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Open Access

Peer-reviewed

Research Article

Synthesis of the Tetrasaccharide Motif and Its Structural Analog Corresponding to the Lipopolysaccharide of Escherichia coli O75

Abstract

Background

Extraintestinal pathogenic E. coli are mostly responsible for a diverse spectrum of invasive human and animal infections leading to the urinary tract infections. Bacterial lipopolysaccharides are responsible for their pathogenicity and their interactions with host immune responses. In spite of several breakthroughs in the development of therapeutics to combat urinary tract infections and related diseases, the emergence of multidrug-resistant bacterial strains is a serious concern. Lipopolysaccharides are attractive targets for the development of long-term therapeutic agents to eradicate the infections. Since the natural sources cannot provide the required amount of oligosaccharides, development of chemical synthetic strategies for their synthesis is relevant to gain access to a reservoir of oligosaccharides and their close analogs.

Methodology

Two tetrasaccharide derivatives were synthesized from a single disaccharide intermediate. β-d-mannoside moiety was prepared from β-d-glucoside moiety following oxidation–reduction methodology. A [2+2] stereoselective block glycosylation strategy has been adopted for the preparation of tetrasaccharide derivative. α-d-Glucosamine moiety was prepared from α-d-mannosidic moiety following triflate formation at C-2 and SN2 substitution. A one-pot iterative glycosylation exploiting the orthogonal property of thioglycoside was carried out during the synthesis of tetrasaccharide analog.

Results

Synthesis of the tetrasaccharide motif (1) and its structural analog (2) corresponding to the lipopolysaccharide of Escherichia coli O75 was successfully achieved in excellent yield. Most of the reactions are clean and high yielding. Both compounds 1 and 2 were synthesized as their 4-methoxyphenyl glycoside, which can act as a temporary anomeric protecting group for further use of these tetrasaccharides in the preparation of glycoconjugates.

Citation: Sau A, Misra AK (2012) Synthesis of the Tetrasaccharide Motif and Its Structural Analog Corresponding to the Lipopolysaccharide of Escherichia coli O75. PLoS ONE 7(5): e37291. https://doi.org/10.1371/journal.pone.0037291

Editor: Joseph J. Barchi, National Cancer Institute at Frederick, United States of America

Received: February 2, 2012; Accepted: April 17, 2012; Published: May 25, 2012

Copyright: © 2012 Sau, Misra. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the Department of Science and Technology (DST), New Delhi (Project No. SR/S1/OC-83/2010) and the Bose Institute, Kolkata. AS thanks CSIR, New Delhi, for providing a Senior Research Fellowship. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Escherichia coli (E. coli) are opportunistic pathogen belong to the Gram-negative Enterobacteriaceae. Pathogenic E. coli are the major causative agents for a number of extra intestinal infections in humans and animals [1]. One of the most frequently occurring E. coli infections is urinary tract infections. Escherichia coli strains are responsible for 60 to 80% of community acquired urinary tract infections in children and adults [2], [3]. Extra intestinal pathogenic E. coli are mostly responsible for a diverse spectrum of invasive human and animal infections leading to pyelonephritis in the developing and developed countries [4], [5], [6]. The mechanism of such kind of ascending urinary tract infections has been explained based on the interactions between E. coli adhesion and their uroepithelial receptor ligands [7]. The most of the urinary tract infections found in human are caused by a small number of E. coli O-serogroups e.g. O4, O6, O14, O22, O75 and O83 [8]. Furthermore, they have phenotypes that are epidemiologically associated with cystitis and acute pyelonephritis in the normal urinary tract [9], [10]. In this context, a revised structure of the E. coli O75 lipopolysaccharide has been reported by Erbing et. al. [11] (Figure 1). Bacterial lipopolysaccharides play vital roles for their pathogenicity and their interactions with host immune responses.

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Figure 1. Tetrasaccharide repeating unit corresponding to the O-specific lipopolysaccharide of Escherichia coli O75.

https://doi.org/10.1371/journal.pone.0037291.g001

In spite of several breakthroughs in the development of therapeutics to combat urinary tract infections and related diseases, the emergence of multi drug resistant bacterial strains is a serious concern. The epidemiological data for the urinary tract infections caused by multi-drug resistant E. coli O75 and other strains in the developed and developing countries have been well documented [12]–[14]. Bacterial lipopolysaccharides and their fragments have been used to prepare several glycoconjugate derivatives towards the development of long term therapeutic agents to eradicate the infections [15]–[17]. In order to establish a clear understanding on the biological potential of the lipooligosaccharide of a particular strain and its glycoconjugates, it is essential to carry out several biological experiments which require pure oligosaccharide in large quantity. Since the natural sources can not provide the required amount of the oligosaccharides, development of chemical synthetic strategies for their synthesis are relevant to get access to a reservoir of oligosaccharides and their close analogs. As a part of the ongoing studies on the synthesis of oligosaccharides of bacterial origin for their use in the preparation of glycoconjugate derivatives, concise chemical synthetic strategies for the synthesis of tetrasaccharide repeating unit (1) (Figure 2) corresponding to the lipopolysaccharide of Escherichia coli O75 and its close tetrasaccharide analog (2) (Figure 3) are reported herein. The difference between tetrasaccharides 1 and 2 is that the d-glucosamine moiety is 1,2-trans linked in compound 1 whereas it is 1,2-cis linked in compound 2. Both tetrasaccharides 1 and 2 were synthesized as their 4-methoxyphenyl (PMP) glycosides.

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Figure 2. Structure of the tetrasaccharide repeating unit corresponding to the O-specific lipopolysaccharide of Escherichia coli O75.

https://doi.org/10.1371/journal.pone.0037291.g002

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Figure 3. Structure of the tetrasaccharide analog related to the repeating unit of the O-specific lipopolysaccharide of Escherichia coli O75.

https://doi.org/10.1371/journal.pone.0037291.g003

Results and Discussion

The synthesis of the tetrasaccharide 1 and its close structural analog 2 as their 4-methoxyphenyl glycosides was achieved by series of stereoselective glycosylations of a number of suitably functionalized monosaccharide derivatives 4, 5 [18], 6, 7, 8 [19], and 9 [20] prepared from the commercially available reducing sugars using synthetic methodologies reported earlier. Since, the preparation of β-linked d-mannose moiety from d-mannose and α-d-glucosamine moiety from d-glucosamine are challenging issues, these two moieties are successfully prepared using β-d-glucosyl moiety and α-d-mannosyl moiety respectively as the precursors after completion of the glycosylations with required stereochemical outcome. The key features of this synthetic strategy include, (a) use of a common disaccharide derivative 11 for the preparation of both 1 and 2; (b) convenient conversion of β-d-glucoside moiety to β-d-mannoside moiety using Dess-Martin periodinane oxidation of C-2 followed by sodium borohydride reduction of the keto- group; (c) [2+2] stereoselective block glycosylation; (d) use of α-d-mannosidic moiety as a precursor for α-d-glucosamine moiety; (e) triflate formation followed by SN2 substitution by azido group at C-2 position of α-d-mannosidic moiety; (f) high yield in most of the intermediate steps.

