Convergent synthesis of a hexasaccharide corresponding to the cell wall O-antigen of Escherichia coli O41

Abstract

A convergent synthetic strategy has been developed for the synthesis of a hexasaccharide corresponding to the O-antigen of E. coli O41 using stereoselective [3 + 3] block glycosylation strategy. The target compound was synthesized assembling a series of suitably protected monosaccharide intermediates. Thioglycoside derivatives have been used as glycosyl donors in most of the glycosylation reactions. All intermediate steps are high yielding and the glycosylation steps are highly stereoselective. A number of recently developed methodologies have been used in the synthesis.

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Introduction

Escherichia coli is a facultative anaerobic gram-negative rod that belongs to the family Enterobacteriaceae.¹ Although most of the E. coli strains found in the human colonic flora are usually non-pathogenic, certain strains acquire virulence and are responsible for a number of infections in humans.² In general three types of E. coli infections are found in humans, which include (i) diarrhoea; (ii) sepsis/meningitis and (iii) urinary tract infections.³ Diarrhoeal infections are a serious concern in the developed countries with inadequate sanitation.⁴E. coli strains associated with diarrhoeal infections are broadly classified in five subgroups based on their mechanism of actions,⁵ which include enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC). EHEC strains are also termed as Shiga-toxin producing E. coli (STEC) and verotoxin producing E. coli (VTEC), which act through the production of Shiga-like toxin during the initial stage of infection to the host.⁶ Among various subgroups, STEC or VTEC are responsible for several diarrhoeal outbreaks as well as haemorrhagic colitis and haemorrhagic uremic syndromes.^7,8 The most well-documented E. coli strain associated STEC is E. coli O157, which has been the cause of several diarrhoeal outbreaks in the developing countries and Japan.⁹ The oligosaccharide repeating units of the cell wall O-antigen of the pathogenic bacterial strains play a pivotal role in the bacterial infection due to its presence in the outer layer of the cell wall.¹⁰E. coli O41 strain belongs to the STEC or VTEC category and has been associated with diarrhoeal infections and colitis.¹¹ Zhu et al. established the structure of the hexasaccharide repeating unit of the O-antigen of E. coli O41, which is composed of two α-linked L-frucose, two β-linked D-glucosamine, α-linked D-galactose and β-linked D-glucuronic acid moieties.¹² In the current scenario, the emergence of multidrug resistant bacterial strains demands for the development of novel effective antibacterial agents. In the past, glycoconjugate derivatives related to the cell wall oligosaccharides (O-antigens) have been evaluated in the development of antimicrobial agents.^13–15 For the detailed biological studies of the cell wall oligosaccharides and glycoconjugate derivatives, considerable amounts of oligosaccharides are required, which cannot be accessible from the natural source and hence chemical synthesis of the oligosaccharide repeating units becomes pertinent. In this report, a convergent synthetic strategy is presented for the synthesis of a hexasaccharide corresponding to the O-antigen of E. coli O41 as its p-methoxyphenyl glycoside (Fig. 1).

Structure of the hexasaccharide repeating unit of the O-antigen of Escherichia coli O41:

→3)-[β-D-GlcpA-(1→4)]-α-L-Fucp-(1→4)-β-D-GlcpNAc-(1→3)-α-L-Fucp-(1→3)-β-D-GlcpNAc-(1→3)-α-D-Galp-(1→

Fig. 1 Structure of the synthesized hexasaccharide as its p-methoxyphenyl glycoside and its synthetic intermediates.

Results and discussion

The target hexasaccharide 1 was synthesized as its p-methoxyphenyl (PMP) glycoside leaving the possibility for further glycoconjugate preparation by the oxidative deprotection of PMP group from compound 1. The synthetic strategy has been designed with the minimum number of steps required for achieving the target molecule. Thus a [3 + 3] block glycosylation strategy has been adopted involving stereoselective coupling of a trisaccharide glycosyl acceptor (11) with a trisaccharide thioglycoside donor (14). The retrosynthetic analysis of compound 1 led to a number of suitably functionalized monosaccharide intermediates 2,¹⁶3,¹⁷4,¹⁸5,¹⁶6¹⁹ and 7,²⁰ which were prepared from the commercially available reducing sugars following reported reaction methodologies (Fig. 1). The notable features of the synthetic strategy include (a) [3 + 3] block glycosylation; (b) formation of a number of stereoselective 1,2-cis-glycosyl linkages; (c) generalized reaction conditions for the glycosylations using thioglycoside derivatives as glycosyl donors; (d) TEMPO mediated selective oxidation of primary hydroxyl group to carboxyl group at a late-stage of the synthesis, etc.

