13, 2.07, 2.03, 2.01, 2.00 and 1.86 (18H, 6×Ac), 1.65–1.59 (4H, m, 2×-CH₂-), 1.40–1.26 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 170.50, 170.47, 170.4, 170.2, 170.1, 170.0, 164.8, 164.6, 137.63, 137.55, 133.0, 132.7, 129.8, 129.53 × 2, 129.52 × 2, 129.0, 128.7, 128.3, 128.2 × 2, 128.1 × 2, 128.0 × 2, 127.8 × 2, 126.2 × 2, 126.0 × 2, 101.3 (C-1 of Gal a), 100.8 (PhCH), 100.6 (C-1 of Gal b), 100.3 (PhCH), 99.9 (C-1 og Gal c), 98.8 (C-1 of GalN), 76.1, 76.0, 75.7, 75.6, 72.5, 72.23, 72.17, 70.7, 69.0 × 2, 68.9, 68.5, 68.2, 67.9, 67.2, 67.1, 66.7, 63.1, 61.6, 60.8, 58.6, 61.5, 33.8, 29.0, 25.6, 24.6, 20.8, 20.73, 20.70, 20.66, 20.63, 20.61. ESI-HRMS: calcd for C₇₁H₈₁N₃O₃₀Na, 1478.4803 m/z; found, 1478.4878 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-acetyl-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-di-O-acetyl-β-d-galactopyranosyl-(1→3)-2-azido-4,6-di-O-acetyl-2-deoxy-α-d-galactopyranoside (38)

A solution of 37 (106 mg, 72.8 μmol) in 80% AcOH (5.0 mL) was stirred at 70 °C for 6 h. Toluene was added and co-evaporated several times. The residue was acetylated with acetic anhydride (1.0 mL) in pyridine (1.0 mL). After the reaction was quenched with MeOH (20 mL) at 0 °C, the reaction mixture was added toluene and concentrated. The residue was purified by silica gel column chromatography using 1:1 CHCl₃—EtOAc as eluent to give 38 (78 mg, 74%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +104.5 (c 1.3, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 7.67–7.16 (10H, m, 2×Ph), 5.60 (1H, d, J_{3, 4} = 2.5 Hz, H-4 of Galc), 5.53–5.50 (2H, m, H-4 of Gala, H-3of Gal c), 5.44 (1H, d, J_{3, 4} = 2.9 Hz, H-4 of GalN), 5.35–5.31 (2H, m, H-2 of Gal a,b), 5.01 (1H, d, J_{1, 2} = 3.7 Hz, H-1 of Gal c), 4.82 (1H, d, J_{1, 2} = 3.7 Hz, H-1 of GalN), 4.77 (1H, d, J_{1, 2} = 7.8 Hz, H-1 of Gal a), 4.74 (1H, d, J_{1, 2} = 3.5 Hz, H-1 of Gal b). 3.66 (3H, s, OMe), 2.31 (2H, t,-CH₂-), 2.36, 2.19, 2.15, 2.13, 2.11, 2.09. 2.04, 2.02, 1.99 and 1.84 (30H, 10×Ac), 1.64–1.58 (m, 4H, 2×-CH₂-), 1.39–1.26 (m, 2H, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 173.9, 170.8, 170.7, 170.6, 170.5 × 3, 170.1, 169.9, 169.7, 169.4, 164.5, 164.4, 132.8, 129.41 × 2, 129.38 × 2, 129.3, 129.02, 128.21 × 2, 128.16 × 2, 128.1, 101.5 (C-1 of Gal a), 101.2 (C-1 of Gal b), 99.0 (C-1 of Gal c), 97.9 (C-1 of GalN), 76.6, 76.3, 73.4, 72.1, 72.0, 71.5, 70.9, 69.5, 69.2, 69.1, 68.8, 68.2, 68.1, 67.4, 67.2, 67.1, 62.8, 62.3, 61.6, 60.7, 59.2, 51.5, 33.8, 28.9, 25.6, 24.5, 21.5, 20.82, 20.75, 20.73 × 2, 20.69 × 2, 20.66 × 2, 20.6. ESI-HRMS: calcd for C₆₅H₈₁N₃O₃₄Na, 1470.4599 m/z; found, 1470.4568 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-acetyl-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-di-O-acetyl-β-d-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-galactopyranoside (39)