4-Methoxyphenyl 3-O-allyl-2,6-di-O-benzyl-α-d-galactopyranoside (4) was prepared from 4-methoxyphenyl 2,3,4,6-tetra-O-acetyl-α-d-galactopyranoside (3) [21] using a series of reactions involving (a) deacetylation using sodium methoxide; (b) 3,4-O-isopropylidene ketal formation using 2,2-dimethoxypropane and p-toluenesulfonic acid [22]; (c) benzylation using benzyl bromide and sodium hydroxide [23]; (d) acidic hydrolysis of isopropylidene ketal and (e) selective 3-O-allylation via stannylidene acetal formation [24] in 70% overall yield (Scheme S1).

Stereo selective glycosylation of compound 4 with glucosyl trichloroacetimidate derivative 5 [18] in the presence of trifluoromethane sulfonic acid (TfOH) [25] furnished 4-methoxyphenyl (2-O-acetyl-3,4,6-tri-O-benzyl-β-d-glucopyranosyl)-(1→4)-3-O-allyl-2,6-di-O-benzyl-α-d-galactopyranoside (10) in 72% yield. Formation of compound 10 was confirmed from its spectral analysis [δ 5.24 (d, J = 3.5 Hz, H-1A), 4.61 (d, J = 8.0 Hz, H-1B) in the 1H NMR and δ 101.8 (C-1B), 97.4 (C-1A) in the 13C NMR spectra]. The coupling constant value (J1,2 = 8.0 Hz) unambiguously confirmed the β-linkage of the d-glucose moiety in compound 10. The d-glucopyranosyl moiety in compound 10 was converted into a d-mannopyranosyl moiety by epimerization at C-2. For this purpose, removal of acetyl group in compound 10 using sodium methoxide and oxidation of the hydroxyl group using Dess-Martin Periodinane [26] followed by reduction of the resulting keto group using sodium borohydride [27] resulted in the β-d-mannopyranosyl moiety, which was acetylated to give compound 11 in 76% overall yield. The spectral analysis of compound 11 supported its formation [δ 5.19 (d, J = 3.0 Hz, H-1A), 4.81 (br s, H-1B) in the 1H NMR and δ 99.1 (C-1B), 97.8 (C-1A) in the 13C NMR spectra]. The formation of β-d-mannosidic residue in compound 11 was unambiguously confirmed from the NMR spectral data [δ 4.81 (br s, H-1B) and δ 99.1 (C-1B) in the 1H NMR and 13C NMR spectra respectively]. Since the presence of β-d-glucosidic moiety in compound 10 was unambiguously confirmed from the NMR spectra and the β-d-mannosidic moiety of the compound 11 was prepared from the β-d-glucosidic moiety of compound 10 by oxidation-reduction at the C-2 center without affecting the stereochemistry at the glycosyl linkages, the glycosyl linkage of the d-mannosidic moiety in compound 11 remained as β-linked (1,2-cis). Compound 11 was treated with palladium chloride [28] to remove allyl group to give compound 12 in 76% yield. In another set of experiment, the O-acetyl group of compound 11 was transformed into benzyl group on treatment with benzyl bromide in the presence of solid sodium hydroxide [23] to give compound 13 in 90% yield, which was treated with palladium chloride [28] to furnish compound 14 in 78% yield. Spectral analysis of compound 14 supported its formation [δ 5.46 (d, J = 3.2 Hz, H-1A), 4.77 (br s, H-1B) in 1H NMR and δ 101.8 (C-1B), 96.8 (C-1A) in the 13C NMR spectra] (Scheme S2).

Stereoselective glycosylation of thioglycoside derivative 6 with thioglycoside derivative 7 in the presence of a combination of N-iodosuccinimide (NIS) and trifluoromethane sulfonic acid (TfOH) [29], [30] furnished disaccharide thioglycoside derivative 15 [31] in 77% yield exploiting the concept of “Relative reactivity values” of thioglycosides discussed in the earlier report [31] (Scheme S3).

Using block synthetic strategy, stereoselective glycosylation of disaccharide thioglycoside 15 with disaccharide acceptor 12 in the presence of a combination of NIS and TfOH [29], [30] furnished tetrasaccharide derivative 16 in 72% yield. Spectral analysis of compound 16 confirmed its formation [δ 5.49 (s, PhCH), 5.43 (d, J = 8.0 Hz, H-1C), 4.90 (br s, H-1B), 4.66 (d, J = 3.5 Hz, H-1A) in the 1H NMR and δ 102.1 (PhCH), 99.5 (C-1C), 98.8 (C-1B), 97.3 (2 C, C-1A, C-1D) in the 13C NMR spectra]. Compound 16 was subjected to a series of reactions involving (a) transformation of N-phthalimido group to acetamido group by hydrazinolysis [31] followed by N-acetylation; (b) removal of benzyl groups and benzylidene acetal by exhaustive hydrogenolysis over pearlman's catalyst [32] and finally, (c) saponification using sodium methoxide to furnish target tetrasaccharide 1 in 57% overall yield. Spectral analysis of compound 1 unambiguously supported its formation [ δ 5.30 (d, J = 8.5 Hz, H-1C), 5.11 (d, J = 3.5 Hz, H-1A), 4.80 (br s, H-1D), 4.46 (br s, H-1B) in the 1H NMR and δ 100.6 (C-1B), 100.2 (C-1D), 99.0 (C-1C), 98.4 (C-1A) in the 13C NMR spectra] (Scheme S4).

In another synthetic strategy for the synthesis of compound 2, a tetrasaccharide derivative 18 was prepared using a convergent reaction protocol, which involves iodonium ion promoted iterative stereoselective glycosylations in one-pot. Stereoselective condensation of thioglycoside 8 and thioglycoside 9 in the first step of the iterative glycosylation using NIS-TfOH [29], [30] as promoter furnished disaccharide thioglycoside derivative 17 following the principle of “armed-disarmed” glycosylation concept [33], [34]. Immediate reaction of the in situ generated disaccharide thioglycoside derivative 17 with the disaccharide acceptor 14 in the presence of same activator present in the reaction pot led to the formation of tetrasaccharide 18 in 71% overall yield together with a minor quantity (∼8%) of other isomeric product generated from the first step of glycosylation, which was separated by column chromatography. In the first step of the iterative glycosylation, although both compound 8 and 9 are thioethyl glycosides, presence of electron donating benzyl group at C-2 makes compound 9 activated or armed to act as glycosyl donor, whereas compound 8 acted as glycosyl acceptor because of the deactivation due to the presence of electron withdrawing O-acetyl group at C-2. In the second step, required 1,2-trans glycosylated product was obtained due to the presence of O-acetyl group at C-2 of the in situ formed disaccharide donor 17. The formation of tetrasaccharide derivative 18 was unambiguously confirmed from its spectral analysis [δ 5.58 (s, PhCH), 5.41 (d, J = 3.0 Hz, H-1A), 5.04 (br s, H-1D), 5.02 (br s, H-1C), 4.63 (br s, 1 H, H-1B) in the 1H NMR and δ 101.5 (C-1B), 101.1 (PhCH), 97.6 (C-1A), 95.0 (C-1D), 94.0 (C-1C) in the 13C NMR spectra]. The stereochemistry of the anomeric centers were further confirmed from the JC-1/H-1 values in the proton coupled 13C NMR spectrum [Appearance JC-1/H-1: 172.0 Hz (α-Rhap), 174.0 (α-Manp), 168.0 Hz (α-Galp), 156.0 Hz (β-Manp)] [35], [36]. The D-mannose moiety in compound 18 was converted to the d-glucosamine moiety following a series of reactions involving (a) removal of O-acetyl group using sodium methoxide; (b) treatment of the resulting hydroxyl group with triflic anhydride to form triflate derivative; (c) treatment of the triflate derivative with sodium azide to substitute triflate group with azido group by SN2 substitution [37]. Finally, removal of the benzyl groups and transformation of the azido group to acetamido group by hydrogenolysis followed by N-acetylation furnished compound 2 in 51% overall yield. Spectral analysis of compound 2 unambiguously supported its formation [δ 5.38 (d, J = 3.5 Hz, H-1A), 5.10 (d, J = 1.5 Hz, H-1C), 4.90 (d, J = 1.5 Hz, H-1D), 4.61 (br s, H-1B) in the 1H NMR and δ 102.6 (C-1B), 100.7 (C-1A), 99.6 (C-1C), 98.0 (C-1D) in the 13C NMR spectra] (Scheme S5).

