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Publicly Available Published by De Gruyter January 20, 2017

Synthesis of p-methoxyphenyl sulfated β-GalNAc derivatives with inhibitory activity against Japanese encephalitis virus

  • Miho Sakuragi EMAIL logo , Ryoko Suzuki , Kazuya I.P.J. Hidari , Takashi Yamanaka and Hirofumi Nakano EMAIL logo

Abstract

The N-acetylgalactosamine (GalNAc) residue is one of the units of chondroitin sulfate E (CS-E) which has been reported to have inhibitory activity against Japanese encephalitis virus (JEV). Herein, we describe the synthesis of a series of p-methoxyphenyl β-GalNAc derivatives with a sulfate group at 3-, 4-, and/or 6-positions using an efficient route through a common synthetic intermediate. By measuring the inhibition activity of these compounds that bear different numbers and positions of sulfate groups, the effect of position specificity for interaction with the virus was determined. From these results, GalNAc6S and GalNAc4S6S derivatives inhibited JEV infections well; we suggest the 6-O-sulfate group is necessary for selective recognition by the virus.

Introduction

Japanese encephalitis virus (JEV) is a member of the Flavivirus genus within the Flaviviridae family. Similarly to West Nile and dengue viruses that belong to the same genus, JEV infection is considered a major public health concern. JEV has caused a major outbreak of viral encephalitis in south, southeast, and east areas of Asia [1], [2], [3], [4], [5]. More than 67,000 cases, including approximately 20,000 deaths annually, occur from the viral infection [6]. In the northeastern part of Australia, JEV infection was first reported in 1995. Distribution areas of JEV are now growing inward in Australia [7], [8].

JEV, an arthropod-borne virus, is transmitted through swine to humans by Culex tritaeniorhynchus mosquitoes [9], [10], [11]. Pigs are considered to be important amplifying hosts because they develop an asymptomatic infection with a high level of viremia during for several days post-infection. Although JEV infection in humans and horses do not result in a level of viremia high enough for human-to-human transmission by mosquitoes, they develop clinical manifestations such as high fever, headache, diarrhea, and vomiting, followed by seizure, flaccid paralysis, meningitis, and encephalitis in severe cases. Therefore, humans and horses are considered to be dead-end hosts [3]. JEV almost results in asymptomatic infections in humans with an incidence rate that varies from 0.1 to 4 % [12]; however, the mortality ratio in symptomatic cases is more than 30 % and a high population of survivors has severe and long-term neurological sequelae [12], [13].

Vaccination is the most effective way to prevent JEV infection. Inactivated vaccines have been developed using Vero cells and widely introduced with routine immunization programs, however, all healthy people who are immunized have some risk of adverse effects. In addition, JEV is not fully controlled in areas with relatively lower vaccine coverage [6], [11], [14]. Since there are no clinically-approved anti-JEV drugs available, patients are confined to symptomatic alleviation and supportive care. Therefore, therapeutic use of anti-JEV drugs need to be developed.

JEV is an envelope virus with an icosahedral lipid bilayer [15]. The virus has a positive-strand RNA genome that encodes a single polypeptide, including three structural (Capsid, Membrane, and Envelope) and seven non-structural proteins. The Envelope (E) protein, expressed in a heterodimer form in mature virus particles, consists of three functional domains, I, II, and III. The E protein is involved in early events of JEV infection, such as adsorption of the virus to host receptors and engagement of fusion between viral and host cell membranes [16], [17], [18]. This protein also induces production of neutralizing antibodies in humans [19]. Domain III of the E protein is critical for virus adsorption to receptors expressed on the host cell surface [20], [21].

In JEV-infected cells, low pH conditions in endosomes triggers conformational alterations of the E protein and receptor-mediated endocytosis of the virus particles, inducing fusion between viral and host cell membranes. After the viral RNA is released into the cytosol, viral genome replication occurs [22].

Glycosaminoglycans (GAGs), a family of complex glycoconjugates, consist of a linear polysaccharide chain composed of a repeating disaccharide unit. The repeating unit consists of uronic acid, glucuronic or iduronic acid, and amino sugar, N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc). GAGs have many isomers and diverse structures formed from the combination of the constituent sugars and position and amount of sulfate groups; for example, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate.

The core structures of GAGs depend on their derived tissues that result from different sulfation positions on sugars. GAGs exist in the extracellular matrix (ECM) and on the cell surface. Interaction of GAGs with GAG-binding proteins induces cell-extracellular adhesion, cell growth, cell differentiation, and regulation of growth factor function.

A virus binding to a host cell surface is mediated through specific receptor molecules that play a significant role in viral infections. These specific interactions account for the definition of the tissue and host tropism [23]. Heparin and chondroitin sulfate E (CS-E), classified as sulfated GAGs, are involved in host cell recognition in the early stage of JEV infection as host co-receptors [24], [25].

In this study, we focused on the structure of GalNAc in CS-E because CS-E shows strong inhibitory activity against JEV infection. We designed, chemically synthesized, and characterized GalNAc derivatives with different numbers and positions of sulfate groups that showed effective inhibitory activity against JEV infection. The compounds that prevent JEV from attaching to cultured cells may contribute to elucidation of the molecular mechanisms for JEV-host recognition, as well as development of anti-JEV drugs.

Results and discussion

The position of the sulfate group, which effected inhibitory activity of JEV, is an important research area. To investigate the suitable position of a sulfate group for interaction with the virus, we synthesized compounds 1–7. The compounds bear sulfate groups at the 3-, 4-, and/or 6-positions, and the 1- and 2-positions had p-methoxyphenyl (MP) and N-acetyl groups, respectively (Fig. 1). The MP group was chosen in this study to allow for easy detection of reaction progress through the aromatic ring that absorbs UV light. The MP group can be removed using diammonium cerium(IV) nitrate, an advantage for forming a variety of aglycons in the future syntheses, after which we will use a click reaction such as the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction [26], [27]. After deprotection of the MP group, β-selective introduction of an azide group at an anomeric position will be performed by reaction with sodium azide and 2-chloro-1,3-dimethylimidazolinium chloride (DMC), developed by Shoda and co-workers [28]. Then, the CuAAC reaction will be used to introduce various substituents in the final step without any protecting groups. We assumed the influence of the configuration of the glycon on the interaction with the virus was stronger than that of aglycons. So, we first sought to find a suitable glycon structure before optimizing their aglycons.

Fig. 1: 
					Target sulfated GalNAc derivatives 1–7.
Fig. 1:

Target sulfated GalNAc derivatives 1–7.

Retrosynthetic analysis gave strategies with simple synthetic steps for 7 types of targeted sulfated GalNAc derivatives from the non-sulfated GalNAc derivative 11. Target compounds 1 and 6 were prepared from compound 8, which was protected at the 4- and 6-positions using a benzylidene group. GalNAc4S6S derivative 6 was obtained by sulfation after protecting with an acyl group at the 3-position of 8 and removal of the benzylidene group. The 4-O-sulfated galactopyranoside 2 was synthesized from compound 10, prepared by selective 3,6-di-O-pivaloylation [29] of 11. Compounds 3 and 4 were synthesized from compound 9, whose 6-position was selectively protected by a trityl group. Benzoylation at the 3- and 4-positions afforded fully a protected GalNAc derivative, then the 6-position was subjected to deprotection under acidic conditions, followed by sulfation to give GalNAc6S 3. Targets 5 and 7 were obtained in one step from the synthetic intermediate 11 by controlled sulfation (Scheme 1).

Scheme 1: 
					Retrosynthetic analysis of target compounds 1–7.
Scheme 1:

Retrosynthetic analysis of target compounds 1–7.

The synthetic intermediate GalNAc 11 was synthesized as depicted in Scheme 2. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-galactopyratnoside 12 [30] was used as the starting material. The azide group at the 2-position of 12 was converted to an acetamido group using thioacetic acid and pyridine [31], [32] to give compound 13 in 85 % yield. The GalNAc derivative 11, with free hydroxy groups at the 3-, 4-, and 6-positions was obtained by treating 13 with sodium methoxide in methanol.

Scheme 2: 
					Synthetic route to common synthetic intermediate 11.
Scheme 2:

Synthetic route to common synthetic intermediate 11.

GalNAc3S 1 was prepared in 3 steps from 11 (Scheme 3). A solution of 11 was treated with benzaldehyde dimethyl acetal (BDA) and D-camphor-10-sulfonic acid (CSA) to give compound 8. Target 1 was then obtained by sulfation of 8 with sulfur trioxide-pyridine complex in pyridine [33], [34], followed by deprotection of the benzylidene group under acidic reaction conditions. The low yield of synthetic intermediate 8 was due to competitive formation of methyl 2-acetamido-4,6-O-benzylidene-2-deoxy-β-D-galactopyranoside by glycosylation of 8 or 11 with the methanol byproduct under the acidic conditions. The formation of the methyl glycoside was confirmed by the 1H NMR spectrum of the reaction mixture.