p-Methoxyphenyl 4,6-O-benzylidene-2-O-benzyl-α-D-galactopyranoside (2)¹⁶ was allowed to couple stereoselectively with ethyl 3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-1-thio-β-D-glucopyranoside (3)¹⁷ in the presence of a combination of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf)^21,22 in dichloromethane to furnish disaccharide derivative (8) in 77% yield. The formation of compound 8 was confirmed from its spectral analysis [signals at δ 5.83 (d, J = 8.5 Hz, H-1_B), 5.55, 5.52 (2 s, 2 PhCH), 5.11 (d, J = 3.5 Hz, H-1_A) in the ¹H NMR and δ 101.7 (PhCH), 100.5 (PhCH), 100.0 (C-1_B), 96.8 (C-1_A) in the ¹³C NMR spectra]. De-O-acetylation of compound 8 using sodium methoxide²³ resulted in the formation of disaccharide acceptor 9 in 94% yield. Stereoselective 1,2-cis-glycosylation of compound 9 with ethyl 3-O-acetyl-2,4-di-O-benzyl-1-thio-β-L-fucopyranoside (4)¹⁸ in the presence of a combination of NIS–TMSOTf^21,22 in the mixed solvent dichloromethane and diethyl ether furnished trisaccharide derivative 10 in 74% yield together with a minor quantity (∼8%) of its other isomer, which was separated by column chromatography. Stereochemistry at the glycosyl linkages in compound 10 was confirmed from the NMR spectral analysis [δ 5.67 (d, J = 8.5 Hz, H-1_B), 5.57, 5.52 (2 s, 2 PhCH), 5.08 (d, J = 3.5 Hz, H-1_A), 4.86 (d, J = 3.5 Hz, H-1_C) in the ¹H NMR spectra and δ 101.7 (PhCH), 100.3 (PhCH), 100.2 (C-1_B), 98.6 (C-1_C), 96.9 (C-1_A) in the ¹³C NMR spectra]. Formation of a new 1,2-cis-glycosyl linkage in compound 10 was unambiguously confirmed using gated ¹H coupled ¹³C NMR spectral analysis. Appearance of J_C1–H1 coupling constants 169.0 Hz, 170.0 Hz and 156.0 Hz supported the presence of two axial (1,2-cis) and one equatorial (1,2-trans) glycosyl bonds in the molecule.²⁴ Compound 10 was treated with sodium methoxide²³ to give trisaccharide acceptor 11 in 96% yield, which has been used in the block glycosylation step for the preparation of hexasaccharide derivative (Scheme 1).

Scheme 1 Reagents: (a) N-iodosuccinimide (NIS), TMSOTf, MS-4Å, CH₂Cl₂, −15 °C, 30 min, 77%; (b) 0.05 M CH₃ONa, CH₃OH, room temperature, 2 h, 94% for compound 9 and 96% for compound 11; (c) NIS, TMSOTf, MS-4Å, CH₂Cl₂–Et₂O (1 : 3; v/v), −10 °C, 30 min, 74%.

In another experiment, stereoselective glycosylation of p-methoxyphenyl 2,3-di-O-benzyl-α-L-fucopyranoside (5)¹⁶ and ethyl 2,3,4,6-tetra-O-benzoyl-1-thio-β-D-glucopyranoside (6)¹⁹ in the presence of NIS–TMSOTf^21,22 furnished disaccharide derivative 12 in 73% yield. Formation of compound 12 was confirmed from its spectral analysis [signals at δ 5.17 (d, J = 3.5 Hz, H-1_E), 5.09 (d, J = 8.0 Hz, H-1_F) in the ¹H NMR and δ 101.7 (C-1_F), 97.5 (C-1_E) in the ¹³C NMR spectra]. Oxidative removal of the p-methoxyphenyl group from compound 12 using ammonium cerium(IV) nitrate (CAN)²⁵ followed by treatment of the hemiacetal derivative with trichloroacetonitrile in the presence of DBU²⁶ led to the formation of disaccharide trichloroacetimidate derivative 13 in 70% yield. Stereoselective 1,2-cis-glycosylation of the trichloroacetimidate derivative 13 with ethyl 3-O-acetyl-6-O-benzyl-2-deoxy-2-N-phthalimido-1-thio-β-D-glucopyranoside (7)²⁰ following a newly developed reaction condition using nitrosyl tetrafluoroborate²⁷ as glycosyl activator in the mixed solvent dichloromethane–diethyl ether (1 : 2) furnished trisaccharide thioglycoside derivative 14 in 70% yield together with a minor quantity (∼6%) of its other isomer, which was separated using column chromatography. The stereochemistry of the glycosyl linkages in compound 14 was established using NMR spectral analysis [signals at δ 5.43 (d, J = 10.5 Hz, H-1_D), 4.96 (d, J = 8.0 Hz, H-1_F), 4.73 (d, J = 3.0 Hz, H-1_E) in the ¹H NMR and δ 101.9 (C-1_F) (J_C1–H1 = 156.0 Hz), 100.5 (C-1_E) (J_C1–H1 = 169.0 Hz), 80.6 (C-1_D) (J_C1–H1 = 157.0 Hz) in the ¹³C NMR spectra] (Scheme 2). The presence of a newly formed 1,2-cis-glycosyl linkage in compound 14 was unambiguously confirmed from the J_C1–H1 coupling constants in the gated ¹H coupled ¹³C NMR spectral analysis.²⁴

Scheme 2 Reagents: (a) NIS, TMSOTf, MS-4Å, CH₂Cl₂, −20 °C, 30 min, 73%; (b) CAN, CH₃CN–H₂O (9 : 1), 0–5 °C, 3 h; (c) CCl₃CN, DBU, CH₂Cl₂, −5 °C, 1 h, 70%; (d) NOBF₄, CH₂Cl₂–Et₂O (1 : 2; v/v), −10 °C, 1 h, 70%.