In summary, synthesis of the tetrasaccharide motif (1) and its structural analog (2) corresponding to the O-specific lipopolysaccharide of Escherichia coli O75 was successfully achieved in excellent yield. A number of notable features are present in the synthetic strategies, which include (a) preparation of β-d-mannosyl moiety from β-d-glucosyl moiety using oxidation-stereoselective reduction approach; (b) preparation of α-d-glucosaminyl moiety from α-d-mannosyl moiety by SN2 substitution of triflate with azido group; (c) one-pot two iterative glycosylations; (d) [2+2] block glycosylation; (e) exploitation of orthogonal property of thioglycosides. Most of the reactions are clean and high yielding.

Materials and Methods

General methods

All reactions were monitored by thin layer chromatography over silica gel coated TLC plates. The spots on TLC were visualized by warming ceric sulphate (2% Ce(SO4)2 in 2N H2SO4) sprayed plates in hot plate. Silica gel 230–400 mesh was used for column chromatography. 1D and 2D NMR spectra were recorded on Bruker Avance 500 and 600 MHz spectrometer using CDCl3 and CD3OD as solvents and TMS as internal reference unless stated otherwise. Chemical shift value is expressed in δ ppm. ESI-MS were recorded on a Micromass mass spectrometer. Elementary analysis was carried out on Carlo Erba analyzer. Optical rotations were measured at 25 °C on a Jasco-P 2000 polarimeter. Commercially available organic solvents of adequate purity are used in all reactions.

4-Methoxyphenyl 3-O-allyl-2,6-di-O-benzyl-α-d-galactopyranoside (4)

A solution of compound 3 (5.0 g, 11.0 mmol) in 0.1 M CH3ONa in CH3OH (50 mL) was stirred at room temperature for 3 h and neutralized with Amberlite IR 120 (H+) resin. The reaction mixture was filtered and concentrated under reduced pressure. To a solution of the crude product in anhydrous DMF (15 mL) were added 2,2-dimethoxypropane (3 mL, 24.4 mmol) and p-TsOH (250.0 mg) and reaction mixture was allowed to stir at room temperature for 12 h. The reaction mixture was cooled to 0 °C and powdered NaOH (2.0 g, 50.0 mmol) was added to it followed by benzyl bromide (2.6 mL, 21.9 mmol) and the reaction was stirred at room temperature for 4 h. The reaction mixture was diluted with water (150 mL) and the extracted with EtOAc (2×100 mL). The organic layer was washed with water, dried (Na2SO4) and concentrated. A solution of the crude benzylated product in 80% AcOH (100 mL) was stirred at 80 °C for 1.5 h. The solvents were evaporated under reduced pressure and co-evaporated with toluene and the crude product was passed through a short pad of SiO2. To a solution of the dihydroxyl compound in anhydrous CH3OH (150 mL) was added Bu2SnO (4.0 g, 16.07 mmol) and the reaction was allowed to stir at 70 °C for 3 h. The solvents were removed under reduced pressure and the stannylidene acetal was dissolved in dry DMF (10 mL). To the solution of the crude product were added allyl bromide (1.4 mL, 16.2 mmol) and Bu4NBr (500 mg) and the reaction mixture was allowed to stir at 60 °C for 8 h. The reaction mixture was diluted with water and extracted with EtOAc (2×100 mL). The organic layer was washed with 1 N HCl, satd. NaHCO3 and water in succession, dried (Na2SO4) and concentrated. The crude product was purified over SiO2 using hexane-EtOAc (4∶1) as eluant to give pure compound 4 (3.9 g, 70%). Yellow oil; [α]D25+110.5 (c 1.2, CHCl3); IR (neat): 3475, 2928, 1504, 1216, 1097, 1040, 756, 698 cm−1; 1H NMR (CDCl3, 500 MHz): δ 7.34-7.23 (m, 10 H, Ar-H), 7.03 (d, J = 9.0 Hz, 2 H, Ar-H), 6.77 (d, J = 9.0 Hz, 2 H, Ar-H), 6.00-5.93 (m, 1 H, CH = CH2), 5.35-5.31 (m, 1 H, CH = CH2), 5.33 (d, J = 3.0 Hz, 1 H, H-1), 5.21-5.19 (m, 1 H, CH = CH2), 4.80 (d, J = 12.0 Hz, 1 H, PhCH2), 4.67 (d, J = 12.0 Hz, 1 H, PhCH2), 4.49 (s, 2 H, PhCH2), 4.29-4.25 (m, 2 H, H-5, O-CH2-), 4.15-4.12 (m, 2 H, H-4, O-CH2), 3.94-3.93 (m, 2 H, H-6ab), 3.77 (dd, J = 10.0, 5.4 Hz, 1 H, H-2), 3.73 (s, 3 H, OCH3), 3.66 (dd, J = 10.0, 6.1 Hz, 1 H, H-3); ESI-MS: 529.2 [M+Na]+; Anal. Calcd. for C30H34O7 (506.23): C, 71.13; H, 6.76%; found: 70.90; H, 7.00%.

4-Methoxyphenyl (2-O-acetyl-3,4,6-tri-O-benzyl-β-d-glucopyranosyl)-(1→4)-3-O-allyl-2,6-di-O-benzyl-α-d-galactopyranoside (10)