Scheme 3: 
					Synthetic route of GalNAc3S derivative 1.
Scheme 3:

Synthetic route of GalNAc3S derivative 1.

The synthetic route for GalNAc4S 2 is described in Scheme 4. Galactosamine 10 was synthesized by selective protection with a pivaloyl group at the 3- and 6-positions of 11 [29]. Then, compound 10 was subjected to sulfation by treating with sulfur trioxide-pyridine complex to afford 15. De-O-pivaloylation with sodium methoxide in methanol gave compound 2 in 81 % yield.

Scheme 4: 
					Synthesis of GalNAc4S derivative 2.
Scheme 4:

Synthesis of GalNAc4S derivative 2.

Compound 16 was obtained by selective 6-O-tritylation of 11 followed by benzoylation at the 3- and 4-positions using benzoyl chloride and pyridine. After removal of the trityl group, sulfation at the 6-position gave 18. Benzoyl groups were deprotected by treating with sodium methoxide to give GalNAc6S 3 in good yield (Scheme 5).

Scheme 5: 
					Synthesis of GalNAc6S derivative 3.
Scheme 5:

Synthesis of GalNAc6S derivative 3.

In order to get the di-O-sulfated saccharide 19, the common intermediate 11 was subjected to a SN1 reaction with trityl chloride in pyridine, followed by sulfation of the free hydroxy groups. Target compound 4 was obtained by removal of the trityl group using catalytic hydrogenation with a Pd-C catalyst (Scheme 6).

Scheme 6: 
					Synthesis of GalNAc3S4S derivative 4.
Scheme 6:

Synthesis of GalNAc3S4S derivative 4.

Protection of 11 at the 4- and 6-positions with benzylidene acetal, followed by pivaloylation afforded compound 20. Deprotection of the benzylidene group with 80 % AcOH-H2O followed by sulfation at the 4- and 6-positions gave 22. Then, sulfated compound 22 was subjected to removal of the pivaloyl group with sodium methoxide to give target compound 6 quantitatively (Scheme 7).

Scheme 7: 
					Synthesis of GalNAc4S6S derivative 6.
Scheme 7:

Synthesis of GalNAc4S6S derivative 6.

GalNa3S4S6S 5 and GalNAc3S6S 7 were simultaneously obtained from compound 11 by sulfation without any protection (Scheme 8). This reaction afforded di-sulfated saccharide 5 in good yield because the highly reactive 6-hydroxy group was sulfated at first, and then sulfation at the 3-position occurred due to the repulsion of their negative charge and steric hindrance.

Scheme 8: 
					Synthesis of GalNAc3S6S derivative 5 and GalNAc3S4S6S derivative 7.
Scheme 8:

Synthesis of GalNAc3S6S derivative 5 and GalNAc3S4S6S derivative 7.

Now, we have completed the synthesis of N-acetylgalactosamine derivatives with sulfate group at the 3- to 6-positions. The 1H and 13C NMR spectral data of compounds 17 and 11 are summarized in Table 1.

Table 1:

The chemical shifts in the 1H and 13C NMR spectra for synthetic intermediate 11 and target sulfated GalNAc derivatives 17.

GalNAc

11
GalNAc

3S

1
GalNAc

4S

2
GalNAc

6S

3
GalNAc

3S4S

4
GalNAc

3S6S

5
GalNAc

4S6S

6
GalNAc

3S4S6S

7
H-1 4.86 5.03 4.93 4.85 5.12 5.00 4.92 5.09
H-2 4.03 4.18 4.04 4.04 4.13 4.17 4.06 4.17
H-3 3.70a 4.40 3.87 3.72 4.49 4.40 3.89 4.51
H-4 3.88 4.21 4.64 3.93 4.88 4.24 4.69 4.91
H-5 3.69a 3.76 3.83 3.96 3.88 4.02 4.14 4.17
H-6a 3.69a 3.71a 3.71 4.11 3.72 4.12 4.15 4.18
H-6b 3.73a 3.76 4.15 3.76 4.17 4.27 4.27
C-1 101.12 100.66 100.90 101.06 100.32 100.54 100.87 100.27
C-2 52.40 50.62 52.72 52.28 51.08 50.48 52.60 50.96
C-3 70.73 77.51 69.72 70.56 74.99 77.21 69.70 74.91
C-4 67.60 66.14 75.44 67.33 73.99 65.92 75.27 73.81
C-5 75.36 75.04 74.64 72.89 74.67 72.66 72.44 72.49
C-6 60.74 60.67 60.75 66.97 60.76 67.02 67.66 67.69
  1. aChemical shift was determined by peak picking of HSQC spectra.

Comparing the signal in the 1H NMR spectrum between the GalNAc derivatives bearing some sulfate groups and the corresponding precursor with no sulfate group reveals a downfield shift in the spectrum of sulfated compounds. Of note, the H-3 signal was slightly affected by the sulfate group located at the 4-position and the H-4 signal of GalNAc3S 1 and GalNAc3S6S 5 shifted downfield at least 0.3 ppm compared to that of compound 11. In GalNAc4S6S 6 and GalNAc3S4S6S 7 derivatives, the signal from H-5 shifted downfield more than 0.4 ppm.

In the 13C NMR spectrum of the GalNAc derivatives, the signal of the carbon atoms bearing a O-sulfate group was also shifted downfield relative to that of the corresponding carbon atoms without a sulfate group. The C-5 signal for GalNAc derivatives having a sulfate group at the 6-position showed a 2.5–2.9 ppm upfield shift compared to the corresponding signal from 11.

The coupling constants in the 1H NMR for the GalNAc derivatives showed values (J1,2=8.5 Hz, J3,4=3.1 Hz, and J4,5=0.0 Hz) typical of a galactosamine configuration. A conformation change due to the position of the sulfate groups was not observed.

Compounds 16 were tested as inhibitors against JEV infection in Vero cells. In the presence of each compound in a final concentration of 0.1 mM, the inhibitory activity relative to a control treatment without the compound is shown in Fig. 2. GalNAc4S6S 6 and GalNAc6S 3 reduced JEV infection by approximately 35 %. Since the sugar structure of 6 constitutes the CS-E, the result is reasonable for strong inhibitory activity against JEV infection. Compound 3 also inhibited JEV infection in spite of bearing only one sulfate group, suggesting that the sulfate group at the 6-position is necessary for JEV to interact with a host cell, however, GalNAc3S6S 5 showed inhibitory activity to a lower extent. This indicates that the 3-O-sufate group prevented the saccharide from binding to virus due to repulsion from the non-natural position of the negative charge. In contrast, a sulfate group at the 4-position might help produce an effective interaction by leading the 6-O-sulfate group to the appropriate binding site. In the presence of each compound up to 0.1 mM, a cytotoxic effect was not significantly observed (Fig. 3), meaning that the compounds did not affect the cell viability.

Fig. 2: 
					Inhibitory effect of the compounds on JEV infection of Vero cells was evaluated using a real-time RT-PCR assay. The assay was performed as described in the Experimental section. Values indicate means±SE of relative infectivity in the presence of compound at the indicated concentration to the virus alone as a control. Bars show standard deviation in the experiment.
Fig. 2:

Inhibitory effect of the compounds on JEV infection of Vero cells was evaluated using a real-time RT-PCR assay. The assay was performed as described in the Experimental section. Values indicate means±SE of relative infectivity in the presence of compound at the indicated concentration to the virus alone as a control. Bars show standard deviation in the experiment.

Fig. 3: 
					Cytotoxicity of compounds 1–6. Values were calculated after normalizing data to control cell viability based on vehicle (DMSO) treatment. Bars show standard deviation of triplicate measurements in each experiment.
Fig. 3:

Cytotoxicity of compounds 1–6. Values were calculated after normalizing data to control cell viability based on vehicle (DMSO) treatment. Bars show standard deviation of triplicate measurements in each experiment.

Conclusions

In the present study, an exhaustive synthisis of p-methoxyphenyl sulfated β-GalNAc derivatives was accomplished using a simple synthetic route from common synthetic intermediate 11. Moreover, their inhibitory activities against JEV were evaluated, and we indentified GalNAc6S 3 and GalNAc4S6S 6 as novel monosaccharide JEV inhibitors. We found a key factor in JEV infection is that the virus may require a 6-O-sulfated saccharide on the host cell surface. These results contribute to the development of anti-JEV compounds with low molecular weights.