To a solution of 38 (77 mg, 53 μmol) in THF—H₂O (6:1, 3.5 mL) triphenylphosphine (PPh₃) (15.8 mg, 60 μmol) was added. The mixture was stirred at 70 °C for 6 h. After completion of the reaction, the mixture was diluted with EtOAc, washed with saturated aqueous NaHCO₃ and water, dried (MgSO₄), and concentrated. The residue was acetylated with acetic anhydride (2.0 mL) in pyridine (3.0 mL). After the reaction was quenched with MeOH, toluene was added and co-evaporated several times. The product was purified by silica gel column chromatography (2:1 toluene-acetone) to give 39 (65 mg, 83%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +102.8 (c 1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 7.66–7.15 (10H, m, 2×Ph), 5.60 (1H, d, J_{3, 4} = 2.5 Hz, H-4 of Galc), 5.54–5.26 (7H, m, H-4 of GalN, H-2,4 of Gala, H-2,3 of Galb, H-3,4 of Galc), 5.18 (1H, dd, J_{1, 2} = 3.5 J_{2, 3} = 11.0Hz, H-2 of GalN), 5.01 (1H, d, J_{1, 2} = 3.9 Hz, H-1 of Gal c), 4.88 (1H, d, J_{1, 2} = 3.2 Hz, H-1 of GalN), 4.73 (1H, d, J_{1, 2} = 7.3 Hz, H-1 of Gal a), 4.68 (1H, d, J_{1, 2} = 7.8 Hz, H-1 of Gal b), 3.68 (3H, s, OMe), 2.26 (2H, t, -CH₂-), 2.19, 2.14, 2.13, 2.11, 2.10, 2.03, 2.01, 1.99, 1.83, 1.66 and 1.54 (33H, 10×Ac), 1.57–1.53 (4H, m, 2×-CH₂-), 1.46–1.34 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 170.8, 170.7, 170.58, 170.55, 170.5, 170.4, 170.10, 170.07, 170.0, 169.9, 169.5, 164.6, 164.4, 132.3, 132.8, 129.42 × 2, 129.37 × 2, 129.0, 128.9, 128.4 × 2, 128.2 × 2, 101.2 (C-1 of Gal a), 99.7 (C-1 of Gal b), 99.1 (C-1 of Gal c), 97.0 (C-1 of GalN), 76.3, 74.1, 72.1, 71.9, 71.8, 69.2, 69.0, 68.1, 67.8, 67.6, 67.2, 67.12, 67.14, 67.12, 63.0, 62.5, 61.5, 60.7 × 2, 51.5 × 2, 49.1, 33.8, 28.7, 25.6, 24.5, 22.5, 20.82, 20.79 × 2, 20.76, 20.73 × 2, 20.68 × 2, 20.6 × 2. ESI-HRMS: calcd for C₆₇H₈₅NO₃₅Na, 1486.4780 m/z; found, 1486.4758 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl α-d-galactopyranosyl-(1→4)-β-d-galactopyranosyl-(1→3)-β-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-α-d-galactopyranoside (18)

To a solution of 39 (62 mg, 42.3 μmol) in MeOH (1.0 mL) 1,4-dioxane (1.0 mL) and NaOMe (18 mg) was added at 45 °C. The mixture was stirred for 17 h and then neutralized with Amberlite IR 120 [H⁺]. The mixture was filtered and concentrated. The product was purified by Sephadex LH-20 column chromatography in MeOH to give 18 (34.5 mg, 98%). Spectral data is described in the experimental part for synthesizing 17 to 18.

• Biotinylated tetrasaccharide (B)

Compound 18 (23.6 mg, 28.2 μmol) was dissolved in neat anhydrous ethylenediamine (4.8 mL) and heated at 70 °C for 64 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 derivative was dissolved in DMF (6 mL), and the pH was adjusted to 8–9 using DIPEA. Biotin-NHS (14.4 mg, 41.4 μmol) was added and the reaction was stirred at room temperature for 19 h. 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 B (29.0 mg, 94%). Spectral data is described above B.

• 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-acetyl-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside (40)

Compound 40 was prepared from 28 (436 mg, 0.39 mmol) and 36 (813 mg, 0.96 mmol) as described for preparation of 37. The product was purified by silica gel column chromatography (1:1 CHCl₃—EtOAc) to give 40 (342 mg, 49%). ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ +107.1 (c 1.2, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 7.96–7.12 (30H, m, 6×Ph), 5.47. 5.46 and 5.31 (3H, each s, 3×PhCH), 4.97 (1H, d, J_{1, 2} = 3.7 Hz, H-1 of Gal d), 4.94 (1H, d, J_{1, 2} = 7.3 Hz, H-1 of Gal c), 4.93 (1H, d, J_{1, 2} = 7.3 Hz, H-1 of Gal a), 4.90 (1H, d, J_{1, 2} = 3.3 Hz, H-1 of GalN), 4.83 (1H, d, J_{1, 2} = 7.5 Hz, H-1 of Gal b), 3.65 (3H, s, OMe), 2.29 (2H, t, -CH₂-), 2.11, 2.03, 1.99, 1.98, 1.97 and 1.82 (18H, 6×Ac), 1.63–1.60 (4H, m, 2×-CH₂-), 1.41–1.26 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.1, 170.51, 170.47, 170.4, 170.2, 170.1, 169.7, 164.8×2, 164.7, 137.74, 137.70, 137.6, 133.0, 132.7, 132.6, 130.3, 129.7 × 2, 129.6, 129.55 × 2, 129.50 × 2, 129.4, 129.0, 128.8 × 2, 128.43, 128.36 × 2, 128.27 × 2, 128.12 × 2, 128.12 × 2, 128.05 × 2, 127.8 × 2, 126.3 × 2, 126.2 × 2, 126.1 × 2, 101.5 (C-1 of Gal a), 100.8 (C-1 of Gal b), 100.7 (PhCH), 100.6 (PhCH), 100.4 (PhCH), 99.6 (C-1 of Gal c), 98.9 (C-1 of Gal d), 98.8 (C-1 of GalN), 76.1, 75.74, 75.70, 75.4, 73.8, 72.5, 72.4, 72.2, 70.8, 70.3, 69.1 × 2, 68.8, 68.6, 68.5, 68.2, 67.9, 67.2, 67.1, 66.81, 66.77, 63.1, 61.5, 60.8, 58.6, 51.5, 33.9, 29.7, 29.0, 25.6, 24.6, 20.8, 20.68, 20.65 × 2, 20.6. ESI-HRMS: calcd for C₉₁H₉₉N₃O₃₆K, 1848.5645 m/z; found, 1848.5612 m/z [M + K]⁺.