A solution of compound 4 (2.0 g, 3.95 mmol) and compound 5 (3.0 g, 4.71 mmol) in anhydrous CH2Cl2 (25 mL) was cooled to −25°C. To the cooled reaction mixture was added TfOH (100 µL) and the reaction mixture was allowed to stir at same temperature for 1 h. The reaction mixture was diluted with CH2Cl2 (150 mL) and successively washed with satd. NaHCO3 and water, dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified over SiO2 using hexane-EtOAc (4∶1) as eluant to give pure compound 10 (2.8 g, 72%). Colorless oil; [α]D25+38.8 (c 1.2, CHCl3); IR (neat): 2823, 2260, 1542, 1344, 1152, 1213, 1104, 1070, 737, 567 cm−1; 1H NMR (CDCl3, 500 MHz): δ 7.28-7.10 (m, 25 H, Ar-H), 6.95 (d, J = 9.0 Hz, 2 H, Ar-H), 6.67 (d, J = 9.0 Hz, 2 H, Ar-H), 5.91-5.80 (m, 1 H, CH = CH2), 5.32-5.28 (m, 1 H, CH = CH2), 5.24 (d, J = 3.5 Hz, 1 H, H-1A), 5.11-5.08 (m, 1 H, CH = CH2), 4.94 (t, J = 9.5 Hz each, 1 H, H-2B), 4.74-4.63 (4 d, J = 11.8 Hz each, 4 H, PhCH2), 4.61 (d, J = 8.0 Hz, 1 H, H-1B), 4.56 (d, J = 11.5 Hz, 1 H, PhCH2), 4.51 (d, J = 11.5 Hz, 1 H, PhCH2), 4.45 (d, J = 12.0 Hz, 1 H, PhCH2), 4.38 (d, J = 12.0 Hz, 1 H, PhCH2), 4.35 (d, J = 12.0 Hz, 1 H, PhCH2), 4.33 (d, J = 12.0 Hz, 1 H, PhCH2), 4.27-4.10 (m, 2 H, O-CH2), 4.08-4.06 (m, 2 H, H-4A, H-5A), 3.84 (dd, J = 10.0, 2.5 Hz, 1 H, H-2A), 3.80 (dd, J = 10.0, 3.5 Hz, 1 H, H-3A), 3.77-3.74 (m, 1 H, H-6aB), 3.68 (dd, J = 9.5, 3.5 Hz, 1 H, H-3B), 3.67 (s, 3 H, OCH3), 3.65-3.56 (m, 4 H, H-4B, H-6bB, H-6abA), 3.39-3.37 (m, 1 H, H-5B), 1.92 (s, 3 H, OCH3); 13C NMR (CDCl3, 125 MHz): δ 169.8 (COCH3), 155.1-114.4 (Ar-C, CH = CH2), 101.8 (C-1B), 97.4 (C-1A), 83.0 (C-3B), 77.8 (C-4B), 77.6 (C-5B), 76.7 (C-4A), 75.3 (C-2A), 75.1 (PhCH2), 75.0 (PhCH2), 74.8 (C-3A), 73.8 (C-2B), 73.5 (PhCH2), 73.4 (PhCH2), 73.0 (PhCH2), 71.9 (O-CH2), 70.2 (C-5B), 71.1 (C-6B), 69.0 (C-6A), 55.6 (OCH3), 21.2 (COCH3); ESI-MS: 1003.4 [M+Na]+; Anal. Calcd. for C59H64O13 (980.43): C, 72.23; H, 6.57%; found: 72.00; H, 6.85%.

4-Methoxyphenyl (2-O-acetyl-3,4,6-tri-O-benzyl-β-d-mannopyranosyl)-(1→4)-3-O-allyl-2,6-di-O-benzyl-α-d-galactopyranoside (11)

A solution of compound 10 (2.5 g, 2.55 mmol) in 0.1 M CH3ONa in CH3OH (25 mL) was stirred at room temperature for 2 h and neutralized with Amberlite IR 120 (H+) resin. The reaction mixture was filtered and concentrated. To a solution of the deacetylated product in anhydrous CH2Cl2 (15 mL) was added Dess-Martin Periodinane (2.0 g, 4.72 mmol) and the reaction mixture was allowed to stir at room temperature for 1 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and the organic layer was successively washed with 5% Na2S2O3, satd. NaHCO3 and water, dried (Na2SO4) and concentrated under reduced pressure. To a solution of the crude keto product in CH3OH (50 mL) was added NaBH4 (1.5 g, 39.65 mmol) and the reaction mixture was allowed to stir at room temperature for 12 h. The solvents were removed under reduced pressure and the crude mass was dissolved in CH2Cl2 (100 mL). The organic layer was successively washed with 1 N HCl, satd. NaHCO3 and water, dried (Na2SO4) and evaporated to dryness. A solution of the crude epimerized product in acetic anhydride-pyridine (10 mL, 1∶1 v/v) was kept at room temperature for 2 h. The solvents were removed under reduced pressure and the crude product was purified over SiO2 using hexane-EtOAc (5∶1) as eluant to give pure compound 11 (1.9 g, 76%). Yellow oil; [α]D25+27.6 (c 1.2, CHCl3); IR (neat): 2818, 2220, 1549, 1365, 1159, 1235, 1100, 739 cm−1; 1H NMR (CDCl3, 125 MHz): δ 7.33-7.11 (m, 25 H, Ar-H), 6.94 (d, J = 9.0 Hz, 2 H, Ar-H), 6.66 (d, J = 9.0 Hz, 2 H, Ar-H), 5.84-5.78 (m, 1 H, CH = CH2), 5.72 (d, J = 2.5 Hz, 1 H, H-2B), 5.23-5.20 (m, 1 H, CH = CH2), 5.19 (d, J = 3.0 Hz, 1 H, H-1A), 5.05-5.03 (m, 1 H, CH = CH2), 4.81 (br s, 1 H, H-1B), 4.78-4.31 (10 d, J = 11.8 Hz each, 10 H, PhCH2), 4.26-4.23 (m, 1 H, O-CH2), 4.19 (br s, 1 H, H-4A), 4.10-4.06 (m, 2 H, H-5A, O-CH2), 3.93 (dd, J = 10.0, 3.5 Hz, 1 H, H-2A), 3.88 (dd, J = 10.0, 2.5 Hz, 1 H, H-3A), 3.71-3.63 (m, 4 H, H 4B, H-6abA, H-6aB), 3.64 (s, 3 H, OCH3), 3.58-3.53 (m, 2 H, H-3B, H-6bB), 3.37-3.34 (m, 1 H, H-5B), 2.09 (s, 3 H, COCH3); 13C NMR (CDCl3, 125 MHz): δ 170.6 (COCH3), 155.1-114.4 (Ar-C, CH = CH2), 99.1 (C-1B), 97.8 (C-1A), 80.4 (C-3B), 77.9 (C-3A), 76.4 (C-2A), 75.6 (C-5B), 75.2 (PhCH2), 74.4 (C-4B), 74.2 (C-4A), 73.7 (PhCH2), 73.5 (PhCH2), 73.0 (PhCH2), 71.9 (PhCH2), 71.4 (PhCH2), 70.0 (C-5A), 69.9 (C-6B), 69.4 (C-6A), 68.1 (C-2B), 55.6 (OCH3), 21.1 (COCH3); ESI-MS: 1003.4 [M+Na]+; Anal. Calcd. for C59H64O13 (980.43): C, 72.23; H, 6.57%; found: 72.04; H, 6.80%.