Experimental section

General

Compound 12 was prepared as previously reported [30]. All solvents were of reagent grade quality and purchased commercially. Structures of synthetic compounds were confirmed by 1H NMR, 13C NMR, and two-dimensional NMR (COSY, HSQC, HMBC, and NOESY) spectroscopy. 1H and 13C NMR spectra were recorded with a Bruker AVANCE III instrument operating at 400.13 and 100.62 MHz, respectively. Chemical shifts were referenced to TMS in CDCl3 and δ values (ppm) of water in D2O (1H: δ=4.70) as internal standard. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) mass spectra were obtained on a Bruker Daltonics micrOTOF-QII. Thin layer chromatography (TLC) was performed on precoated Silica gel 60 F254 plates. Column chromatography was performed on silica gel 60 N (spherical neutral) purchased from Kanto Chemical Company, Japan.

Synthesis of p-methoxyphenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranoside (13)

A solution of 12 (1.283 g, 2.93 mmol, 1.0 equiv) in dry pyridine (14.67 mL, 0.20 M) was cooled by ice-water bath, and then thioacetic acid (6.30 mL, 6.71 g, 88.1 mmol, 30 equiv) was added to the solution dropwisely. The mixture was stirred at room temperature for 4 h and then at 40°C for 20 h. The reaction mixture was diluted with chloroform (100 mL). The organic layer was washed with 5 % aq HCl, saturated aq NaHCO3, and saturated aq NaCl successively, dried over Na2SO4, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography eluting with 1 % methanol-chloroform to give 13 (1.126 g, 2.48 mmol, 85 %) as a colorless solid. Rf=0.50 (10 % methanol-chloroform); 1H NMR (CDCl3, 400.13 MHz): δ 1.98 (s, 3 H, 2-NHCOCH3), 2.03 (s, 3 H, 3-OCOCH3), 2.05 (s, 3 H, 6-OCOCH3), 2.17 (s, 3 H, 4-OCOCH3), 3.77 (s, 3 H, p-OCH3), 4.03 (t, 1 H, J5,6b=6.8 Hz, J5,6a=6.6 Hz, H-5), 4.15 (dd, 1 H, J6a,6b=11.3 Hz, J5,6a=6.6 Hz, H-6a), 4.13–4.24 (m, 1 H, H-2), 4.21 (dd, 1 H, J6a,6b=11.3 Hz, J5,6b=6.8 Hz, H-6b), 5.18 (d, 1 H, J1,2=8.4 Hz, H-1), 5.40 (dd, 1 H, J2,3=12.8 Hz, J3,4=3.3 Hz, H-3), 5.41 (d, 1 H, J3,4=3.3 Hz, H-4), 5.56 (d, 1 H, J2,NH=8.6 Hz, 2-NHCOCH3), 6.78–6.83 (m, 2 H, m-arom. H), 6.94–6.98 (m, 2 H, o-arom. H); 13C NMR (CDCl3, 100.62 MHz): δ 20.63 (q, OCOCH3X2), 20.66 (q, OCOCH3), 23.44 (q, 2-NHCOCH3), 51.79 (d, C-2), 55.62 (q, p-OCH3), 61.46 (t, C-6), 66.63 (d, C-4), 69.61 (d, C-3), 70.83 (d, C-5), 100.34 (d, C-1), 114.50 (d, m-arom. CH), 118.51 (d, o-arom. CH), 151.14 (s, arom. C), 155.58 (s, p-arom. C), 170.24 (s, 4-OCOCH3), 170.36 (s, 6-OCOCH3), 170.41 (s, 3-OCOCH3), 170.44 (s, 2-NHCOCH3); HRMS (APCI) m/z [M+H]+ calcd for C21H28NO10+ 454.1708, found 454.1704.

Synthesis of p-methoxyphenyl 2-acetamido-2-deoxy-β-D-galactopyranoside (11)

A suspension of compound 13 (1.126 g, 2.48 mmol, 1.0 equiv) in dry methanol (24.83 mL, 0.10 M) was treated with 1.0 M sodium methoxide in methanol (0.37 mL, 0.37 mmol, 0.15 equiv) and stirred at room temperature for 5 h. The mixture was neutralized with Amberlite IRC-50 H+ resin and concentrated under diminished pressure to give 11 (789 mg, 2.41 mmol, 97 %) as a colorless solid. Rf=0.56 (30 % methanol-chloroform); 1H NMR (D2O, 400.13 MHz): δ 1.92 (s, 3 H, 2-NHCOCH3), 3.66–3.72 (m, 4 H, H-5, H-6, H-3), 3.69 (s, 3 H, p-OCH3), 3.88 (d, 1 H, J3,4=3.3 Hz, H-4), 4.03 (dd, 1 H, J2,3=10.9 Hz, J1,2=8.5 Hz, H-2), 4.86 (d, 1 H, J1,2=8.5 Hz, H-1), 6.83–6.87 (m, 2 H, m-arom. H), 6.92–6.96 (m, 2 H, o-arom. H); 13C NMR (D2O, 100.62 MHz): δ 22.11 (q, 2-NHCOCH3), 52.40 (d, C-2), 55.73 (q, p-OCH3), 60.74 (t, C-6), 67.60 (d, C-4), 70.73 (d, C-3), 75.36 (d, C-5), 101.12 (d, C-1), 115.00 (d, m-arom. CH), 118.21 (d, o-arom. CH), 151.21 (s, arom. C), 154.71 (s, p-arom. C), 175.01 (s, 2-NHCOCH3); HRMS (APCI) m/z [M+H]+ calcd for C15H22NO7+ 328.1391, found 328.1386.

Synthesis of p-methoxyphenyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-sulfonato-β-D-galactopyranoside sodium salt (14)

Benzaldehyde dimethyl acetal (0.13 mL, 0.132 g, 0.866 mmol, 3.2 equiv) and D-camphor-10-sulfonic acid (6.8 mg, 29.2 μmol, 0.11 equiv) were added to a suspension of 11 (87.5 mg, 0.267 mmol, 1.0 equiv) in dry acetonitrile (3.8 mL, 70 mM), the mixture was stirred at 40°C for 15.5 h. After cooling, the reaction was quenched with triethyl amine (1.0 mL), and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 2 % methanol-chloroform to give 8 as colorless solid. Sulfur trioxide-pyridine complex (130.2 mg, 0.818 mmol, 3.1 equiv) was added to a solution of 8 in dry pyridine (1.66 mL, 0.16 M), and the mixture was stirred for 36.5 h at 40°C. Methanol (2.0 mL) was added to the reaction mixture, and the resulting solution was passed through the column of Dowex 50WX8 Na+ resin. The effluent was concentrated under diminished pressure. The residue was separated by reversed phase column chromatography eluting with 50 % water-methanol to give 14 (21.0 mg, 40.6 μmol, 15 %) as colorless solid. Rf=0.45 (30 % methanol-chloroform); 1H NMR (D2O, 400.13 MHz): δ 1.93 (s, 3 H, 2-NHCOCH3), 3.72 (s, 3 H, p-OCH3), 3.87 (s, 1 H, H-5), 4.19 (d, 1 H, J6a,6b=14.6 Hz, H-6a), 4.22 (d, 1 H, J6a,6b=14.6 Hz, H-6b), 4.29 (dd, 1 H, J2,3=10.9 Hz, J1,2=8.7 Hz, H-2), 4.58 (dd, 1 H, J2,3=10.9 Hz, J3,4=3.0 Hz, H-3), 4.63 (d, 1 H, J3,4=3.0 Hz, H-4), 5.19 (d, 1 H, J1,2=8.7 Hz, H-1), 5.73 (s, 1 H, 4,6-OCHC6H5), 6.88–6.90 (m, 2 H, m-arom. H of 1-OMP), 7.02–7.04 (m, 2 H, o-arom. H of 1-OMP), 7.39–7.41 (m, 3 H, p-arom. H and m-arom. H of benzylidene), 7.51–7.54 (m, 2 H, o-arom. H of benzylidene); 13C NMR (D2O, 100.62 MHz): δ 22.18 (q, 2-NHCOCH3), 50.28 (d, C-2), 55.81 (q, p-OCH3), 66.54 (d, C-5), 68.66 (t, C-6), 73.38 (d, C-4), 75.43 (d, C-3), 100.37 (d, C-1), 101.28 (d, 4,6-OCHC6H5), 115.05 (d, m-arom. CH of 1-OMP), 118.70 (d, o-arom. CH of 1-OMP), 126.53 (d, o-arom. CH of benzylidene), 128.70 (d, m-arom. CH of benzylidene), 129.94 (d, p-arom. CH of benzylidene), 136.68 (s, arom. C of benzylidene), 150.88 (s, arom. C of 1-OMP), 154.96 (s, p-arom. C of 1-OMP), 174.95 (s, 2-NHCOCH3); HRMS (ESI) m/z [M-Na] calcd for C22H24NO10S 494.1126, found 494.1121.