Finally, a [3 + 3] block glycosylation strategy has been adopted for the synthesis of hexasaccharide derivative (15) by the stereoselective glycosylation of compound 11 and compound 14. For this purpose, compound 11 was reacted with thioglycoside derivative 14 in the presence of NIS–TMSOTf^21,22 in dichloromethane to furnish hexasaccharide derivative 15 in 66% yield. Formation of compound 15 was confirmed from its spectral analysis [signals at δ 5.48 (d, J = 8.5 Hz, H-1_B), 5.46 (d, J = 8.5 Hz, H-1_D), 5.05 (d, J = 3.0 Hz, H-1_A), 4.97 (d, J = 8.0 Hz, H-1_F), 4.71 (d, J = 3.0 Hz, H-1_C), 4.51 (d, J = 3.0 Hz, 1H, H-1_E) in the ¹H NMR and δ 101.8 (C-1_F), 100.4 (C-1_B), 100.3 (C-1_C), 99.2 (C-1_E), 96.8 (C-1_A), 95.0 (C-1_D) in the ¹³C NMR spectra]. In order to achieve compound 1 containing D-glucuronic acid moiety, compound 15 was subjected to a series of synthetic transformations involving (a) conversion of N-phthaloyl group to acetamido group by the removal of phthaloyl group²⁸ using hydrazine monohydrate followed by N- and O-acetylation using acetic anhydride and pyridine and de-O-acylation using sodium methoxide; (b) TEMPO mediated selective oxidation of the primary hydroxyl group to the carboxylic group;²⁹ and (c) catalytic transfer hydrogenation³⁰ of the oxidized hexasaccharide derivative using triethylsilane and 10% Pd–C to furnish compound 1, which was passed through a column of Dowex 50W X (Na⁺) and then through a Sephadex LH-20 gel column to give pure compound 1 as its sodium salt and p-methoxyphenyl glycoside in 53% overall yield. Formation of compound 1 was unambiguously confirmed from its spectral analysis [signals at δ 5.34 (br s, H-1_A), 4.99, 4.95 (2 d, J = 8.5 Hz each, H-1_B, H-1_D), 4.62 (d, J = 8.0 Hz, H-1_F), 4.35, 4.34 (2 d, J = 3.0 Hz each, H-1_C, H-1_E) in the ¹H NMR and δ 103.1 (3C, C-1_C, C-1_E, C-1_F), 100.6 (C-1_B), 100.1 (C-1_D), 98.5 (C-1_A) in the ¹³C NMR spectra] (Scheme 3).

Scheme 3 Reagents: (a) NIS, TMSOTf, MS-4Å, CH₂Cl₂, −10 °C, 45 min, 66%; (b) NH₂NH₂·H₂O, EtOH, 80 °C, 10 h; (c) acetic anhydride, pyridine, room temperature, 3 h; (d) 0.1 M CH₃ONa, CH₃OH, room temperature, 2 h; (e) (i) TEMPO, NaBr, TBAB, NaOCl, CH₂Cl₂, H₂O, NaHCO₃, 0–5 °C, 3 h; (ii) tert-butanol, 2-methyl-but-2-ene, NaClO₂, NaH₂PO₄, room temperature, 3 h; (f) Et₃SiH, 10% Pd–C, CH₃OH–CHCl₃ (5 : 1; v/v), room temperature, 10 h, overall 53%.

Conclusions

In summary, a hexasaccharide (1) corresponding to the O-antigen of the cell wall polysaccharide of E. coli O41 has been synthesized as its p-methoxyphenyl glycoside and sodium salt in excellent yield using [3 + 3] block glycosylation approach. During the synthesis of the target molecule, a number of novel reaction conditions have been applied for the functional group manipulations. In most of the glycosylation reactions, thioglycoside derivatives have been used as selective glycosyl donors. The stereochemistries of the glycosyl linkages were confirmed by the NMR spectral analysis. All synthetic intermediates were solid and fully characterized using spectral analysis. A late stage TEMPO mediated selective oxidation of the primary hydroxyl group in a biphasic reaction condition efficiently furnished D-glucuronic acid moiety in the hexasaccharide derivative. All intermediate glycosylation steps were high yielding with excellent stereo control.