4-Methoxyphenyl (2-O-acetyl-3,4,6-tri-O-benzyl-β-d-mannopyranosyl)-(1→4)-2,6-di-O-benzyl-α-d-galactopyranoside (12)

To a solution of compound 11 (900.0 mg, 0.92 mmol) in anhydrous CH3OH (5 mL) was added PdCl2 (100.0 mg, 0.56 mmol) and the reaction mixture was allowed to stir at room temperature for 1 h. The solvents were removed under reduced pressure and the crude product was purified over SiO2 using hexane-EtOAc (4∶1) to give pure compound 12 (660.0 mg, 76%). Yellow oil; [α]D25+40 (c 1.2, CHCl3); IR (neat): 2834, 2297, 1534, 1371, 1156, 1218, 1080, 736 cm−1; 1H NMR (CDCl3, 500 MHz): δ 7.35-7.0 (m, 25 H, Ar-H), 6.91 (d, J = 9.0 Hz, 2 H, Ar-H), 6.67 (d, J = 9.0 Hz, 2 H, Ar-H), 5.62 (d, J = 3.0 Hz, 1 H, H-2B), 5.21 (d, J = 3.5 Hz, 1 H, H-1A), 4.78 (d, J = 10.5 Hz, 1 H, PhCH2), 4.69 (br s, 1 H, H-1B), 4.67-4.61 (3 d, J = 11.0 Hz each, 3 H, PhCH2), 4.50-4.23 (6 d, J = 11.0 Hz each, 6 H, PhCH2), 4.13-4.11 (m, 2 H, H-4A, H-5A), 4.09-4.06 (m, 1 H, H-6aA), 3.76-3.68 (m, 4 H, H-2A, H-4B, H-6aB, H-6bA), 3.67 (s, 3 H, OCH3), 3.65-3.61 (m, 1 H, H-3B), 3.59 (dd, J = 10.0, 2.6 Hz, 1 H, H-3A), 3.54-3.51 (m, 1 H, H-6bB), 3.34-3.31 (m, 1 H, H-5B), 2.10 (s, 3 H, COCH3); 13C NMR (CDCl3, 125 MHz): δ 171.5 (COCH3), 155.1-114.5 (Ar-C), 100.5 (C-1B), 96.9 (C-1A), 80.1 (C-3B), 78.5 (C-3A), 76.6 (C-2A), 75.5 (C-5B), 75.2 (PhCH2), 74.3 (C-4B), 73.5 (C-4A), 73.0 (PhCH2), 72.8 (PhCH2), 71.5 (PhCH2), 69.8 (PhCH2), 69.5 (C-5A), 69.4 (C-6B), 69.3 (C-6A), 68.7 (C-2B), 55.6 (OCH3), 21.3 (COCH3); ESI-MS: 963.4 [M+Na]+; Anal. Calcd. for C56H60O13 (940.40): C, 71.47; H, 6.43%; found: 71.26; H, 6.60%.

4-Methoxyphenyl (2,3,4,6-tetra-O-benzyl-β-d-mannopyranosyl)-(1→4)-3-O-allyl-2,6-di-O-benzyl-α-d-galactopyranoside (13)

To a solution of compound 11 (900.0 mg, 0.92 mmol) in THF (5 mL) were added powdered NaOH (0.2 g, 5.0 mmol), benzyl bromide (250 µL, 2.10 mmol) and Bu4NBr (25.0 mg) and reaction mixture was allowed to stir at room temperature for 3 h. The reaction mixture was diluted with CH2Cl2 (50 mL) and the organic layer was washed with water, dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified over SiO2 using hexane-EtOAc (7∶1) as eluant to give pure compound 13 (850.0 mg, 90%). Yellow oil; [α]D25+20 (c 1.2, CHCl3); IR (neat): 3443, 2923, 2860, 1508, 1454, 1362, 1213, 1114, 1070, 1027, 737, 697 cm−1; 1H NMR (CDCl3, 500 MHz): δ.7.55-7.14 (m, 30 H, Ar-H), 7.10 (d, J = 9.0 Hz, 2 H, Ar-H), 6.75 (d, J = 9.0 Hz, 2 H, Ar-H), 5.90-5.84 (m, 1 H, CH = CH2), 5.40 (d, J = 3.0 Hz, 1 H, H-1A), 5.25-5.09 (m, 2 H, CH = CH2), 4.93 (d, J = 12.0 Hz, 1 H, PhCH2), 4.89 (d, J = 12.0 Hz, 1 H, PhCH2), 4.79 (d, J = 12.0 Hz, 1 H, PhCH2), 4.76 (br s, 1 H, H-1B), 4.74 (d, J = 12.0 Hz, 1 H, PhCH2), 4.68 (d, J = 12.0 Hz, 1 H, PhCH2), 4.61 (d, J = 12.0 Hz, 1 H, PhCH2), 4.54 (d, J = 12.0 Hz, 1 H, PhCH2), 4.52 (d, J = 12.0 Hz, 1 H, PhCH2), 4.48 (d, J = 12.0 Hz, 1 H, PhCH2), 4.39 (br s, 2 H, PhCH2), 4.30-4.28 (m, 1 H, OCH2-), 4.27 (br s, 1 H, H-4A), 4.25-4.23 (m, 1 H, H-5A), 4.15-4.12 (m, 1 H, OCH2-), 4.01 (d, J = 2.4 Hz, 1 H, H-2B), 3.97 (dd, J = 10.2, 3.0 Hz, 1 H, H-3B), 3.93-3.88 (m, 2 H, H-2A, H-4B), 3.85 (dd, J = 10.8, 4.2 Hz, 1 H, H-6aB), 3.79-3.75 (m, 3 H, H-6bB, H-6abA), 3.73 (s, 3 H, OCH3), 3.51 (dd, J = 9.6, 3.0 Hz, 1 H, H-3B), 3.42-3.40 (m, 1 H, H-5B); 13C NMR (CDCl3, 125 MHz): δ 155.6-114.4 (Ar-C, -CH = CH2), 101.6 (C-1B), 97.8 (C-1A), 82.5 (C-3B), 77.9 (C-3A), 76.0 (C-2A), 75.9 (C-5B), 75.2 (PhCH2), 74.8 (C-4B), 73.9 (PhCH2), 73.8 (C-2B), 73.7 (C-4A), 73.4 (PhCH2), 73.1 (PhCH2), 73.0 (PhCH2), 71.7 (OCH2-), 71.4 (PhCH2), 70.4 (C-6B), 70.3 (C-4A), 69.7 (C-6A), 55.5 (OCH3); ESI-MS: 1051.4 [M+Na]+; Anal. Calcd. for C64H68O12 (1028.47): C, 74.69; H, 6.66%; found: 74.90; H, 6.90%.

4-Methoxyphenyl (2,3,4,6-tetra-O-benzyl-β-d-mannopyranosyl)-(1→4)-2,6-di-O-benzyl-α-d-galactopyranoside (14)