Synthesis of p-methoxyphenyl 2-acetamido-2-deoxy-3-O-sulfonato-β- D-galactopyranoside sodium salt (1)

A solution of 14 (17.8 mg, 34.4 μmol, 1.0 equiv) in 80 % acetic acid-water (0.40 mL) was stirred at room temperature for 3 h. 80 % acetic acid-water (0.20 mL) was added to the mixture, and then it was stirred at 40°C for 3 h. The mixture was concentrated under reduced pressure. The residue was diluted with methanol and passed through the column of Dowex 50WX8 Na+ resin. The effluent was evaporated under reduced pressure. The residue was separated by reversed phase column chromatography eluting with 85 % water-methanol to give 1 (9.9 mg, 23.1 μmol, 67 %) as a colorless solid. Rf=0.13 (30 % methanol-chloroform); 1H NMR (D2O, 400.13 MHz): δ 1.92 (s, 3 H, 2-NHCOCH3), 3.71 (s, 3 H, p-OCH3), 3.67–3.76 (m, 2 H, H-6), 3.76 (dd, 1 H, J5,6b=9.9 Hz, J5,6a=5.9 Hz, H-5), 4.18 (dd, 1 H, J2,3=11.0 Hz, J1,2=8.6 Hz, H-2), 4.21 (d, 1 H, J3,4=3.1 Hz, H-4), 4.40 (dd, 1 H, J2,3=11.0 Hz, J3,4=3.1 Hz, H-3), 5.03 (d, 1 H, J1,2=8.6 Hz, H-1), 6.85–6.89 (m, 2 H, m-arom. H), 6.95–6.99 (m, 2 H, o-arom. H); 13C NMR (D2O, 100.62 MHz): δ 22.18 (q, 2-NHCOCH3), 50.62 (d, C-2), 55.79 (q, p-OCH3), 60.67 (t, C-6), 66.14 (d, C-4), 75.04 (d, C-5), 77.51 (d, C-3), 100.66 (d, C-1), 115.05 (d, m-arom. CH), 118.43 (d, o-arom. CH), 151.06 (s, arom. C), 154.85 (s, p-arom. C), 174.93 (s, 2-NHCOCH3); HRMS (ESI) m/z [M-Na] calcd for C15H20NO10S 406.0813, found 406.0830.

Synthesis of p-methoxyphenyl 2-acetamido-2-deoxy-3,6-di-O-pivaloyl-β- D-galactopyranoside (10)

Pivaloyl chloride (0.12 mL, 118 mg, 0.975 mmol, 3.9 equiv) was added to a solution of 11 (82.7 mg, 0.253 mmol, 1.0 equiv) in dry pyridine (3.53 mL, 72 mM), and the mixture was stirred at −10°C for 2.5 h. After addition of methanol (1.0 mL), the mixture was concentrated under diminished pressure. The residue was diluted with chloroform (20 mL) and washed with 2.5 % aq HCl (20 mL). The aqueous phase was extracted with chloroform (5 mL×3). The combined organic phase was washed with 2.5 % aq HCl (15 mL×2), saturated aq NaHCO3 (15 mL×2), and saturated aq NaCl (15 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was separated by silica gel chromatography eluting with chloroform to afford 10 (111.6 mg, 0.225 mmol, 89 %) as a colorless amorphous. Rf=0.26 (50 % ethyl acetate-hexane); 1H NMR (CDCl3, 400.13 MHz): δ 1.16 (s, 9 H, 6-OCOC(CH3)3), 1.21 (s, 9 H, 3-OCOC(CH3)3), 1.93 (s, 3 H, 2-NHCOCH3), 2.99 (d, 1 H, J4,OH=6.0 Hz, 4-OH), 3.73 (s, 3 H, p-OCH3), 3.91 (dd, 1 H, J5,6b=7.3 Hz, J5,6a=4.8 Hz, H-5), 4.02 (dd, 1 H, J4,OH=6.0 Hz, J3,4=3.1 Hz, H-4), 4.28 (dd, 1 H, J6a,6b=11.6 Hz, J5,6a=4.8 Hz, H-6a), 4.31 (dd, 1 H, J6a,6b=11.6 Hz, J5,6b=7.3 Hz, H-6b), 4.51 (dt, 1 H, J2,3=11.1 Hz, J2,NH=9.0 Hz, J1,2=8.8 Hz, H-2), 4.97 (d, 1 H, J1,2=8.8 Hz, H-1), 5.19 (dd, 1 H, J2,3=11.1 Hz, J3,4=3.1 Hz, H-3), 6.42 (d, 1 H, J2,NH=9.0 Hz, 2-NHCOCH3), 6.66–6.69 (m, 2 H, m-arom. H), 6.87–6.89 (m, 2 H, o-arom. H); 13C NMR (CDCl3, 100.62 MHz): δ 23.14 (q, 2-NHCOCH3), 26.97 (q, 3-OCOC(CH3)3), 27.06 (q, 6-OCOC(CH3)3), 38.66 (s, 6-OCOC(CH3)3), 39.02 (s, 3-OCOC(CH3)3), 50.59 (d, C-2), 55.52 (q, p-OCH3), 63.13 (t, C-6), 66.97 (d, C-4), 72.40 (d, C-3), 72.58 (d, C-5), 100.93 (d, C-1), 114.33 (d, m-arom. CH), 118.55 (d, o-arom. CH), 151.39 (s, arom. C), 155.32 (s, p-arom. C), 170.45 (s, 2-NHCOCH3), 178.21 (s, 6-OCOC(CH3)3), 178.50 (s, 3-OCOC(CH3)3); HRMS (FAB) m/z [M+H]+ calcd for C25H38NO9+ 496.2541, found 496.2547.

Synthesis p-methoxyphenyl 2-acetamido-2-deoxy-3,6-di-O-pivaloyl-4-O-sulfonato-β-D-galactopyranoside sodium salt (15)

A mixture of 10 (63 mg, 0.127 mmol, 1.0 equiv) and sulfur trioxide-pyridine complex (101 mg, 0.635 mmol, 5.0 equiv) in dry pyridine (3.5 mL, 36 mM) was stirred for 49 h at room temperature. Methanol (5.0 mL) was then added, and the mixture was passed through the column of Dowex 50WX8 Na+ resin. The effluent was concentrated under reduced pressure. The residue was separated by silica gel column chromatography eluting with 15 % methanol-chloroform to give 15 (69 mg, 0.115 mmol, 91 %) as colorless crystals. Rf=0.50 (20 % methanol-chloroform); 1H NMR (CD3OD, 400.13 MHz): δ 1.20 (s, 9 H, 6-OCOC(CH3)3), 1.24 (s, 9 H, 3-OCOC(CH3)3), 1.95 (s, 3 H, 2-NHCOCH3), 3.75 (s, 3 H, p-OCH3), 4.04 (dd, 1 H, J5,6a=8.2 Hz, J5,6b=3.7 Hz, H-5), 4.39 (dd, 1 H, J6a,6b=11.9 Hz, J5,6a=8.2 Hz, H-6a), 4.36–4.41 (m, 1 H, H-2), 4.44 (dd, 1 H, J6a,6b=11.9 Hz, J5,6b=3.7 Hz, H-6b), 4.85 (d, 1 H, J3,4=3.3 Hz, H-4), 5.07 (d, 1 H, J1,2=8.7 Hz, H-1), 5.06–5.09 (m, 1 H, H-3), 6.79–6.84 (m, 2 H, m-arom. H), 6.98–7.02 (m, 2 H, o-arom. H); 13C NMR (CD3OD, 100.62 MHz): δ 21.47 (q, 2-NHCOCH3), 26.18 (q, 6-OCOC(CH3)3), 26.23 (q, 3-OCOC(CH3)3), 38.36 (s, 6-OCOC(CH3)3), 38.56 (s, 3-OCOC(CH3)3), 50.48 (d, C-2), 54.68 (q, p-OCH3), 64.25 (t, C-6), 70.95 (d, C-3), 71.79 (d, C-4), 72.49 (d, C-5), 100.41 (d, C-1), 114.10 (d, m-arom. CH), 118.12 (d, o-arom. CH), 151.56 (s, arom. C), 155.53 (s, p-arom. C), 171.97 (s, 2-NHCOCH3), 178.36 (s, 6-OCOC(CH3)3), 178.41 (s, 3-OCOC(CH3)3); HRMS (FAB) m/z [M+Na]+ calcd for C25H36NNa2O12S+ 620.1748, found 620.1755.