Experimental

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 sulfate [2% Ce(SO₄)₂ in 2 N H₂SO₄]-sprayed plates on a hot plate. Silica gel 230–400 mesh was used for column chromatography. ¹H and ¹³C NMR, DEPT 135, 2D COSY and 2D HSQC NMR spectra were recorded on a Bruker Avance DRX 500 MHz spectrometer using CDCl₃ and D₂O as solvents and TMS as internal reference unless stated otherwise. Chemical shift values are expressed in δ ppm. MALDI-MS were recorded on a Bruker Daltronics 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 grades of organic solvents of adequate purity are used in all reactions.

p-Methoxyphenyl (3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl)-(1→3)-2-O-benzyl-4,6-O-benzylidene-α-D-galactopyranoside (8)

To a solution of compound 2 (1.5 g, 3.23 mmol) and compound 3 (1.7 g, 3.51 mmol) in anhydrous CH₂Cl₂ (20 mL) was added MS-4Å (2 g) and the reaction mixture was allowed to stir at room temperature for 30 min under argon. The reaction mixture was cooled to −15 °C. To the cooled reaction mixture were added N-iodosuccinimide (NIS; 830 mg, 3.69 mmol) and TMSOTf (15 μL) and it was stirred at the same temperature for 30 min. The reaction mixture was diluted with CH₂Cl₂ (100 mL) and filtered through a Celite® bed and washed with CH₂Cl₂. The combined organic layer was successively washed with 5% Na₂S₂O₃, satd. NaHCO₃ and water, dried (Na₂SO₄) and concentrated. The crude product was purified over SiO₂ using hexane–EtOAc (5 : 1) as eluent to give pure compound 8 (2.2 g, 77%). White solid; m.p. 108–110 °C [EtOH]; [α]²⁵_D +32.4 (c 1.2, CHCH₃); IR (KBr): 3651, 2924, 1777, 1742, 1721, 1507, 1387, 1223, 1102, 1040, 995, 796 cm⁻¹; ¹H NMR (500 MHz, CDCl₃): δ 7.75–6.94 (m, 19H, Ar–H), 6.85, 6.74 (2 d, J = 9.0 Hz each, 4H, Ar–H), 5.95 (t, J = 9.5 Hz each, 1H, H-3_B), 5.83 (d, J = 8.5 Hz, 1H, H-1_B), 5.55, 5.52 (2 s, 2H, 2 PhCH), 5.11 (d, J = 3.5 Hz, 1H, H-1_A), 4.47 (t, J = 8.5 Hz each, 1H, H-2_B), 4.45–4.43 (m, 1H, H-5_B), 4.34 (d, J = 3.0 Hz, 1H, H-4_A), 4.29 (dd, J = 10.5, 3.0 Hz, 1H, H-3_A), 4.24 (d, J = 12.5 Hz, 1H, PhCH₂), 4.14 (d, J = 12.0 Hz, 1H, H-6_aA), 3.99 (d, J = 12.0 Hz, 1H, H-6_bA), 3.95 (d, J = 12.5 Hz, 1H, PhCH₂), 3.90–3.80 (m, 4H, H-2_A, H-4_B, H-6_abB), 3.74 (s, 3H, OCH₃), 3.73–3.72 (m, 1H, H-5_A), 1.92 (s, 3H, COCH₃); ¹³C NMR (125 MHz, CDCl₃): δ 169.9 (COCH₃), 167.4 (Phth), 154.8–114.4 (Ar–C), 101.7 (PhCH), 100.5 (PhCH), 100.0 (C-1_B), 96.8 (C-1_A), 79.2 (C-2_A), 76.5 (C-3_A), 76.3 (C-4_B), 74.9 (C-4_A), 73.1 (PhCH₂), 69.8 (C-3_B), 69.1 (C-6_A), 68.7 (C-6_B), 66.0 (C-5_B), 63.1 (C-5_A), 55.6 (C-2_B), 55.5 (OCH₃), 20.6 (COCH₃); ESI-MS: 908.3 [M + Na]⁺; anal. calcd for C₅₀H₄₇NO₁₄ (885.30): C, 67.79; H, 5.35; found: C, 67.60; H, 5.50.

p-Methoxyphenyl (4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl)-(1→3)-2-O-benzyl-4,6-O-benzylidene-α-D-galactopyranoside (9)