To a solution of compound 13 (800.0 mg, 0.78 mmol) in anhydrous CH3OH (5 mL) was added PdCl2 (90.0 mg, 0.51 mmol) and the reaction mixture was allowed to stir at room temperature for 1 h. The solvents were removed under reduced pressure and crude product was purified over SiO2 using hexane-EtOAc (4∶1) to give pure compound 14 (600.0 mg, 78%). White solid; m.p. 132–133 °C (EtOH); [α]D25+38 (c 1.2, CHCl3); IR (KBr): 3440, 2936, 2854, 1512, 1466, 1345, 1209, 1121, 1078, 1037, 697 cm−1; 1H NMR (CDCl3, 500 MHz): δ 7.47-7.20 (m, 30 H, Ar-H), 7.08 (d, J = 9.0 Hz, 2 H, Ar-H), 6.77 (d, J = 9.0 Hz, 2 H, Ar-H), 5.46 (d, J = 3.2 Hz, 1 H, H-1A), 4.91-4.85 (3 d, J = 12.0 Hz each, 3 H, PhCH2), 4.77 (br s, 1 H, H-1B), 4.69 (d, J = 11.5 Hz, 1 H, PhCH2), 4.62-4.52 (m, 5 H, PhCH2), 4.47 (d, J = 11.5 Hz, 1 H, PhCH2), 4.43 (br s, 2 H, PhCH2), 4.25-4.22 (m, 2 H, H-3A, H-5A), 4.21 (br s, 1 H, H-4A), 3.97 (d, J = 2.6 Hz, 1 H, H-2B), 3.93-3.74 (m, 6 H, H-2A, H-4B, H-6abA, H-6abB), 3.73 (s, 3 H, OCH3), 3.54 (dd, J = 9.2, 2.7 Hz, 1 H, H-3B), 3.48-3.46 (m, 1 H, H-5B); 13C NMR (CDCl3, 125 MHz): δ 155.3-114.6 (Ar-C), 101.8 (C-1B), 96.8 (C-1A), 82.6 (C-3B), 75.8 (C-5B), 75.3 (C-2A), 75.0 (PhCH2), 74.9 (PhCH2), 74.0 (C-4B), 73.7 (C-3A), 73.5 (PhCH2), 73.0 (PhCH2), 72.5 (C-2B), 71.7 (PhCH2), 70.7 (C-4A), 70.5 (C-5A), 69.8 (C-6B), 69.7 (C-6A), 55.6 (OCH3); ESI-MS: 1011.4 [M+Na]+; Anal. Calcd. for C61H64O12 (988.44): C, 74.07; H, 6.52%; found: 74.25; H, 6.75%.

4-Methoxyphenyl (2,3,4-tri-O-acetyl-α-l-rhamnopyranosyl)-(1→3)-(4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)-[2-O-acetyl-3,4,6-tri-O-benzyl-β-d-mannopyranosyl-(1→4)]-2,6-di-O-benzyl-α-d-galactopyranoside (16)

To a solution of compound 12 (600.0 mg, 0.64 mmol) and compound 15 (550.0 mg, 0.77 mmol) in anhydrous CH2Cl2 (10 mL) was added MS 4 Å (1.5 g) and the reaction mixture was stirred under argon at room temperature for 30 min. The reaction mixture was cooled to −30 °C and NIS (210.0 mg, 0.93 mmol) and TfOH (2 µL) were added to it. After stirring at same temperature for 1 h the reaction mixture was filtered through a Celite® bed and washed with CH2Cl2 (50 mL). The organic layer was successively washed with 5% Na2S2O3, satd. NaHCO3 and water, dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified over SiO2 using hexane-EtOAc (4∶1) as eluant to give pure compound 16 (730 mg, 72%). White solid; m.p. 98–100 °C (EtOH); [α]D25+10 (c 1.2, CHCl3); IR (KBr): 2929, 1745, 1454, 1373, 1233, 1100, 1069, 754, 698 cm−1; 1H NMR (CDCl3, 500 MHz): δ 7.76-6.99 (m, 34 H, Ar-H), 6.72 (d, J = 9.0 Hz, 2 H, Ar-H), 6.55 (d, J = 9.0 Hz, 2 H, Ar-H), 5.72-5.71 (m, 1 H, H-2B), 5.49 (s, 1 H, PhCH), 5.43 (d, J = 8.0 Hz, 1 H, H-1C), 5.15 (dd, J = 10.0, 3.5 Hz, 1 H, H-3D), 5.03 (d, J = 11.0 Hz, 1 H, PhCH2), 4.90 (br s, 1 H, H-1B), 4.86-4.83 (m, 2 H, PhCH2), 4.80 (t, J = 9.5 Hz each, 1 H, H-3C), 4.75 (t, J = 10.0 Hz each, 1 H, H-4D), 4.66 (d, J = 3.5 Hz, 1 H, H-1A), 4.64-4.63 (m, 1 H, H-2D), 4.53-4.50 (2 d, J = 11.0 Hz each, 2 H, PhCH2), 4.48 (br s, 1 H, H-1D), 4.40 (d, J = 11.0 Hz, 1 H, PhCH2), 4.32 (dd, J = 10.5, 2.0 Hz, 1 H, H-6aC), 4.28-4.21 (2 d, J = 12.0 Hz each, 2 H, PhCH2), 4.20-4.15 (m, 3 H, H-2C, PhCH2), 4.08-4.02 (m, 3 H, H-3B, H-5A, H-6bC), 3.96-3.90 (m, 1 H, H-5D), 3.75-3.63 (m, 7 H, H-4A, H-4B, H-5C, H-6abA, H-6abB), 3.62 (s, 3 H, OCH3), 3.61-3.58 (m, 1 H, H-4C), 3.56 (dd, J = 10.0, 2.5 Hz, 1 H, H-3A), 3.52-3.48 (m, 2 H, H-2A, H-5B), 1.97, 1.86, 1.80, 1.72 (4 s, 12 H, 4 COCH3), 0.52 (d, J = 6.0 Hz, 3 H, CCH3); 13C NMR (CDCl3, 125 MHz): δ 170.7, 169.9, 169.5 (4 COCH3), 168.6, 167.8 (PhthCO), 155.1-114.3 (Ar-C), 102.1 (PhCH), 99.5 (C-1C), 98.8 (C-1B), 97.3 (2 C, C-1A, C-1D), 80.6 (C-5A), 80.5 (C-5C), 76.6 (C-3B), 75.9 (C-3A), 75.4 (C-3C), 75.3 (C-2A), 75.0 (PhCH2), 74.6 (C-5B), 73.8 (C-4B), 73.7 (PhCH2), 73.6 (PhCH2), 72.8 (PhCH2), 71.8 (PhCH2), 71.3 (C-4D), 70.4 (C-4C), 70.2 (C-6B), 69.8 (2 C, C-2D, C-6C), 68.7 (C-6A), 68.4 (C-3D), 68.3 (C-2B), 66.4 (C-4A), 66.2 (C-4D), 56.9 (C-2C), 55.5 (OCH3), 21.1, 20.8, 20.7, 20.6 (COCH3), 16.5 (CCH3); ESI-MS: 1614.6 [M+Na]+; Anal. Calcd. for C89H93NO26 (1591.60): C, 67.12; H, 5.89%; found: 66.92; H, 6.12%.

4-Methoxyphenyl (2,3,4-tri-O-benzyl-α-l-rhamnopyranosyl)-(1→3)-(2-O-acetyl-4,6-O-benzylidene-α-d-mannopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-β-d-mannopyranosyl-(1→4)]-2,6-di-O-benzyl-α-d-galactopyranoside (18)