Synthesis of p-methoxyphenyl 2-acetamido-2-deoxy-4-O-sulfonato-β- D-galactopyranoside sodium salt (2)

A solution of 15 (50 mg, 83.7 μmol, 1.0 equiv) in dry methanol (1.0 mL, 84 mM) was treated with 1.0 M sodium methoxide-methanol solution (0.17 mL, 0.170 mmol, 2.0 equiv) for 20 h at room temperature and then for 6 h at 40°C. The mixture was neutralized with Amberlite IRC-50 H+ resin and concentrated under diminished pressure. The residue was purified by reversed phase column chromatography eluting with 90 % and 85 % water-methanol to give 2 (29.0 mg, 67.5 μmol, 81 %) as a colorless powder. Rf=0.49 (30 % methanol-chloroform); 1H NMR (D2O, 400.13 MHz): δ 1.93 (s, 3 H, 2-NHCOCH3), 3.70 (s, 3 H, p-OCH3), 3.71 (dd, 1 H, J6a,6b=12.0 Hz, J5,6a=7.7 Hz, H-6a), 3.76 (dd, 1 H, J6a,6b=12.0 Hz, J5,6b=4.6 Hz, H-6b), 3.83 (dd, 1 H, J5,6a=7.7 Hz, J5,6b=4.6 Hz, H-5), 3.87 (dd, 1 H, J2,3=11.0 Hz, J3,4=3.2 Hz, H-3), 4.04 (dd, 1 H, J2,3=11.0 Hz, J1,2=8.4 Hz, H-2), 4.64 (d, 1 H, J3,4=3.2 Hz, H-4), 4.93 (d, 1 H, J1,2=8.4 Hz, H-1), 6.84–6.88 (m, 2 H, m-arom. H), 6.93–6.97 (m, 2 H, o-arom. H); 13C NMR (D2O, 100.62 MHz): δ 22.11 (q, 2-NHCOCH3), 52.72 (d, C-2), 55.73 (q, p-OCH3), 60.75 (t, C-6), 69.72 (d, C-3), 74.64 (d, C-5), 75.44 (d, C-4), 100.90 (d, C-1), 115.00 (d, m-arom. CH), 118.33 (d, o-arom. CH), 151.05 (s, arom. C), 154.80 (s, p-arom. C), 174.99 (s, 2-NHCOCH3); HRMS (FAB) m/z [M+Na]+ calcd for C15H20NNa2O10S+ 452.0598, found 452.0614.

Synthesis of p-methoxyphenyl 2-acetamido-3,4-di-O-benzoyl-2-deoxy-6-O-trityl-β-D-galactopyranoside (16)

A mixture of 11 (355.4 mg, 1.09 mmol, 1.0 equiv) and trityl chloride (956 mg, 3.43 mmol, 3.2 equiv) in dry pyridine (10.88 mL, 0.10 M) was stirred for 24.5 h at 40°C. Methanol (4.0 mL) was added, and the reaction mixture was evaporated under reduced pressure. The residue was dissolved in dry pyridine (5.5 mL) and CH2Cl2 (2.8 mL, 0.13 M), benzoyl chloride (0.277 mL, 0.336 g, 2.39 mmol, 2.2 equiv) was added to the solution. The mixture was stirred at 0°C for 3 h and then 5°C for 14 h. Methanol (1.0 mL) was then added, the reaction mixture was diluted with chloroform (50 mL). The organic phase was washed with 2.5 % aq HCl (30 mL×2), saturated aq NaHCO3 (30 mL×2), and saturated aq NaCl (30 mL×1), dried over Na2SO4, and concentrated under diminished pressure. The residue was separated by silica gel column chromatography eluting with 40 % ethyl acetate-hexane to give 16 (678.8 mg, 0.873 mmol, 80 %) as a colorless amorphous. Rf=0.43 (50 % ethyl acetate-hexane); 1H NMR (CDCl3, 400.13 MHz): δ 1.88 (s, 3 H, 2-NHCOCH3), 3.26 (dd, 1 H, J6a, 6b=9.5 Hz, J5,6a=6.8 Hz, H-6a), 3.51 (dd, 1 H, J6a,6b=9.5 Hz, J5,6b=6.5 Hz, H-6b), 3.76 (s, 3 H, p-OCH3), 4.01 (t, 1 H, J5,6a=6.8 Hz, J5,6b=6.5 Hz, H-5), 4.45 (dt, 1 H, J2,3=11.0 Hz, J2,NH=8.7 Hz, J1,2=8.6 Hz, H-2), 5.21 (d, 1 H, J1,2=8.6 Hz, H-1), 5.54–5.61 (m, 1 H, 2-NHCOCH3), 5.66 (dd, 1 H, J2,3=11.0 Hz, J3,4=3.1 Hz, H-3), 5.86 (d, 1 H, J3,4=3.1 Hz, H-4), 6.77–6.82 (m, 2 H, m-arom. H of 1-OMP), 7.02–7.06 (m, 2 H, o-arom. H of 1-OMP), 7.12–7.16 (m, 3 H, p-arom. H of 6-OTr), 7.16–7.20 (m, 6 H, m-arom. H of 6-OTr), 7.30–7.33 (m, 2 H, m-arom. H of 3-OBz), 7.36–7.38 (m, 6 H, o-arom. H of 6-OTr), 7.40–7.44 (m, 2 H, m-arom. H of 4-OBz), 7.48–7.52 (m, 1 H, p-arom. H of 3-OBz), 7.57–7.61 (m, 1 H, p-arom. H of 4-OBz), 7.83–7.85 (m, 2 H, o-arom. H of 3-OBz), 7.93–7.95 (m, 2 H, o-arom. H of 4-OBz); 13C NMR (CDCl3, 100.62 MHz): δ 23.46 (q, 2-NHCOCH3), 52.21 (d, C-2), 55.65 (q, p-OCH3), 61.57 (t, C-6), 67.80 (d, C-4), 70.89 (d, C-3), 72.88 (d, C-5), 87.03 (s, 6-OCPh3), 100.81 (d, C-1), 114.56 (d, m-arom. CH of 1-OMP), 118.47 (d, o-arom. CH of 1-OMP), 127.04 (d, p-arom. CH of 6-OTr), 127.83 (d, m-arom. CH of 6-OTr), 128.38 (d, m-arom. CH of 3-OBz), 128.43 (d, m-arom. CH of 4-OBz), 128.58 (d, o-arom. CH of 6-OTr), 129.03 (s, arom. C of 3-OBz), 129.42 (s, arom. C of 4-OBz), 129.92 (d, o-arom. CH of 3-OBz), 130.00 (d, o-arom. CH of 4-OBz), 133.25 (d, p-arom. CH of 4-OBz), 133.37 (d, p-arom. CH of 3-OBz), 143.43 (s, arom. C of 6-OTr), 151.44 (s, arom. C of 1-OMP), 155.47 (s, p-arom. C of 1-OMP), 165.41 (s, 4-OCOC6H5), 166.17 (s, 3-OCOC6H5), 170.36 (s, 2-NHCOCH3); HRMS (APCI) m/z [M+H]+ calcd for C48H44NO9+ 778.3011, found 778.3009.

Synthesis of p-methoxyphenyl 2-acetamido-3,4-di-O-benzoyl-2-deoxy-6-O-sulfonato-β-D-galactopyranoside sodium salt (18)