A solution of compound 8 (2 g, 2.26 mmol) in 0.05 M CH₃ONa in CH₃OH (30 mL) was allowed to stir at room temperature for 2 h. The reaction mixture was neutralized with Dowex 50W-X8 (H⁺) resin, filtered and concentrated. The crude product was passed through a small pad of SiO₂ using hexane–EtOAc (1 : 1) as eluent to give pure compound 9 (1.8 g, 94%). White solid; m.p. 122–124 °C [EtOH]; [α]²⁵_D +25 (c 1.2, CHCH₃); IR (KBr): 3476, 2924, 2865, 1775, 1724, 1507, 1486, 1390, 1210, 1175, 1095, 1044, 993, 798 cm⁻¹; ¹H NMR (500 MHz, CDCl₃): δ 7.75–6.94 (m, 19H, Ar–H), 6.84, 6.72 (2 d, J = 9.0 Hz each, 4H, Ar–H), 5.66 (d, J = 8.5 Hz, 1H, H-1_B), 5.58, 5.52 (2 s, 2H, 2 PhCH), 5.09 (d, J = 3.0 Hz, 1H, H-1_A), 4.73 (t, J = 10.0 Hz each, 1H, H-3_B), 4.42–4.38 (m, 2H, H-2_B, H-5_B), 4.33 (d, J = 2.5 Hz, 1H, H-4_A), 4.29 (dd, J = 10.5, 3.0 Hz, 1H, H-3_A), 4.24 (d, J = 12.5 Hz, 1H, PhCH₂), 4.11 (d, J = 12.5 Hz, 1H, H-6_aA), 3.99 (d, J = 12.5 Hz, 1H, H-6_bA), 3.93 (d, J = 12.5 Hz, 1H, PhCH₂), 3.90–3.80 (m, 2H, H-2_A, H-6_aB), 3.74 (s, 3H, OCH₃), 3.73–3.70 (m, 2H, H-5_A, H-6_bB), 3.66 (t, J = 10.0 Hz each, 1H, H-4_B); ¹³C NMR (125 MHz, CDCl₃): δ 167.5 (Phth), 155.6–114.1 (Ar–C), 101.9 (PhCH), 100.3 (PhCH), 100.2 (C-1_B), 96.8 (C-1_A), 82.2 (C-4_B), 76.5 (C-4_A), 75.8 (C-3_A), 75.2 (C-2_A), 73.2 (PhCH₂), 69.0 (C-6_A), 68.7 (C-6_B), 68.4 (C-5_B), 66.0 (C-3_B), 63.2 (C-5_A), 56.7 (C-2_B), 55.5 (OCH₃); ESI-MS: 866.2 [M + Na]⁺; anal. calcd for C₄₈H₄₅NO₁₃ (843.29): C, 68.32; H, 5.37; found: C, 68.12; H, 5.55.

p-Methoxyphenyl (3-O-acetyl-2,4-di-O-benzyl-α-L-fucopyranosyl)-(1→3)-(4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl)-(1→3)-2-O-benzyl-4,6-O-benzylidene-α-D-galactopyranoside (10)

To a solution of compound 9 (1.5 g, 1.78 mmol) and compound 4 (830 mg, 1.93 mmol) in anhydrous CH₂Cl₂–Et₂O (15 mL; 1 : 3, v/v) was added MS-4Å (2 g) and the reaction mixture was allowed to stir at room temperature for 30 min under argon. The reaction mixture was cooled to −10 °C. To the cooled reaction mixture were added NIS (450 mg, 2.0 mmol) and TMSOTf (5 μL) and it was stirred at the same temperature for 30 min. The reaction mixture was diluted with CH₂Cl₂ (50 mL) and filtered through a Celite® bed and washed with CH₂Cl₂. The combined organic layer was successively washed with 5% Na₂S₂O₃, satd. NaHCO₃ and water, dried (Na₂SO₄) and concentrated. The crude product was purified over SiO₂ using hexane–EtOAc (5 : 1) as eluent to give pure compound 10 (1.6 g, 74%). White solid; m.p. 118–120 °C [EtOH]; [α]²⁵_D −18 (c 1.2, CHCH₃); IR (KBr): 3477, 2933, 1777, 1742, 1718, 1507, 1387, 1391, 1244, 1099, 1044, 995, 797 cm⁻¹; ¹H NMR (500 MHz, CDCl₃): δ 7.59–6.94 (m, 29H, Ar–H), 6.84, 6.71 (2 d, J = 9.0 Hz each, 4H, Ar–H), 5.67 (d, J = 8.5 Hz, 1H, H-1_B), 5.57, 5.52 (2 s, 2H, 2 PhCH), 5.08 (d, J = 3.5 Hz, 1H, H-1_A), 4.96 (dd, J = 10.5, 3.5 Hz, 1H, H-3_C), 4.86 (d, J = 3.5 Hz, 1H, H-1_C), 4.76 (t, J = 9.5 Hz each, 1H, H-3_B), 4.54 (t, J = 8.5 Hz each, 1H, H-2_B), 4.45–4.40 (m, 3H, H-5_B, PhCH₂), 4.34 (d, J = 3.5 Hz, 1H, H-4_A), 4.28 (dd, J = 10.0, 3.5 Hz, 1H, H-3_A), 4.24 (d, J = 12.0 Hz, 1H, PhCH₂), 4.16–4.10 (m, 3H, H-6_aA, PhCH₂), 3.98–3.91 (m, 3H, H-6_bA, H-6_aB, PhCH₂), 3.88–3.82 (m, 2H, H-2_A, H-5_C), 3.79–3.66 (m, 2H, H-4_B, H-4_C), 3.73 (s, 3H, OCH₃), 3.69 (dd, J = 10.5, 3.5 Hz, 1H, H-2_C), 3.59–3.57 (m, 1H, H-5_A), 1.63 (s, 3H, COCH₃), 0.75 (d, J = 6.5 Hz, 3H, CCH₃); ¹³C NMR (125 MHz, CDCl₃): δ 169.9 (COCH₃), 167.6 (Phth), 154.8–114.4 (Ar–C), 101.7 (PhCH), 100.3 (PhCH), 100.2 (C-1_B), 98.6 (C-1_C), 96.9 (C-1_A), 81.6 (C-4_B), 78.6 (C-5_A), 76.5 (C-4_A), 75.9 (C-3_A), 75.5 (PhCH₂), 75.0 (C-3_B), 74.9 (C-5_C), 73.4 (C-3_C), 73.2 (PhCH₂), 73.1 (C-2_C), 72.5 (PhCH₂), 69.1 (C-6_A), 68.8 (C-6_B), 66.6 (C-5_B), 66.2 (C-2_A), 63.2 (C-4_C), 56.0 (C-2_B), 55.5 (OCH₃), 20.7 (COCH₃), 15.9 (CCH₃); MALDI-MS: 1234.3 [M + Na]⁺; anal. calcd for C₇₀H₆₉NO₁₈ (1211.45): C, 69.35; H, 5.74; found: C, 69.18; H, 5.95.