To a solution of compound 8 (275.0 mg, 0.77 mmol) and compound 9 (375.0 mg, 0.78 mmol) in anhydrous CH2Cl2 (5 mL) was added MS 4 Å (1.0 g) and the reaction mixture was stirred under argon at room temperature for 30 min. The reaction mixture was cooled to −30 °C and NIS (190.0 mg, 0.84 mmol) and TfOH (5 µL) were added to it and the reaction was stirred at same temperature for 30 min. Thin layer chromatography (TLC; hexane-EtOAc, 5∶1) showed complete disappearance of the starting materials. To the reaction mixture were added compound 14 (650.0 mg, 0.66 mmol) and NIS (140 mg, 0.62 mmol) and the stirring reaction mixture was kept at same temperature for another 30 min. The reaction mixture was filtered through a Celite® bed and washed with CH2Cl2 (50 mL). The organic layer was successively washed with 5% Na2S2O3, satd. NaHCO3 and water, dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified over SiO2 using hexane-EtOAc (4∶1) as eluant to give pure compound 18 (700.0 mg, 71%). White solid; m.p. 58–60 °C (EtOH); [α]D25+16 (c 1.2, CHCl3); IR (KBr): 2934, 1757, 1462, 1384, 1243, 1108, 1078, 757, 699 cm−1; 1H NMR (CDCl3, 500 MHz): δ 7.47-7.07 (m, 50 H, Ar-H), 6.77 (d, J = 9.0 Hz, Ar-H), 5.58 (s, 1 H, PhCH), 5.41 (d, J = 3.0 Hz, 1 H, H-1A), 5.28 (br s, 1 H, H-2C), 5.04 (br s, 1 H, H-1D), 5.02 (br s, 1 H, H-1C), 4.98-4.82 (m, 4 H, PhCH2), 4.71 (d, J = 12.0 Hz, PhCH2), 4.63 (br s, 1 H, H-1B), 4.62-4.46 (m, 11 H, PhCH2), 4.40-4.34 (m, 4 H, H-3C, H-5C, PhCH2), 4.32-4.28 (m, 3 H, H-4A, H-3D, H- 6aC), 4.26-4.22 (m, 1 H, H-5A), 4.13 (br s, 1 H, H-2B), 4.10-4.04 (m, 1 H, H-5D), 3.95-3.83 (m, H-2A, H-3A, H-4B, H-6aB, H-6bC), 3.82-3.76 (m, 4 H, H-4C, H-6bB, H-6abA), 3.75 (s, 3 H, OCH3), 3.70-3.66 (m, 1 H, H-3B), 3.63 (br s, 1 H, H-2D), 3.59-3.50 (m, 2 H, H-4D, H-5B), 2.06 (s, 3 H, COCH3), 1.07 (d, J = 5.8 Hz, 3 H, CCH3); 13C NMR (CDCl3, 125 MHz): δ 170.0 (COCH3), 155.3-114.4 (Ar-C), 101.5 (C-1B), 101.1 (PhCH), 97.6 (C-1A), 95.0 (C-1D), 94.0 (C-1C), 83.2 (C-2D), 80.4 (C-4D), 79.5 (C-4C), 76.5 (C-3D), 75.6 (C-5B), 74.9 (C-2A), 74.7 (C-3A), 74.6 (C-2B), 74.5 (C-3B), 74.4 (PhCH2), 74.3 (2 C, C-4B, PhCH2), 73.4 (PhCH2), 73.0 (PhCH2), 72.9 (2 C, 2 PhCH2), 72.2 (PhCH2), 71.9 (PhCH2), 71.8 (PhCH2), 71.5 (C-4A), 70.5 (C-3C), 70.1 (C-5A), 69.9 (C-6A), 68.8 (C-6B), 68.5 (C-6C), 68.0 (2 C, C-2C, C-5D), 63.9 (C-5C), 55.6 (OCH3); ESI-MS: 1719.7 [M+Na]+; Anal. Calcd. for C103H108O22 (1696.73): C, 72.86; H, 6.41%; found: 73.04; H, 6.65%.

4-Methoxyphenyl (α-l-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→3)-[β-d-mannopyranosyl-(1→4)]-α-d-galactopyranoside (1)

To a solution of compound 16 (700.0 mg, 0.44 mmol) in EtOH (30 mL) was added hydrazine monohydrate (0.3 mL, 6.18 mmol) and the reaction mixture was allowed to stir at 80 °C for 6 h. After removal of the solvents the crude mass was dissolved in acetic anhydride-pyridine (5 mL; 1∶1 v/v) and kept at room temperature for 1 h. The solvents were removed under reduced pressure to give the crude acetylated product. To a solution of the crude mass in CH3OH (15 mL) was added 20% Pd(OH)2-C (200.0 mg) and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was filtered through a Celite® bed and evaporated to dryness. A solution of the crude product in 0.1 M CH3ONa in CH3OH (15 mL) was allowed to stir at room temperature for 4 h and neutralized with Dowex 50W X8 (H+) resin. The reaction mixture was filtered and concentrated to dryness to give compound 1, which was passed through a Sephadex® LH-20 column using CH3OH-H2O (4∶1) as eluant to give pure compound 1 (200.0 mg, 57%). Glass; [α]D25+46 (c 1.2, CH3OH); IR (KBr): 2929, 1763, 1458, 1392, 1257, 1100, 1082, 759, 697 cm−1; 1H NMR (CD3OD, 500 MHz): δ 6.85 (d, J = 9.0 Hz, 2 H, Ar-H), 6.77 (d, J = 9.0 Hz, 2 H, Ar-H), 5.30 (d, J = 8.5 Hz, 1 H, H-1C), 5.11 (d, J = 3.5 Hz, 1 H, H-1A), 4.80 (br s, 1 H, H-1D), 4.46 (br s, 1 H, H-1B), 4.33 (dd, J = 8.5 Hz each, 1 H, H-3C), 4.24 (br s, 1 H, H-2D), 4.06 (dd, J = 10.0, 2.5 Hz, H-3D), 3.93 (t, J = 8.5 Hz each, 1 H, H-2C), 3.48-3.80 (m, 1 H, H-6aB), 3.78-3.74 (m, 3 H, H-5A, H-5D, H-6bB), 3.66-3.56 (m, 2 H, H-4A, H-6aA), 3.60 (s, 3 H, OCH3), 3.55-3.51 (m, 4 H, H-2A, H-3B, H-6ac, H-6bA), 3.49-3.42 (m, 3 H, H-3A, H-5C, H-6bC), 3.40-3.36 (m, 2 H, H-2B, H-4C), 3.25-3.20 (m, 1 H, H-5B), 3.71 (t, J = 9.0 Hz each, 1 H, H-4D), 2.04 (s, 3 H, COCH3), 1.02 (d, J = 6.0 Hz, 3 H, CCH3); 13C NMR (CD3OD, 125 MHz): δ 173.0 (COCH3), 154.6-114.9 (Ar-C), 100.6 (C-1B), 100.2 (C-1D), 99.0 (C-1C), 98.4 (C-1A), 77.9 (C-2C), 77.0 (C-3D), 76.1 (2 C, C-2D, C-5B), 75.6 (C-3A), 73.1 (C-4D), 71.8 (C-4C), 70.9 (C-3B), 70.6 (2 C, C-4A, C-4B), 70.0 (C-2B), 69.4 (C-5D), 68.8 (C-5C), 67.8 (C-5A), 67.1 (C-2A), 61.1 (C-6B), 60.8 (C-6A), 60.5 (C-6C), 56.4 (C-2C), 55.8 (OCH3), 23.2 (COCH3), 16.4 (CCH3); ESI-MS: 820.3 [M+Na]+; Anal. Calcd. for C33H51NO21 (797.30): C, 49.68; H, 6.44%; found: 49.46; H, 6.69%.