• 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-acetyl-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-di-O-acetyl-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-di-O-acetyl-β-d-galactopyranosyl-(1→3)-2-azido-4,6-di-O-acetyl-2-deoxy-α-d-galactopyranoside (41)

Compound 41 was prepared from 40 (363 mg, 0.20 mmol) by the same method described for preparation of 38. The product was purified by silica gel column chromatography (1:1 CHCl₃—EtOAc) to give 41 (295 mg, 82%). ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ +91.6 (c 1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 7.71–7.04 (15H, m, 3×Ph), 4.98 (1H, d, J_{1, 2} = 3.4 Hz, H-1 of Gal d), 4.80 (1H, d, J_{1, 2} = 3.7 Hz, H-1 of GalN), 4.69 (1H, d, J_{1, 2} = 7.8 Hz, H-1 of Gal a), 4.63 (1H, d, J_{1, 2} = 7.8 Hz, H-1 of Gal c), 4.58 (1H, d, J_{1, 2} = 7.6 Hz, H-1 of Gal b), 3.65 (3H, s, OMe), 2.31 (2H, t, -CH₂-), 2.17, 2.12×2, 2.07, 2.063, 2.056, 2.045. 2.02, 2.01, 2.00, 1.98 and 1.80 (36H, 12×Ac), 1.64–1.56 (4H, m, 2×-CH₂-), 1.36–1.25 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 170.8, 170.79, 170.74, 170.66, 170.6 × 2, 170.5, 170.2, 170.1, 170.0, 169.8, 169.5, 164.5, 164.3, 164.1, 133.0, 132.8, 132.6, 129.6 × 3, 129.4 × 2, 129.3 × 2, 129.1, 129.0, 128.3 × 2, 128.2 × 2, 128.1 × 2, 101.7 (C-1 of Gal a), 101.1 (C-1 of Gal b), 101.0 (C-1 of Gal c), 99.1 (C-1 of Gal d), 98.0 (C-1 of GalN), 76.3 × 2, 75.4, 73.5, 72.1, 71.8, 71.5, 71.4, 71.0, 69.6, 69.2 × 2, 69.1, 68.9, 68.3, 68.2, 67.4, 67.3, 67.2, 62.9, 62.4, 62.1, 61.6, 60.8, 60.5, 59.3, 51.6, 33.9, 29.0, 25.7, 24.6, 20.89 × 2, 20.87, 20.80 × 2, 20.78 × 2, 20.75 × 3, 20.69, 20.68. ESI-HRMS: calcd for C₈₂H₉₉N₃O₄₂Na, 1820.5601 m/z; found, 1820.5710 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-acetyl-β-d-galactopyranosyl-(1→3)-2-O-benzoyl-4,6-di-O-acetyl-β-d-galacto-pyranosyl-(1→3)-2-O-benzoyl-4,6-di-O-acetyl-β-d-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-galactopyranoside (42)

Compound 42 was prepared from 41 (279 mg, 0.16 mmol) by the same method described for preparation of 39. The product was purified by silica gel column chromatography (3:2 toluene-acetone) to give 42 (232 mg, 82%). [α]_D +98.0 (c 1.2, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 7.69–7.03 (15H, m, 3×Ph), 4.98 (1H, d, J_{1, 2} = 3.5 Hz, H-1 of Gal d), 4.85 (1H, d, J_{1, 2} = 3.7 Hz, H-1 of GalN), 4.62 (1H, d, J_{1, 2} = 8.0 Hz, H-1 of Gal c), 4.58 (2H, m, H-1 of Gal a, H-1 of Gal b), 3.67 (3H, s, OMe), 2.35 (2H, t, -CH₂-), 2.17, 2.12×2, 2.08, 2.07, 2.06, 2.01. 2.004, 1.997, 1.98, 1.80 and 1.51 (36H, each s, 12×Ac), 1.58–1.52 (4H, m, 2×-CH₂-), 1.38–1.20 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.0, 170.7 × 2, 170.5 × 2, 170.4 × 2, 170.1, 170.0 × 2, 169.9 × 2, 169.8, 169.4, 164.4 × 2, 163.9, 133.3, 132.7, 132.5, 129.5 × 2, 129.3 × 2, 129.13 × 2, 129.1, 128.8, 128.5 × 2, 128.2, 128.1 × 2, 128.0 × 2, 100.1 (C-1 of Gal a), 100.8 (C-1 of Gal b), 99.8 (C-1 of Gal c), 99.0 (C-1 of Gal d), 96.9 (C-1 of GalN), 76.1 × 2, 75.6, 74.1, 72.2, 72.0, 71.7, 71.6, 71.4, 70.9, 69.1, 69.0, 68.8 × 2, 68.1, 67.7, 67.6, 67.2, 67.1 × 2, 63.0, 62.2, 62.1, 61.4, 60.6, 51.5, 49.1, 33.7, 28.7, 25.5, 24.4, 22.5, 20.8 × 3, 20.71, 20.67, 20.63 × 2, 20.61 × 3, 20.56 × 2. ESI-HRMS: calcd for C₈₄H₁₀₃NO₄₃Na, 1836.5802 m/z; found, 1836.5949 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl α-d-galactopyranosyl-(1→4)-β-d-galactopyranosyl-(1→3)β-d-galactopyranosyl-(1→3)-β-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-α-d-galactopyranoside (43)