p-Methoxyphenyl (2,4-di-O-benzyl-α-L-fucopyranosyl)-(1→3)-(4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl)-(1→3)-2-O-benzyl-4,6-O-benzylidene-α-D-galactopyranoside (11)

A solution of compound 10 (1.5 g, 1.24 mmol) in 0.05 M CH₃ONa in CH₃OH (25 mL) was allowed to stir at room temperature for 2 h. The reaction mixture was neutralized with Dowex 50W-X8 (H⁺) resin, filtered and concentrated. The crude product was passed through a small pad of SiO₂ using hexane–EtOAc (1 : 1) as eluent to give pure compound 11 (1.4 g, 96%). White solid; m.p. 80–81 °C [EtOH]; [α]²⁵_D −12.6 (c 1.2, CHCH₃); IR (KBr): 3477, 2931, 2869, 1777, 1716, 1507, 1454, 1391, 1243, 1215, 1101, 1045, 995, 797, 697 cm⁻¹; ¹H NMR (500 MHz, CDCl₃): δ 7.54–6.94 (m, 29H, Ar–H), 6.84, 6.71 (2 d, J = 9.0 Hz each, 4H, Ar–H), 5.69 (d, J = 8.5 Hz, 1H, H-1_B), 5.55, 5.51 (2 s, 2H, 2 PhCH), 5.09 (d, J = 3.5 Hz, 1H, H-1_A), 4.91 (d, J = 3.5 Hz, 1H, H-1_C), 4.74 (t, J = 9.5 Hz each, 1H, H-3_B), 4.61 (d, J = 12.0 Hz, 1H, PhCH₂), 4.53–4.48 (m, 2H, H-2_B, PhCH₂), 4.44–4.41 (m, 1H, H-5_B), 4.34 (d, J = 3.0 Hz, 1H, H-4_A), 4.30–4.22 (m, 3H, H-3_A, PhCH₂), 4.14–4.80 (m, 2H, H-5_C, PhCH₂), 3.99–3.87 (m, 3H, H-4_C, H-6_aA, PhCH₂), 3.86–3.80 (m, 3H, H-2_A, H-3_C, H-6_bA), 3.79–3.73 (m, 3H, H-4_B, H-6_abB), 3.74 (s, 3H, OCH₃), 3.45–3.42 (m, 2H, H-2_C, H-5_A), 0.88 (d, J = 6.5 Hz, 3H, CCH₃); ¹³C NMR (125 MHz, CDCl₃): δ 167.7 (Phth), 154.8–114.4 (Ar–C), 101.5 (PhCH), 100.4 (PhCH), 100.2 (C-1_B), 97.7 (C-1_C), 96.8 (C-1_A), 81.6 (C-4_B), 79.8 (C-5_A), 76.5 (C-2_C), 76.4 (C-4_A), 76.0 (C-3_A), 75.4 (PhCH₂), 75.0 (C-3_B), 74.5 (C-3_C), 73.2 (PhCH₂), 72.2 (PhCH₂), 70.2 (C-5_C), 69.1 (C-6_A), 68.8 (C-6_B), 67.0 (C-5_B), 66.1 (C-2_A), 63.1 (C-4_C), 56.1 (C-2_B), 55.5 (OCH₃), 16.4 (CCH₃); MALDI-MS: 1192.4 [M + Na]⁺; anal. calcd for C₆₈H₆₇NO₁₇ (1169.44): C, 69.79; H, 5.77; found: C, 69.62; H, 5.95.

p-Methoxyphenyl (2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-(1→4)-2,3-di-O-benzyl-α-L-fucopyranoside (12)