4-Methoxyphenyl (α-l-rhamnopyranosyl)-(1→3)-(2-acetamido-2-deoxy-α-d-glucopyranosyl)-(1→3)-[β-d-mannopyranosyl-(1→4)]-α-d-galactopyranoside (2)

A solution of compound 18 (650.0 mg, 0.38 mmol) in 0.1 M CH3ONa in CH3OH (10 mL) was allowed to stir at room temperature for 1 h and neutralized with Amberlite IR 120 (H+) resin. The reaction mixture was filtered and concentrated. To a solution of the deacetylated product in anhydrous CH2Cl2 (5 mL) were added pyridine (2 mL) and triflic anhydride (200 µL, 1.19 mmol) and the reaction mixture was stirred at −10°C for 1 h. The solvents were removed under reduced pressure and triflate derivative was dissolved in HMPT-DMF (6 mL, 2∶1 v/v). To the solution of the triflate derivative was added NaN3 (500.0 mg, 7.69 mmol) and the reaction mixture was allowed to stir at 90 °C for 8 h. The reaction mixture was diluted with water and extracted with EtOAc (100 mL). The organic layer was successively washed with satd. NaHCO3 and water, dried (Na2SO4) and concentrated to give the crude product, which was passed through a short pad of SiO2. To a solution of the azido derivative in CH3OH (15 mL) was added 20% Pd(OH)2-C (200.0 mg) and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was filtered through a Celite® bed and evaporated to dryness. To a solution of the crude product in CH3OH (10 mL) was added acetic anhydride (2 mL) and the reaction mixture was allowed to stir at room temperature for 1 h. The solvents were removed under reduced pressure to give compound 2, which was passed through a Sephadex® LH-20 column using CH3OH-H2O (4∶1) as eluant to give pure compound 2 (155.0 mg, 51%). White powder; [α]D25+32 (c 1.2, CH3OH); IR (KBr): 2932, 1757, 1452, 1388, 1233, 1113, 1086, 759, 697 cm−1; 1H NMR (CD3OD, 500 MHz): δ 7.10 (d, J = 9.0 Hz, 2 H, Ar-H), 6.80 (d, J = 9.0 Hz, 2 H, Ar-H), 5.38 (d, J = 3.5 Hz, 1 H, H-1A), 5.10 (d, J = 1.5 Hz, 1 H, H-1C), 4.90 (d, J = 1.5 Hz, 1 H, H-1D), 4.61 (br s, 1 H, H-1B), 4.43 (d, J = 2.5 Hz, 1 H, H-2D), 4.20 (dd, J = 10.5, 3.0 Hz, 1 H, H-3A), 4.09 (dd, J = 10.0, 3.5 Hz, 1 H, H-2A), 4.06-4.02 (m, 1 H, H-5A), 4.01-3.99 (m, 2 H, H-3B, H-5D), 3.98-3.95 (m, 1 H, H-3D), 3.94-3.92 (m, 3 H, H-3C, H-4B, H-6aB), 3.91-3.89 (m, 3 H, H-5C, H-6aA, H-6aC), 3.81 (dd, J = 9.0, 3.0 Hz, 1 H, H-2C), 3.77-3.74 (m, 2 H, H-4C, H-6bA), 3.73 (s, 3 H, OCH3), 3.65 (dd, J = 11.5, 8.0 Hz, 1 H, H-6bC), 3.54 (dd, J = 11.0, 6.0 Hz, 1 H, H-6bB), 3.50-3.46 (m, 2 H, H-2B, H-4A), 3.41 (t, J = 9.5 Hz each, 1 H, H-4D), 3.29-3.24 (m, 1 H, H-5B), 1.99 (s, 3 H, COCH3), 1.29 (d, J = 6.5 Hz, 3 H, CCH3); 13C NMR (CD3OD, 125 MHz): δ 169.8 (COCH3), 156.7-115.5 (Ar-C), 102.6 (C-1B), 100.7 (C-1A), 99.6 (C-1C), 98.0 (C-1D), 78.2 (C-5B), 77.4 (C-3A), 76.3 (C-3B), 75.1 (C-2B), 74.6 (C-5A), 74.2 (C-4D), 73.9 (C-2D), 72.3 (C-2C), 72.2 (2 C, C-3C, C-5D), 72.1 (C-4B), 70.1 (C-5C), 69.0 (C-4A), 68.8 (C-2A), 68.2 (C-3D), 66.7 (C-4C), 63.2 (C-6B), 62.9 (C-6A), 61.3 (C-6C), 56.0 (OCH3), 22.4 (COCH3), 18.1 (CCH3); ESI-MS: 820.3 [M+Na]+; Anal. Calcd. for C33H51NO21 (797.30): C, 49.68; H, 6.44%; found: 49.44; H, 6.70%.

Supporting Information

Scheme S1.

Reagents: (a) 0.1 M CH3ONa, CH3OH, room temperature, 3 h; (b) 2,2-dimethoxypropane, p-TsOH, DMF, room temperature, 12 h; (c) benzyl bromide, NaOH, DMF, room temperature, 4 h; (d) 80% aq. AcOH, 80 °C, 1.5 h; (e) Bu2SnO, MeOH, 70 °C, 3 h; (f) allyl bromide, DMF, TBAB, 60 °C, 8 h, 70% in six steps.

https://doi.org/10.1371/journal.pone.0037291.s001

(TIF)

Scheme S2.

Reagents: (a) TfOH, CH2Cl2, −25 °C, 1 h, 72%; (b) 0.1 M CH3ONa, CH3OH, room temperature, 2 h; (c) Dess-Martin Periodinane, CH2Cl2, room temperature, 1 h; (d) NaBH4, CH3OH, room temperature, 12 h; (e) acetic anhydride, pyridine, room temperature, 2 h, 76% in four steps; (f) PdCl2, CH3OH, room temperature, 1 h, 76% for 12 and 78% for 14; (g) benzyl bromide, NaOH, Bu4NBr, THF, room temperature, 3 h, 90%.

https://doi.org/10.1371/journal.pone.0037291.s002

(TIF)

Scheme S3.

Reagents: (a) N-Iodosuccinimide, TfOH, CH2Cl2, MS 4 Å, −30 °C, 1 h, 77%.

https://doi.org/10.1371/journal.pone.0037291.s003

(TIF)

Scheme S4.

Reagents: (a) N-Iodosuccinimide, TfOH, CH2Cl2, MS 4 Å, −30 °C, 1 h, 72%; (b) NH2NH2·H2O, EtOH, 80 °C, 6 h; (c) acetic anhydride, pyridine, room temperature, 1 h; (d) H2, 20% Pd(OH)2-C, CH3OH, room temperature, 24 h; (e) 0.1 M CH3ONa, CH3OH, room temperature, 4 h, 57% in four steps.

https://doi.org/10.1371/journal.pone.0037291.s004

(TIF)

Scheme S5.

Reagents: (a) a) N-Iodosuccinimide, TfOH, CH2Cl2, MS 4 Å, −30 °C, 30 min, then compound 14 followed by NIS and TfOH, −30 °C, 30 min, 71%; (b) 0.1 M CH3ONa, CH3OH, room temperature, 1 h; (c) triflic anhydride, pyridine, CH2Cl2, −10 °C, 1 h; (d) NaN3, HMPT-DMF, 90 °C, 8 h; (e) H2, 20% Pd(OH)2-C, CH3OH, room temperature, 24 h; (f) acetic anhydride, CH3OH, room temperature, 1 h, 51% in four steps.

https://doi.org/10.1371/journal.pone.0037291.s005

(TIF)

Author Contributions

Conceived and designed the experiments: AKM AS. Performed the experiments: AS. Analyzed the data: AKM AS. Contributed reagents/materials/analysis tools: AKM AS. Wrote the paper: AKM AS.

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