Compound 43 was prepared from 42 (215 mg, 0.12 mmo) by the same method described for preparation of 18 from 39. The product was purified by Sephadex LH-20 column chromatography in MeOH to give 43 (107 mg, 90%). ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ +96.2 (c 1.0, H₂O). ¹H-NMR (500 MHz, D₂O): δ 4.96 (1H, d, J_{1, 2} = 3.7 Hz, H-1 of Gal d), 4.88 (1H, d, J_{1, 2} = 3.7 Hz, H-1 of GalN), 4.69 (1H, d, J_{1, 2} = 7.6 Hz, H-1 of Gal a), 4.66 (1H, d, J_{1, 2} = 7.6 Hz, H-1 of Gal c), 4.52 (1H, d, J_{1, 2} = 7.9 Hz, H-1 of Gal b), 3.69 (3H, s, OMe), 2.41 (2H, t, -CH₂-), 2.01 (3H, Ac), 1.66–1.60 (4H, m, 2×-CH₂-), 1.42–1.32 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, D₂O): δ 178.16, 175.04, 104.99 × 2 (C-1 of Gal a, C-1 of Gal b), 104.6 (C-1 of Gal c), 100.9 (C-1 of Gal d), 97.6 (C-1 of GalN), 82.5, 82.3, 78.1, 77.9, 75.7, 75.3, 75.2, 72.7, 71.6, 71.4, 71.1, 70.9, 70.4, 69.7, 69.5, 69.3, 69.2, 69.02, 68.95, 68.4, 61.8, 61.51, 61.45, 61.1, 60.8, 52.7, 49.3, 34.2, 28.7, 25.5, 24.6, 22.6. ESI-HRMS: calcd for C₃₉H₆₇NO₂₈Na, 1020.3747 m/z; found, 1020.3753 m/z [M + Na]⁺.

• Biotinylated pentasaccharide (C)

Compound C was prepared from 43 (49 mg, 48.8 μmol) as described for preparation of B, yielding 52 mg (85%). ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ +85.5 (c 1.0, H₂O). ¹H-NMR (500 MHz, D₂O): δ 4.86 (1H, d, J_{1, 2} = 4.1 Hz, H-1 of Gal d), 4.88 (1H, d, J_{1, 2} = 3.7 Hz, H-1 of GalN), 4.70 (1H, d, J_{1, 2} = 7.6 Hz, H-1 of Gal a), 4.66 (1H, d, J_{1, 2} = 7.6 Hz, H-1 of Gal c), 4.52 (1H, d, J_{1, 2} = 7.8 Hz, H-1 of Gal b), 3.32 (3H, s, OMe). ¹³C-NMR (125 MHz, D₂O): δ 177.7, 177.6, 175.0, 165.9, 105.0×2 (C-1 of Gal a, C-1 of Gal b), 104.6 (C-1 of Gal c), 100.9 (C-1 of Gal d), 97.6 (C-1 of GalN), 82.6, 82.4, 78.2, 77.9, 75.7, 75.3, 75.2, 72.7, 71.6, 71.4, 71.1, 70.9, 70.4, 69.7, 69.6, 69.2, 69.2, 69.02, 68.96, 68.5, 62.7, 61.8, 61.52, 61.46, 61.1, 60.84, 60.78, 55.9, 49.3, 40.3, 39.2, 39.1, 36.4, 36.1, 28.8, 28.5, 28.3, 25.7, 25.5, 22.6, 20.6. ESI-HRMS: calcd for C₅₀H₈₅N₅O₂₉SNa, 1274.4949 m/z; found, 1274.4942 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 4,6-di-O-acetyl-2,3-di-O-benzyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl-(1→4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2- (2,2,2-trichloroethoxycarbonyl-amino)-β-d-glucopyranoside (45)