To a solution of compound 5 (1.2 g, 2.66 mmol) and compound 6 (1.8 g, 2.81 mmol) in anhydrous CH₂Cl₂ (25 mL) was added MS-4Å (3 g) and the reaction mixture was allowed to stir at room temperature for 30 min under argon. The reaction mixture was cooled to −20 °C. To the cooled reaction mixture were added NIS (660 mg, 2.93 mmol) and TMSOTf (10 μL) and it was stirred at the same temperature for 30 min. The reaction mixture was diluted with CH₂Cl₂ (100 mL) and filtered through a Celite® bed and washed with CH₂Cl₂. The combined organic layer was successively washed with 5% Na₂S₂O₃, satd. NaHCO₃ and water, dried (Na₂SO₄) and concentrated. The crude product was purified over SiO₂ using hexane–EtOAc (5 : 1) as eluent to give pure compound 12 (2 g, 73%). White solid; m.p. 60–62 °C [EtOH]; [α]²⁵_D −22.4 (c 1.2, CHCH₃); IR (KBr): 3445, 2918, 1736, 1507, 1452, 1263, 1072, 1068, 1026, 709 cm⁻¹; ¹H NMR (500 MHz, CDCl₃): δ 7.98–7.19 (m, 30H, Ar–H), 6.87, 6.74 (2 d, J = 9.0 Hz each, 4H, Ar–H), 5.78 (t, J = 9.5 Hz each, 1H, H-3_F), 5.66 (t, J = 10.0 Hz each, 1H, H-4_F), 5.60 (t, J = 9.0 Hz each, 1H, H-2_F), 5.17 (d, J = 3.5 Hz, 1H, H-1_E), 5.09 (d, J = 8.0 Hz, 1H, H-1_F), 4.86–4.78 (m, 3H, PhCH₂), 4.65 (d, J = 12.0 Hz, 1H, PhCH₂), 4.46 (dd, J = 12.0, 3.0 Hz, 1H, H-6_aF), 4.25 (dd, J = 12.0, 4.0 Hz, 1H, H-6_bF), 4.14 (dd, J = 10.0, 3.5 Hz, 1H, H-2_E), 4.06 (dd, J = 10.0, 3.0 Hz, 1H, H-3_E), 4.01 (br s, 1H, H-4_E), 4.0–3.96 (m, 1H, H-5_E), 3.73 (s, 3H, OCH₃), 3.72–3.70 (m, 1H, H-5_F), 0.97 (d, J = 6.5 Hz, 3H, CCH₃); ¹³C NMR (125 MHz, CDCl₃): δ 165.9, 165.8, 164.9, 164.8 (4 COPh), 154.8–114.4 (Ar–C), 101.7 (C-1_F), 97.5 (C-1_E), 78.9 (C-4_E), 77.8 (C-3_E), 75.7 (C-2_E), 73.7 (PhCH₂), 73.4 (C-3_F), 73.1 (PhCH₂), 72.7 (C-2_F), 72.3 (C-5_F), 69.6 (C-4_F), 66.6 (C-5_E), 62.9 (C-6_F), 55.5 (OCH₃), 16.3 (CCH₃); MALDI-MS: 1051.3 [M + Na]⁺; anal. calcd for C₆₁H₅₆O₁₅ (1028.36): C, 71.19; H, 5.48; found: C, 71.00; H, 5.69.

(2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranosyl)-(1→4)-2,3-di-O-benzyl-α-L-fucopyranosyl trichloroacetimidate (13)

To a solution of compound 12 (1.8 g, 1.75 mmol) in CH₃CN–H₂O (25 mL; 9 : 1; v/v) was added ammonium cerium(IV) nitrate (CAN; 2 g; 3.65 mmol) and the reaction mixture was allowed to stir at 0–5 °C for 3 h. The reaction mixture was diluted with CH₂Cl₂ (100 mL) and the organic layer was successively washed with satd. NaHCO₃, water, dried (Na₂SO₄) and concentrated to give the crude product, which was passed through a short pad of SiO₂ using hexane–EtOAc (2 : 1) as eluent to give the hemiacetal derivative. To a solution of the hemiacetal derivative in anhydrous CH₂Cl₂ (20 mL) were added CCl₃CN (1.2 mL, 11.97 mmol) and DBU (50 μL) and the reaction mixture was allowed to stir at −5 °C for 1 h. The solvents were removed under reduced pressure and the crude product was passed through a short pad of SiO₂ using hexane–EtOAc (9 : 1) as eluent to give pure compound 13 (1.3 g, 70%), which was used immediately without further characterization.