A solution of 16 (182.1 mg, 0.234 mmol, 1.0 equiv) in 80 % acetic acid-water (1.17 mL) was stirred at 80°C for 1 h. After cooling, triethylamine (2.6 mL) was added to the mixture. The product was extracted by chloroform (5 mL×3). The organic phase was washed with saturated aq NaHCO3 (30 mL×3) and saturated aq NaCl (30 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was dissolved in dry pyridine (2.34 mL, 0.10 M) and sulfur trioxide-pyridine complex (180 mg, 1.13 mmol, 4.8 equiv) was added to the mixture. The mixture was stirred for 15.5 h at room temperature and then for 25.5 h at 40°C. Methanol (1 mL) was added to the reaction mixture, and the resulting solution was passed through the column of Dowex 50WX8 Na+ resin. The effluent was concentrated under reduced pressure. The residue was separated by reversed phase column chromatography eluting with 40 % water-methanol to afford 18 (101.4 mg, 0.159 mmol, 68 %) as a colorless solid. Rf=0.40 (20 % methanol-chloroform); 1H NMR (D2O, 400.13 MHz): δ 1.79 (s, 3 H, 2-NHCOCH3), 3.72 (s, 3 H, p-OCH3), 4.15 (d, 1 H, J5, 6=6.3 Hz, H-6), 4.48 (t, 1 H, J5,6=6.3 Hz, H-5), 4.60 (dd, 1 H, J2,3=10.8 Hz, J1,2=9.0 Hz, H-2), 5.28 (d, 1 H, J1,2=9.0 Hz, H-1), 5.46 (dd, 1 H, J2,3=10.8 Hz, J3,4=3.0 Hz, H-3), 5.79 (d, 1 H, J3,4=3.0 Hz, H-4), 6.89–6.91 (m, 2 H, m-arom. H of 1-OMP), 7.05–7.07 (m, 2 H, o-arom. H of 1-OMP), 7.27–7.31 (m, 2 H, m-arom. H of 3-OBz), 7.44–7.48 (m, 2 H, m-arom. H of 4-OBz), 7.50–7.53 (m, 1 H, p-arom. H of 3-OBz), 7.61–7.64 (m, 1 H, p-arom. H of 4-OBz), 7.68–7.70 (m, 2 H, o-arom. H of 3-OBz), 7.95–7.97 (m, 2 H, o-arom. H of 4-OBz); 13C NMR (D2O, 100.62 MHz): δ 21.86 (q, 2-NHCOCH3), 50.41 (d, C-2), 55.76 (q, p-OCH3), 65.54 (t, C-6), 67.55 (d, C-4), 71.21 (d, C-5), 71.48 (d, C-3), 100.41 (d, C-1), 115.06 (d, m-arom. CH of 1-OMP), 118.61 (d, o-arom. CH of 1-OMP), 128.06 (s, arom. C of 3-OBz), 128.33 (s, arom. C of 4-OBz), 128.73 (d, m-arom. CH of 3-OBz), 128.84 (d, m-arom. CH of 4-OBz), 129.40 (d, o-arom. CH of 3-OBz), 129.75 (d, o-arom. CH of 4-OBz), 134.21 (d, p-arom. CH of 3-OBz), 134.34 (d, p-arom. CH of 4-OBz), 150.73 (s, arom. C of 1-OMP), 155.04 (s, p-arom. C of 1-OMP), 167.00 (s, 3-OCOC6H5), 167.43 (s, 4-OCOC6H5), 174.68 (s, 2-NHCOCH3); HRMS (ESI) m/z [M-Na] calcd for C29H28NO12S 614.1338, found 614.1325.

Synthesis of p-methoxyphenyl 2-acetamido-2-deoxy-6-O-sulfonato-β-D-galactopyranoside sodium salt (3)

Compound 18 (49.7 mg, 77.9 μmol, 1.0 equiv) was dissolved in dry methanol (1.0 mL, 78 mM) and treated with 1 M sodium methoxide in methanol (0.16 mL, 0.160 mmol, 2.1 equiv) for 7.5 h at room temperature. The mixture was neutralized with Amberlite IRC-50 H+ resin and concentrated under reduced pressure. The residue was purified by reversed phase column chromatography eluting with 90 % and 85 % water-methanol to afforded 3 (33.4 mg, 77.8 μmol, 100 %) as colorless solid. Rf=0.56 (50 % methanol-chloroform); 1H NMR (D2O, 400.13 MHz): δ 1.92 (s, 3 H, 2-NHCOCH3), 3.68 (s, 3 H, p-OCH3), 3.72 (dd, 1 H, J2,3=10.9 Hz, J3,4=3.3 Hz, H-3), 3.93 (d, 1 H, J3,4=3.3 Hz, H-4), 3.96 (dd, 1 H, J5,6a=7.1 Hz, J5,6b=5.3 Hz, H-5), 4.04 (dd, 1 H, J2,3=10.9 Hz, J1,2=8.5 Hz, H-2), 4.11 (dd, 1 H, J6a,6b=10.8 Hz, J5,6a=7.1 Hz, H-6a), 4.15 (dd, 1 H, J6a,6b=10.8 Hz, J5, 6b=5.3 Hz, H-6b), 4.85 (d, 1 H, J1,2=8.5 Hz, H-1), 6.83–6.87 (m, 2 H, m-arom. H), 6.93–6.97 (m, 2 H, o-arom. H); 13C NMR (D2O, 100.62 MHz): δ 22.11 (q, 2-NHCOCH3), 52.28 (d, C-2), 55.73 (q, p-OCH3), 66.97 (t, C-6), 67.33 (d, C-4), 70.56 (d, C-3), 72.89 (d, C-5), 101.06 (d, C-1), 115.00 (d, m-arom. CH), 118.17 (d, o-arom. CH), 151.25 (s, arom. C), 154.70 (s, p-arom. C), 175.00 (s, 2-NHCOCH3); HRMS (ESI) m/z [M-Na] calcd for C15H20NO10S 406.0813, found 406.0835.

Synthesis of p-methoxyphenyl 2-acetamido-2-deoxy-3,4-di-O-sulfonato-6-O-trityl-β-D-galactopyranoside disodium salts (19)

A mixture of 11 (21.8 mg, 66.6 μmol, 1.0 equiv) and trityl chloride (55.7 mg, 0.200 mmol, 3.0 equiv) in dry pyridine (0.61 mL, 0.11 M) was stirred for 19 h at 40°C. Methanol (2.0 mL) was then added, and the mixture was concentrated under reduced pressure. The residue was dissolved in chloroform and purified by silica gel column chromatography eluting with 5 % methanol-chloroform to give 9 as colorless amorphous. Sulfur trioxide-pyridine complex (68.1 mg, 0.428 mmol, 6.4 equiv) was added to a solution of 9 in dry pyridine (1.00 mL, 67 mM), and the mixture was stirred for 14.5 h at room temperature. Methanol (2 mL) was added to the reaction mixture, and the resulting solution was passed through the column of Dowex 50WX8 Na+ resin. The effluent was concentrated under reduced pressure. The residue was separated by reversed phase column chromatography eluting with 50 %, 40 %, 30 %, and 20 % water-methanol to give 19 (24.6 mg, 31.8 μmol, 48 %) as a colorless solid. Rf=0.35 (50 % methanol-chloroform); 1H NMR (D2O, 400.13 MHz): δ 1.95 (s, 3 H, 2-NHCOCH3), 3.08 (d, 1 H, J6a,6b=10.9 Hz, H-6a), 3.57 (dd, 1 H, J6a,6b=10.9 Hz, J5,6b=9.5 Hz, H-6b), 3.62 (s, 3 H, p-OCH3), 3.70 (d, 1 H, J5,6b=9.5 Hz, H-5), 4.09 (dd, 1 H, J2,3=10.9 Hz, J1,2=8.6 Hz, H-2), 4.41 (dd, 1 H, J2,3=10.9 Hz, J3,4=3.1 Hz, H-3), 4.58 (d, 1 H, J3,4=3.1 Hz, H-4), 5.27 (d, 1 H, J1,2=8.6 Hz, H-1), 6.86–6.88 (m, 2 H, m-arom. H of 1-OMP), 7.16–7.19 (m, 2 H, o-arom. H of 1-OMP), 7.20–7.26 (m, 9 H, p- and m-arom. H of 6-OTr), 7.33–7.35 (m, 6 H, o-arom. H of 6-OTr); 13C NMR (D2O, 100.62 MHz): δ 22.29 (q, 2-NHCOCH3), 51.18 (d, C-2), 55.78 (q, p-OCH3), 64.08 (t, C-6), 73.74 (d, C-5), 74.26 (d, C-4), 75.18 (d, C-3), 87.11 (s, 6-OCPh3), 99.17 (d, C-1), 115.01 (d, m-arom. CH of 1-OMP), 118.80 (d, o-arom. CH of 1-OMP), 127.53 (d, p-arom. CH of 6-OTr), 128.19 (d, m-arom. CH of 6-OTr), 128.42 (d, o-arom. CH of 6-OTr), 143.41 (s, arom. C of 6-OTr), 150.34 (s, arom. C of 1-OMP), 154.89 (s, p-arom. C of 1-OMP), 175.02 (s, 2-NHCOCH3); HRMS (ESI) m/z [M-Na] calcd for C34H33NNaO13S2 750.1296 found 750.1282.