To a solution of 44 (100 mg, 64.9 μmol) and methyl 6-hydroxyhexanoate (19 mg, 0.13 mmol) in dry CH₂Cl₂ (1 mL) was added powdered MS AW-300 (120 mg), and the mixture was stirred under Ar atmosphere at room temperature for 3 h, then cooled to −20 °C. NIS (63.3 mg, 0.130 mmol) and TfOH (2.73 μL, 13.0 μmol) were added to the mixture, which was stirred at −20 °C for 1 h, 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 (2:1 hexane-EtOAc) to give 45 (89 mg, 89%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +20.8 (c 1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.01–7.05 (35H, m, 7×Ph), 5.78 (1H, d, J = 9.7 Hz, NH), 5.61 (1H, d, J =2.1 Hz, H-4 of Gal b), 5.44–5.93 (2H, m, H-3 of GlcN, H-2 of Gal a), 4.97 (1H, d, J_{1, 2} = 3.5 Hz, H-1 of Gal b), 4.63 (1H, d, J_{1, 2} = 6.0 Hz, H-1 of Gal a), 4.35 (1H, d, J_{1, 2} = 7.0 Hz, H-1 of GlcN), 3.65 (1H, s, OMe), 2.35 (2H, t, -CH₂-), 1.94 and 1.85 (6H, each s, 2×Ac),1.60–1.50 (4H, m, 2×-CH₂-), 1.32–1.29 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.2, 170.4, 170.2, 165.6, 154.4, 138.5, 138.2, 138.1, 137.9, 137.2, 133.1, 133.0, 130.0, 128.4, 128.3, 128.2, 128.0, 127.6, 127.5, 127.4, 127.3, 101.4, 101.0 (C-1 of Gal a), 100.3 (C-1 of Gal b), 95.4 (C-1 of GlcN), 85.2, 78.5, 76.4, 76.2, 75.1, 74.4, 74.1, 73.9, 73.5, 73.4, 73.0, 72.7, 71.8, 71.6, 71.1, 69.3, 68.0, 67.6, 66.8, 66.5, 62.4, 61.2, 56.3, 51.4, 34.0, 33.9, 33.8, 32.2, 29.6, 29.0, 25.2, 24.5, 24.3, 20.7, 20.6. ESI-HRMS: calcd for C₈₁H₈₈Cl₃NO₂₃Na, 1570.4710 m/z; found, 1570.4782 m/z [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 4,6-di-O-acetyl-2,3-di-O-benzyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl-(1→4)-2-acetamido-3-O-benzoyl-6-O-benzyl-2-deoxy-β-d-glucopyranoside (46)

To a solution of 45 (89 mg, 57.6 μmol) in THF—AcOH—Ac₂O (3:2:1, 4.0 mL) Zn—Cu (0.50 g) was added. The mixture was stirred at room temperature for 30 min. After completion of the reaction, the solid was filtered off. The filtrate was concentrated and purified by silica gel column chromatography (3:1 toluene-acetone) to give 46 (60 mg, 74%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +40.1 (c 1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃) δ 7.99–7.09 (35H, m, 7×Ph), 5.85 (1H, d, J = 9.7 Hz, NH), 5.62 (1H, d, J =2.1 Hz, H-4 of Gal b), 5.44 (1H, dd, J_{1, 2} = 7.9, J_{2, 3} = 10.5Hz, H-2 of Gal), 5.35 (1H, t, J_{1, 2} = J_{2, 3} = 7.9Hz, H-3 of GlcN), 5.00 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of Gal b), 4.62 (d, 1H, H-1 of GlcN), 4.40 (d, 1H, H-1 of Gal a), 3.64 (1H, s, OMe), 2.25 (2H, t, -CH₂-), 2.03, 1.94 and 1.85 (9H, each s, 3×Ac),1.65–1.48 (4H, m, 2×-CH₂-), 1.34–1.27 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.1, 170.3, 170.2, 170.0, 166.0, 165.2, 138.6, 138.2, 138.0, 137.9, 137.2, 133.2, 130.0, 129.8, 129.7, 129.0, 128.4, 128.2, 128.0, 127.7, 127.6, 127.5, 127.4, 125.3, 101.4 (C-1 of GlcN), 100.9 (C-1 of Gal a), 100.4 (C-1 of Gal b), 78.4, 76.4, 75.4, 75.2, 74.5, 73.6, 73.5, 73.3, 73.1, 72.8, 71.8, 71.7, 71.4, 69.0, 68.4, 67.5, 66.9, 66.6, 61.2, 53.1, 51.4, 33.9, 29.0, 25.4, 24.5, 23.1, 21.4, 20.8, 20.7. ESI-HRMS: calcd for C₈₀H₈₉NO₂₂Na, m/z 1438.5774; found, m/z 1438.5842 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl α-d-galactopyranosyl-(1→4)-β-d-galactopyranosyl-(1→4)-2- acetamido-2-deoxy-β-d-glucopyranoside (47)

To a solution of 46 (53 mg, 37.7 μmol) in THF (2.0 mL) was hydrogenolysed in the presence of Pd(OH)₂/C (600 mg) at room temperature for 0.5 h. The mixture was filtered and concentrated, and the residue was acetylated with acetic anhydride (0.3 mL) in pyridine (0.5 mL). After the reaction was quenched with MeOH, toluene was added and co-evaporated several times. The product was purified by silica gel column chromatography (1:1 toluene-acetone) to give an acylated compound (38 mg, 86%). ESI-HRMS: calcd for C₅₅H₆₉NO₂₇Na, m/z 1198.3955; found, m/z 1198.4036 [M + Na]⁺. To a solution of this compound in MeOH (1.0 mL), NaOMe (10 mg) was added and the mixture was stirred at 50 °C for 2 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 47 (19.6 mg, 77%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +27.6 (c 0.50, MeOH). ¹H-NMR (500 MHz, CD₃OD) δ 4.89 (1H, d, J_{1, 2} = 3.6 Hz, H-1 of Gal b), 4.37 (1H, d, J_{1, 2} = 7.3 Hz, H-1 of Gal a), 4.34 (1H, d, J_{1, 2} = 8.1 Hz, H-1 of GlcN), 3.28 (1H, s, OMe), 2.25 (2H, t, -CH₂-), 1.90 (3H, s, Ac),1.56–1.47 (4H, m, 2×-CH₂-), 1.33–1.30 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, CD₃OD): δ 175.9, 173.5, 105.4 (C-1 of Gal a), 102.6 (C-1 of Gal b, GlcN), 81.4, 79.7, 76.6, 76.5, 74.7, 74.2, 72.8, 72.7, 71.3, 71.0, 70.5, 70.3, 62.7, 62.0, 61.4, 56.9, 52.0, 49.8, 49.5, 49.3, 34.8, 30.2, 26.6, 25.7, 23.0. ESI-HRMS: calcd for C₂₇H₄₇NO₁₈Na, m/z 696.2691; found, m/z 696.2714 [M + Na]⁺.

• Biotinylated trisaccharide (D)

Compound D was prepared from 47 (21 mg, 30.9 μmol) as described for preparation of B, yielding 13.5 mg (47%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +27.6 (c 0.50, H₂O). ¹H-NMR (500 MHz, D₂O) δ 4.93 (1H, d, J_{1, 2} = 4.5 Hz, H-1 of Gal b), 4.51 (1H, d, J_{1, 2} = 8.0 Hz, H-1 of GlcN), 4.50 (1H, d, J_{1, 2} = 8.5 Hz, H-1 of Gal a), 2.00 (3H, s, Ac). ¹³C-NMR (125 MHz, D₂O): δ 177.8, 177.7, 175.0, 166.0, 103.9 (C-1 of Gal a), 101.7 (C-1 of GlcN), 100.9 (C-1 of Gal b), 79.4, 77.9, 76.1, 75.5, 73.2, 72.8, 71.6, 71.5, 70.9, 69.8, 69.6, 69.2, 62.8, 61.1, 61.0, 60.9, 60.7, 56.0, 55.9, 40.4, 39.9, 39.2, 36.5, 36.2, 29.0, 28.6, 28.4, 25.8, 25.6, 25.5, 25.3, 22.9. ESI-HRMS: calcd for C₃₈H₆₅N₅O₁₉SNa, m/z 950.3892; found, m/z 950.3971 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-3,6-di-O-benzyl-4-O-chloroacetyl-β-d-galactopyranosyl-(1→3)-2,4,6-tri-O-benzoyl-β-d-galactopyranosyl-(1→3)-2-azido-4-O-benzyl-2-deoxy-α-d-galactopyranoside (48)

To a solution of 10 (468 mg, 0.33mmol)) in dry CH₂Cl₂ (6.5 mL) MS AW-300 (500 mg) was added, and the mixture was stirred at room temperature for 2 h, then cooled to −78 °C. Et₃SiH (160 μL, 0.99 mmol) and PhBCl₂ (0.15 mL 1.12 mmol) were added, and the mixture was stirred for 45 min, then neutralized with Et₃N and added to MeOH. 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 (1:1 hexane—EtOAc) to give 48 (413 mg, 88%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +57.1 (c 0.50, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.07–6.93 (35H, m, 7×Ph), 5.80 (1H, d, J_3,4 = 3.0 Hz, H-4 of Gal b), 5.67 (1H, dd, J_{1, 2} = 8.0, J_{2, 3} = 10Hz, H-2 of Gal a), 5.12 (1H, d, J_{1, 2} = 7.7 Hz, J_{2, 3} = 10Hz, H-2 of Gal b), 5.67 (1H, dd, J_{1, 2} = 8.0, J_{2, 3} = 10Hz, H-2 of Gal a), 4.88 (1H, J_{1, 2} = 7.5 Hz, H-1 of Gal a), 4.75 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of GalN), 4.72 (d, 1H, J_{1, 2} = 7.5 Hz, H-1 of Gal b), 3.64 (1H, s, OMe), 2.25 (2H, t, -CH₂-), 1.73–1.46 (4H, m, 2×-CH₂-), 1.37–1.23 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 174.1, 166.8, 166.0, 164.4, 137.9, 136.8, 133.3, 133.2, 132.7, 132.5, 130.0, 129.6, 129.3, 128.8, 128.6, 128.5, 128.3, 128.2, 128.1, 128.0, 127.9, 127.6, 102.9 (C-1 of Gal a), 101.2 (C-1 of Gal b), 98.1 (C-1 of GalN), 76.2, 75.7, 74.5, 73.7, 72.0, 71.9, 71.3, 70.8, 70.7, 70.5, 70.3, 67.9, 67.5, 67.1, 63.0, 62.3, 59.1, 51.5, 40.6, 33.8, 31.9, 29.7, 29.3, 28.9, 25.6, 24.6, 22.7, 14.1. ESI-HRMS: calcd for C₇₆H₇₈ClN₃O₂₂Na, m/z 1442.4663; found, m/z 1442.4816 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-3,6-di-O-benzyl-4-O-chloroacetyl-β-d-galactopyranosyl-(1→3)-2,4,6-tri-O-benzoyl-β-d-galactopyranosyl-(1→3)-[4,6-di-O-acetyl-2,3-di-O-benzyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl-(1→4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranosyl-(1→6)]-2-azido-4-O-benzyl-2-deoxy-α-d-galactopyranoside (49)