Synthesis of p-methoxyphenyl 2-acetamido-2-deoxy-3,4-di-O-sulfonato-β-D-galactopyranoside disodium salts (4)

To a solution of 19 (10 mg, 12.9 μmol) in 80 % methanol-water (1.7 mL, 7.6 mM) was added 10 % Pd-C (11 mg) and the mixture was stirred for 23 h at room temperature under hydrogen. The reaction mixture was filtered through Celite and concentrated under reduced pressure. The residue was purified by reversed phase column chromatography eluting with 80 % water-methanol and then HPLC (XSelect CSH C18, 4.6×250 mm, 5 μm) eluting with water to give 4 (4.5 mg, 8.47 μmol, 66 %) as a colorless solid. Rf=0.45 (30 % methanol-chloroform); 1H NMR (D2O, 400.13 MHz): δ 1.92 (s, 3 H, 2-NHCOCH3), 3.70 (s, 3 H, p-OCH3), 3.72 (dd, 1 H, J6a,6b=12.1 Hz, J5,6a=7.5 Hz, H-6a), 3.76 (dd, 1 H, J6a,6b=12.1 Hz, J5,6b=4.7 Hz, H-6b), 3.88 (dd, 1 H, J5,6a=7.5 Hz, J5,6b=4.7 Hz, H-5), 4.13 (dd, 1 H, J2,3=11.1 Hz, J1,2=8.5 Hz, H-2), 4.49 (dd, 1 H, J2,3=11.1 Hz, J3,4=3.1 Hz, H-3), 4.88 (d, 1 H, J3,4=3.1 Hz, H-4), 5.12 (d, 1 H, J1,2=8.5 Hz, H-1), 6.85–6.89 (m, 2 H, m-arom. H), 6.96–7.00 (m, 2 H, o-arom. H); 13C NMR (D2O, 100.62 MHz): δ 22.20 (q, 2-NHCOCH3), 51.08 (d, C-2), 55.77 (q, p-OCH3), 60.76 (t, C-6), 73.99 (d, C-4), 74.67 (d, C-5), 74.99 (d, C-3), 100.32 (d, C-1), 115.03 (d, m-arom. CH), 118.49 (d, o-arom. CH), 150.90 (s, arom. C), 154.91 (s, p-arom. C), 174.95 (s, 2-NHCOCH3); HRMS (ESI) m/z [M-Na] calcd for C15H19NNaO13S2 508.0201, found 508.0234.

Synthesis of p-methoxyphenyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-pivaloyl-β-D-galactopyranoside (20)

A mixture of 11 (130 mg, 0.397 mmol, 1.0 equiv), benzaldehyde dimethyl acetal (0.34 mL, 0.345 g, 2.27 mmol, 5.7 equiv), and D-camphor-10-sulfonic acid (9.1 mg, 41.7 μmol, 0.10 equiv) in dry acetonitrile (10.2 mL, 39 mM) was stirred at room temperature for 14 h and then at 40°C for 23.5 h. After cooling, the reaction was quenched with triethylamine (10 μL). The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 5 % methanol-chloroform to give 8 as a colorless solid. Pivaloyl chloride (0.21 mL, 206 mg, 1.71 mmol, 4.3 equiv) and dry pyridine (0.14 mL, 137 mg, 1.73 mmol, 4.4 equiv) were added to a solution of 8 in dry dichloromethane (3.80 mL, 0.10 M). The mixture was stirred for 11 h at room temperature. Methanol (0.2 mL) was added to the mixture. The resulting solution was diluted with chloroform (50 mL), and washed with 5 % aq HCl (30 mL×3), saturated aq NaHCO3 (30 mL×2), and saturated aq NaCl (30 mL). The organic layer was dried over Na2SO4, and concentrated under reduced pressure. Purification was carried out by silica gel column chromatography eluting with chloroform to give 20 (37 mg, 74.1 μmol, 19 %) as a colorless solid. Rf=0.54 (10 % methanol-chloroform); 1H NMR (CDCl3, 400.13 MHz): δ 1.21 (s, 9 H, 3-OCOC(CH3)3), 1.96 (s, 3 H, 2-NHCOCH3), 3.65 (d, 1 H, J5,6b=1.1 Hz, H-5), 3.76 (s, 3 H, p-OCH3), 4.09 (dd, 1 H, J6a,6b=12.4 Hz, J5,6a=1.4 Hz, H-6a), 4.18 (dt, 1 H, J2,3=11.3 Hz, J1,2=8.3 Hz, J2,NH=8.1 Hz, H-2), 4.36 (dd, 1 H, J6a,6b=12.4 Hz, J5,6b=1.1 Hz, H-6b), 4.43 (d, 1 H, J3,4=3.5 Hz, H-4), 5.40 (d, 1 H, J1,2=8.3 Hz, H-1), 5.47 (dd, 1 H, J2,3=11.3 Hz, J3,4=3.5 Hz, H-3), 5.53 (d, 1 H, J2,NH=8.1 Hz, 2-NHCOCH3), 5.56 (s, 1 H, 4,6-OCHC6H5), 6.77–6.81 (m, 2 H, m-arom. H of 1-OMP), 6.98–7.02 (m, 2 H, o-arom. H of 1-OMP), 7.34–7.39 (m, 3 H, p- and m-arom. H of benzylidene), 7.50–7.52 (m, 2 H, o-arom. H of benzylidene); 13C NMR (CDCl3, 100.62 MHz): δ 23.48 (q, 2-NHCOCH3), 27.02 (q, 3-OCOC(CH3)3), 39.01 (s, 3-OCOC(CH3)3), 52.04 (d, C-2), 55.63 (q, p-OCH3), 66.51 (d, C-5), 69.12 (t, C-6), 69.89 (d, C-3), 72.97 (d, C-4), 99.88 (d, C-1), 100.49 (d, 4,6-OCHC6H5), 114.43 (d, m-arom. CH of 1-OMP), 119.12 (d, o-arom. CH of 1-OMP), 126.06 (d, o-arom. CH of benzylidene), 128.09 (d, m-arom. CH of benzylidene), 128.83 (d, p-arom. CH of benzylidene), 137.67 (s, arom. C of benzylidene), 151.29 (s, arom. C of 1-OMP), 155.48 (s, p-arom. C of 1-OMP), 170.25 (s, 2-NHCOCH3), 178.24 (s, 3-OCOC(CH3)3); HRMS (APCI) m/z [M+H]+ calcd for C27H34NO8+ 500.2279, found 500.2270.

Synthesis of p-methoxyphenyl 2-acetamido-2-deoxy-3-O-pivaloyl-4,6-di-O-sulfonato-β-D-galactopyranoside disodium salts (22)

Galactosamine 20 (37 mg, 74.1 μmol, 1.0 equiv) was dissolved in 80 % acetic acid-water (5.0 mL) and stirred at 80°C for 2 h. The mixture was concentrated under reduced pressure. Then, the residue was diluted with chloroform (50 mL) and washed with saturated aq NaHCO3 (20 mL). The aqueous phase was extracted by chloroform (5 mL×3). The combined organic layer was dried over Na2SO4 and evaporated under diminished pressure. The residue was dissolved in dry pyridine (1.0 mL, 74 mM), sulfur trioxide–pyridine complex (142 mg, 0.892 mmol, 12 equiv) was added to the solution, and the mixture was stirred for 64.5 h at 30°C. Methanol (1.0 mL) was added to the reaction mixture, and the resulting solution was passed through the column of Dowex 50WX8 Na+ resin. The effluent was concentrated under diminished pressure. The residue was separated by reversed phase column chromatography eluting with 95 % and 90 % water-methanol to give 22 (38.7 mg, 62.9 μmol, 85 %) as a colorless solid. Rf=0.15 (30 % methanol-chloroform); 1H NMR (D2O, 400.13 MHz): δ 1.08 (s, 9 H, 3-OCOC(CH3)3), 1.90 (s, 3 H, 2-NHCOCH3), 3.71 (s, 3 H, p-OCH3), 4.13–4.28 (m, 3 H, H-6, H-5), 4.28 (dd, 1 H, J2,3=11.3 Hz, J1,2=8.5 Hz, H-2), 4.80 (d, 1 H, J3,4=3.2 Hz, H-4), 4.98 (dd, 1 H, J2,3=11.3 Hz, J3,4=3.2 Hz, H-3), 5.06 (d, 1 H, J1,2=8.5 Hz, H-1), 6.86–6.90 (m, 2 H, m-arom. H), 6.99–7.03 (m, 2 H, o-arom. H); 13C NMR (D2O, 100.62 MHz): δ 21.96 (q, 2-NHCOCH3), 26.22 (q, 3-OCOC(CH3)3), 38.70 (s, 3-OCOC(CH3)3), 50.18 (d, C-2), 55.78 (q, p-OCH3), 67.50 (t, C-6), 70.96 (d, C-3), 72.41 (d, C-5), 72.49 (d, C-4), 100.23 (d, C-1), 115.07 (d, m-arom. CH), 118.42 (d, o-arom. CH), 150.96 (s, arom. C), 154.92 (s, p-arom. C), 174.54 (s, 2-NHCOCH3), 180.64 (s, 3-OCOC(CH3)3); HRMS (ESI) m/z [M-Na] calcd for C20H27NNaO14S2 592.0776, found 592.0736.