Compound 49 was prepared from 44 (298 mg, 0.21mmol) and 48 (355 mg, 0.23mmol) as described for preparation of 7. The product was purified by silica gel column chromatography (8:7 n-hexane—EtOAc) to give 49 (559 mg, 94%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +52.7 (c 1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.01–6.90 (70H, m, 14×Ph), 5.79–5.07 (7H, m, H-2, 4 of Gal a and Gal b, H-2 of Gal c, H-4 of Gal d), 4.97 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of Gal d), 4.90 (1H, d, J = 11.0 Hz, NH), 4.85 (d, 1H, J_{1, 2} = 7.5 Hz, H-1 of Gal a), 4.70 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal b), 4.69 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of GalN), 4.62 (d, 1H, J_{1, 2} = 7.0 Hz, H-1 of GlcN), 4.58 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal c), 3.59 (1H, s, OMe), 2.23 (2H, t, -CH₂-), 2.00 and 1.94 (6H, each s, 2×Ac),1.58–1.54 (4H, m, 2×-CH₂-), 1.31–1.25 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 102.7 (C-1 of Gal a), 101.2 (C-1 of Gal c), 101.0 (C-1 of Gal b), 100.9 (C-1 of GlcN), 100.3 (C-1 of Gal d), 97.8 (C-1 of GalN). ESI-HRMS: calcd for C₁₅₀H₁₅₂Cl₄N₄O₄₂Na, m/z 2843.8533; found, m/z 2843.8428 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-3,6-di-O-benzyl-4-O-chloroacetyl-β-d-galacto-pyranosyl-(1→3)-2,4,6-tri-O-benzoyl-β-d-galactopyranosyl-(1→3)-[4,6-di-O-acetyl-2,3-di-O-benzyl-α-d-galactopyranosyl-(1→4)-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl-(1→4)-2-acetamido-3-O-benzoyl-6-O-benzyl-2-deoxy-β-d-glucopyranosyl-(1→6)]-2- acetamido-4-O-benzyl-2-deoxy-α-d-galactopyranoside (50)

Compound 50 was prepared from 49 (200 mg, 70.8 μmol) by the same method described for preparation of 46. The product was purified by silica gel column chromatography (2:1 toluene-acetone) to give 50 (128 mg, 67%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +48.6 (c 1.0, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): δ 8.01–6.50 (70H, m, 14×Ph), 5.78–5.06 (7H, m, H-2, 4 of Gal a and Gal b, H-2 of Gal c, H-4 of Gal d), 4.98 (d, 1H, J_{1, 2} = 3.0 Hz, H-1 of Gal d), 4.85 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal a), 4.71 (d, 1H, J_{1, 2} = 8.5 Hz, H-1 of GlcN), 4.66 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of GalN) 4.60 (d, 1H, J_{1, 2} = 7.0 Hz, H-1 of Gal a), 4.23 (d, 1H, J_{1, 2} = 7.5 Hz, H-1 of Gal c), 3.61 (1H, s, OMe), 2.23 (2H, t, -CH₂-), 2.01×2, 1.935 ×2 (12H, each s, 4×Ac),1.66–1.64 (4H, m, 2×-CH₂-), 1.32–1.25 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 102.1 (C-1 of Gal a), 101.4 (C-1 of Gal c), 101.2 (C-1 of Gal b), 100.9 (C-1 of GlcN), 100.3 (C-1 of Gal d), 96.8 (C-1 of GalN). ESI-HRMS: calcd for C₁₅₁H₁₅₇ClN₂O₄₂Na, m/z 2727.9797; found, m/z 2727.9705 [M + Na]⁺.