Synthesis of p-methoxyphenyl 2-acetamido-2-deoxy-4,6-di-O-sulfonato-β-D-galactopyranoside disodium salts (6)

Sodium methoxide in methanol (1.0 M) (6.43 mL, 6.43 mmol, 14 equiv) was added to the suspension of 22 (273 mg, 0.444 mmol, 1.0 equiv) in methanol (9.0 mL, 49 mM), and stirred at room temperature for 67.5 h. The reaction mixture was neutralized with Amberlite IRC-50 H+ resin, and concentrated under reduced pressure to afford 5 (234.9 mg, 0.442 mmol, 100 %) as colorless solid. Rf=0.53 (50 % methanol-chloroform); 1H NMR (D2O, 400.13 MHz): δ 1.94 (s, 3 H, 2-NHCOCH3), 3.71 (s, 3 H, p-OCH3), 3.89 (dd, 1 H, J2,3=10.3 Hz, J3,4=2.7 Hz, H-3), 4.06 (dd, 1 H, J2,3=10.3 Hz, J1,2=8.8 Hz, H-2), 4.14 (dd, 1 H, J5,6a=8.2 Hz, J5,6b=7.6 Hz, H-5), 4.15 (dd, 1 H, J6a,6b=15.5 Hz, J5,6a=8.2 Hz, H-6a), 4.27 (dd, 1 H, J6a,6b=15.5 Hz, J5,6b=7.6 Hz, H-6b), 4.69 (d, 1 H, J3,4=2.7 Hz, H-4), 4.92 (d, 1 H, J1,2=8.8 Hz, H-1), 6.86–6.88 (m, 2 H, m-arom. H), 6.98–6.99 (m, 2 H, o-arom. H); 13C NMR (D2O, 100.62 MHz): δ 22.14 (q, 2-NHCOCH3), 52.60 (d, C-2), 55.78 (q, p-OCH3), 67.66 (t, C-6), 69.70 (d, C-3), 72.44 (d, C-5), 75.27 (d, C-4), 100.87 (d, C-1), 115.05 (d, m-arom. CH), 118.27 (d, o-arom. CH), 151.20 (s, arom. C), 154.80 (s, p-arom. C), 175.03 (s, 2-NHCOCH3); HRMS (ESI) m/z [M-Na] calcd for C15H19NNaO13S2 508.0201, found 508.0200.

Synthesis of p-methoxyphenyl 2-acetamido-2-deoxy-3,6-di-O-sulfonato-β-D-galactopyranoside disodium salts (5) and p-methoxyphenyl 2-acetamido-2-deoxy-3,4,6-tri-O-sulfonato-β-D-galactopyranoside trisodium salts (7)

Sulfur trioxide-trimethylamine complex (331 mg, 2.38 mmol, 15.0 equiv) was added to a solution of 11 (51.9 mg, 0.159 mmol, 1.0 equiv) in dry pyridine (1.59 mL, 0.10 M), and the mixture was stirred for 13.5 h at room temperature. After addition of methanol (3.0 mL), the mixture was subjected to Dowex 50WX8 Na+ resin. The effluent was concentrated under diminished pressure. The residue was separated by reversed phase column chromatography eluting with 100 % water-methanol to give 7 (15.4 mg, 23.2 μmol, 15 %) as a colorless solid, then with 97.5 % water-methanol to give 6 (62.9 mg, 0.118 mmol, 75 %) as a colorless solid. 6: Rf=0.18 (50 % methanol-chloroform); 1H NMR (D2O, 400.13 MHz): δ 1.91 (s, 3 H, 2-NHCOCH3), 3.69 (s, 3 H, p-OCH3), 4.02 (dd, 1 H, J5,6a=7.2 Hz J5,6b=5.2 Hz, H-5), 4.12 (dd, 1 H, J6a,6b=10.9 Hz, J5,6a=7.2 Hz, H-6a), 4.17 (dd, 1 H, J6a,6b=10.9 Hz, J5,6b=5.2 Hz, H-6b), 4.17 (dd, 1 H, J2,3=10.9 Hz, J1,2=8.3 Hz, H-2), 4.24 (d, 1 H, J3,4=3.2 Hz, H-4), 4.40 (dd, 1 H, J2,3=10.9 Hz, J3,4=3.2 Hz, H-3), 5.00 (d, 1 H, J1,2=8.3 Hz, H-1), 6.83–6.87 (m, 2 H, m-arom. H), 6.95–6.99 (m, 2 H, o-arom. H); 13C NMR (D2O, 100.62 MHz): δ 22.14 (q, 2-NHCOCH3), 50.48 (d, C-2), 55.72 (q, p-OCH3), 65.92 (d, C-4), 67.02 (t, C-6), 72.66 (d, C-5), 77.21 (d, C-3), 100.54 (d, C-1), 115.00 (d, m-arom. CH), 118.32 (d, o-arom. CH), 151.09 (s, arom. C), 154.80 (s, p-arom. C), 174.90 (s, 2-NHCOCH3); HRMS (ESI) m/z [M-Na] calcd for C15H19NNaO13S2 508.0201, found 508.0196. 7: Rf=0.10 (50 % methanol-chloroform); 1H NMR (D2O, 400.13 MHz): δ 1.93 (s, 3 H, 2-NHCOCH3), 3.71 (s, 3 H, p-OCH3), 4.14–4.20 (m, 3 H, H-2, H-6a, H-5), 4.27 (dd, 1 H, J6a,6b=16.6 Hz, J5,6b=8.9 Hz, H-6b), 4.51 (dd, 1 H, J2,3=11.0 Hz, J3,4=2.9 Hz, H-3), 4.91 (d, 1 H, J3,4=2.9 Hz, H-4), 5.09 (d, 1 H, J1,2=8.5 Hz, H-1), 6.87–6.89 (m, 2 H, m-arom. H), 7.00–7.02 (m, 2 H, o-arom. H); 13C NMR (D2O, 100.62 MHz): δ 22.21 (q, 2-NHCOCH3), 50.96 (d, C-2), 55.79 (q, p-OCH3), 67.69 (t, C-6), 72.49 (d, C-5), 73.81 (d, C-4), 74.91 (d, C-3), 100.27 (d, C-1), 115.07 (d, m-arom. CH), 118.42 (d, o-arom. CH), 151.05 (s, arom. C), 154.88 (s, p-arom. C), 174.96 (s, 2-NHCOCH3); HRMS (ESI) m/z [M-Na] calcd for C15H18NNa2O16S3 609.9589 found 609.9579.

Viral infection

A Japanese encephalitis virus (JEV) strain, Beijing-1, was used in this study. Compounds were dissolved in DMSO at a concentration of 10 mM for a stock solution. Inhibition of JEV infection with compounds was determined by real-time reverse transcription (RT)-PCR targeting the gene encoding viral envelope (E) protein. Vero cells were seeded onto 96-well plastic plates and cultured overnight at 37°C. The virus was premixed in a serum-free medium, Virus Production Serum Free medium (VPSFM) (GIBCO Life Technologies, Carlsbad, CA, USA) on ice with compounds at the indicated concentration. The virus-compound premixtures were immediately inoculated on the cells for 2 h at 37°C. After removal of the premixtures, VPSFM was added, and plates were incubated at 37°C for 20–24 h.

Quantitative real-time RT-PCR

Total RNA was extracted from the infected cells using NucleoSpin RNA (Machery-Nagel, Düren, Germany) according to the manufacturer’s instructions. mRNA was reverse-transcribed using Random 6 mers with a PrimeScript RT reagent kit (Takara Bio Inc., Kyoto). The cellular expression of the genes encoding the E protein and β-actin were quantitatively measured by a real-time RT-PCR method using primers as follows: forward primer (5′-CATAGGGAAAGCTGTTCACCA-3′) and reverse primer (5′-ATTGACCGGTCTCGTGCGTT-3′) for JEV E protein gene amplification, and forward primer (5′-AGAAAATCTGGCACCACACC-3′) and reverse primer (5′-TGCTATCCCTGTACGCCTCT-3′) for β-actin gene amplification. Real-Time PCR was performed using an ABI Prism7000 Sequence Detection System and a Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK). Reaction cycles was performed for 45 cycles of denaturation (95°C, 30 s), anneal-ing (55°C, 30 s), and extension (60°C, 30 s). The PCR efficiency was examined by serially diluting template cDNA and the melting curve data were collected to check the PCR specificity. Values were calculated using the delta Ct method normalizing to β-actin expression for each sample. Relative infectivity (%) was shown in the presence of compound at the indicated concentration to virus treated with DMSO as control.

Cellular cytotoxicity of compounds

The cytotoxicity of compounds was determined by a MTT assay according to the manufacturer’s instructions. Briefly, cells were seeded in 96-well plastic plates and cultured overnight at 37°C. After removal of the media, the compounds were diluted to the indicated concentrations with VPSFM and added onto the cells. Plates were incubated at 37°C for 48 h. Cell viability was then measured by the MTT assay.


Article note

A collection of invited papers based on presentations at the XXVIII International Carbohydrate Symposium (ICS-28), New Orleans, July 17–21 2016.


Acknowledgments

This work was supported by a Cooperative Research Grant (to K.I.P.J.H.) of the Institute of Tropical Medicine, Nagasaki University, 2016.

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Published Online: 2017-01-20
Published in Print: 2017-08-28

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