• 5-(Methoxycarbonyl)pentyl β-d-galactopyranosyl-(1→3)-β-d-galactopyranosyl-(1→3)-[α-d-galactopyranosyl-(1→4)-β-d-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-d-glucopyranosyl-(1→6)]-2-acetamido-2-deoxy-α-d-galactopyranoside (51)

To a solution of 50 (90 mg, 33.2 μmol) in MeOH—dioxane (1:1, 3.0 mL) NaOMe (30 mg) was added at room temperature and the mixture was stirred at 40 °C for 3 h, then neutralized with Amberlite IR 120[H⁺]. The mixture was filtered off and concentrated. The residue was purified by silica gel column chromatography using 20:1 CHCl₃—MeOH as eluent to give the target intermediate (43.7 mg). A solution of the residue in MeOH (6.0 mL) was hydrogenolysed under hydrogen in the presence of 10% Pd/C (50 mg) for 0.5 h at room temperature, then filtered and concentrated. The product was purified by Sephadex LH-20 column chromatography in 1:1 MeOH—H₂O to give 51 (30.4 mg, 2 steps 76%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +39.8 (c 1.0, H₂O). ¹H-NMR (500 MHz, D₂O) δ 4.95 (d, 1H, J_{1, 2} = 3.8 Hz, H-1 of Gal d), 4.86 (d, 1H, J_{1, 2} = 3.8 Hz, H-1 of GalN), 4.61 (d, 1H, J_{1, 2} = 7.1 Hz, H-1 of Gal c), 4.55 (d, 1H, J_{1, 2} = 8.5 Hz, H-1 of GlcN), 4.53 (d, 1H, J_{1, 2} = 7.7 Hz, H-1 of Gal b), 4.52 (d, 1H, J_{1, 2} = 7.7 Hz, H-1 of Gal a), 3.70 (1H, s, OMe), 2.42 (2H, t, -CH₂-), 2.02 and 2.01 (6H, each s, 2×Ac),1.66–1.61 (4H, m, 2×-CH₂-), 1.43–1.39 (2H, m, -CH₂-). ¹³C-NMR (125 MHz, CDCl₃): δ 178.2, 175.1, 174.8, 105.0 (C-1 of Gal a), 104.9 (C-1 of Gal c), 103.9 (C-1 of Gal b), 102.1 (C-1 of GlcN), 100.9 (C-1 of Gal d), 97.4 (C-1 of GalN), 82.6, 79.5, 78.0, 77.9, 76.1, 75.7, 75.5, 75.3, 73.3, 73.2, 72.8, 71.7, 71.6, 71.5, 70.7, 70.5, 69.9, 69.8, 69.6, 69.2, 69.0, 68.0, 61.6, 61.2, 61.0, 60.7, 55.8, 52.8, 49.3, 34.3, 30.9, 28.7, 25.7, 24.7, 22.9, 22.6. ESI-HRMS: calcd for C₄₇H₈₀N₂O₃₃Na, m/z 1223.4541; found, m/z 1223.4639 [M + Na]⁺.

• Biotinylated hexasaccharide (E)

Compound E was prepared from 51 (16 mg, 13.2 μmol) as described for preparation of B, yielding 15.4 mg (79%). ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ +27.9 (c 0.50, H₂O). ¹H-NMR (500 MHz, D₂O): δ 4.95 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of Gal d), 4.86 (d, 1H, J_{1, 2} = 3.5 Hz, H-1 of GalN), 4.60 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal c), 4.55 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of GlcN), 4.53 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal b), 4.52 (d, 1H, J_{1, 2} = 8.0 Hz, H-1 of Gal a). ¹³C-NMR (125 MHz, D₂O): δ 180.1, 177.9, 177.7, 175.1, 174.8, 166.0, 105.1 (C-1 of Gal a), 105.0 (C-1 of Gal c), 103.9 (C-1 of Gal b), 102.1 (C-1 of GlcN), 100.9 (C-1 of Gal d), 97.5 (C-1 of GalN), 82.7, 79.5, 77.9, 76.1, 75.7, 75.5, 75.3, 73.3, 73.2, 72.8, 71.7, 71.6, 71.5, 70.5, 70.0, 69.8, 69.6, 69.2, 68.1, 62.7, 61.6, 61.2, 61.0, 90.9, 56.0, 55.8, 49.3, 40.4, 40.0, 39.3, 36.5, 30.8, 30.5, 28.9, 28.6, 28.4, 25.9, 25.8, 25.5, 23.0, 22.7 ESI-HRMS: calcd for C₅₈H₉₈N₆O₃₄SNa, m/z 1477.5742; found, m/z 1477.5882 [M + Na]⁺.

4.2. Serum Samples

Serum samples of 60 and 45 patients confirmed to have AE and CE, respectively, and those of 60 healthy individuals, which are kept in Hokkaido Institute of Public Health, were used for ELISA assay under the approval of the institute.

4.3. ELISA Protocol

ELISA was performed using as previously described [12] 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 IgG/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 the washing of the wells, bound antibodies were detected by the addition of ABTS peroxidase substrate solution (KPL, Gaithersburg, MD, USA, 200 μL per well). After the incubation period of 8 min at 37 °C, the reaction was stopped by the addition of 1% SDS, and the absorbance (A) values were read at 405 nm on a microplate reader (Model 680; BIORAD, Hercules, CA, USA).