Ethyl (2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-(1→4)-(2,3-di-O-benzyl-α-L-fucopyranosyl)-(1→4)-3-O-acetyl-6-O-benzyl-2-deoxy-2-N-phthalimido-1-thio-β-D-glucopyranoside (14)

To a solution of compound 7 (500 mg, 1.03 mmol) and compound 13 (1.2 g, 1.12 mmol) in anhydrous CH₂Cl₂–Et₂O (10 mL; 1 : 2, v/v) was added NOBF₄ (140 mg, 1.2 mmol) and the reaction mixture was allowed to stir at −10 °C for 1 h under argon. The reaction mixture was diluted with CH₂Cl₂ (50 mL) and filtered through a Celite® bed and washed with CH₂Cl₂. The combined organic layer was successively washed with satd. NaHCO₃ and water, dried (Na₂SO₄) and concentrated. The crude product was purified over SiO₂ using hexane–EtOAc (3 : 1) as eluent to give pure compound 14 (1 g, 70%). White solid; m.p. 88–90 °C [EtOH]; [α]²⁵_D −28.6 (c 1.2, CHCH₃); IR (KBr): 3447, 2929, 1722, 1718, 1452, 1386, 1265, 1094, 710 cm⁻¹; ¹H NMR (500 MHz, CDCl₃): δ 7.94–7.21 (m, 39H, Ar–H), 5.80 (t, J = 9.5 Hz each, 1H, H-3_F), 5.64 (t, J = 10.0 Hz each, 1H, H-4_F), 5.62 (t, J = 9.5 Hz each, 1H, H-2_F), 5.54 (t, J = 9.5 Hz each, 1H, H-3_D), 5.43 (d, J = 10.5 Hz, 1H, H-1_D), 4.96 (d, J = 8.0 Hz, 1H, H-1_F), 4.82 (d, J = 12.0 Hz, 1H, PhCH₂), 4.76 (d, J = 12.0 Hz, 1H, PhCH₂), 4.73 (d, J = 3.0 Hz, 1H, H-1_E), 4.60 (d, J = 12.0 Hz, 1H, PhCH₂), 4.54 (d, J = 12.0 Hz, 1H, PhCH₂), 4.47 (dd, J = 12.0, 2.5 Hz, 1H, H-6_aF), 4.33–4.29 (m, 2H, H-6_bF, PhCH₂), 4.24 (d, J = 12.0 Hz, 1H, PhCH₂), 4.15 (t, J = 10.0 Hz each, 1H, H-2_D), 4.01–3.99 (m, 1H, H-6_aD), 3.91 (br s, 1H, H-4_E), 3.89 (dd, J = 10.0, 3.5 Hz, 1H, H-2_E), 3.85–3.81 (m, 1H, H-5_F), 3.78–3.68 (m, 3H, H-3_E, H-5_D, H-5_E), 3.59–3.52 (m, 2H, H-4_D, H-6_bD), 2.74–2.56 (m, 2H, SCH₂CH₃), 1.42 (s, 3H, COCH₃), 1.19 (t, J = 7.5 Hz each, 3H, SCH₂CH₃), 0.89 (d, J = 6.5 Hz, 3H, CCH₃); ¹³C NMR (125 MHz, CDCl₃): δ 170.9 (COCH₃), 167.9, 167.8 (Phth), 166.0, 165.9, 165.1, 164.7 (4 COPh), 138.7–123.5 (Ar–C), 101.9 (C-1_F), 100.5 (C-1_E), 80.6 (C-1_D), 79.1 (C-4_E), 78.4 (C-3_E), 78.2 (C-4_D), 77.1 (C-2_E), 75.9 (C-5_D), 74.2 (PhCH₂), 73.9 (C-3_D), 73.1 (C-3_F), 73.0 (PhCH₂), 72.3 (2C, C-2_F, C-5_F), 71.9 (PhCH₂), 69.9 (C-6_D), 69.7 (C-4_F), 67.1 (C-5_E), 63.3 (C-6_F), 54.1 (C-2_D), 24.3 (SCH₂CH₃), 20.4 (COCH₃), 15.9 (CCH₃), 15.0 (SCH₂CH₃); MALDI-MS: 1412.4 [M + Na]⁺; anal. calcd for C₇₉H₇₅NO₂₀S (1389.46): C, 68.24; H, 5.44; found: C, 68.05; H, 5.64.

p-Methoxyphenyl (2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-(1→4)-(2,3-di-O-benzyl-α-L-fucopyranosyl)-(1→4)-(3-O-acetyl-6-O-benzyl-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl)-(1→3)-(2,4-di-O-benzyl-α-L-fucopyranosyl)-(1→3)-(4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl)-(1→3)-2-O-benzyl-4,6-O-benzylidene-α-D-galactopyranoside (15)

To a solution of compound 11 (720 mg, 0.61 mmol) and compound 14 (900 mg, 0.65 mmol) in anhydrous CH₂Cl₂ (15 mL) was added MS-4Å (2 g) and the reaction mixture was allowed to stir at room temperature for 30 min under argon and cooled to −10 °C. To the cooled reaction mixture were added NIS (160 mg, 0.71 mmol) and TMSOTf (5 μL) and it was stirred at the same temperature for 45 min. The reaction mixture was diluted with CH₂Cl₂ (100 mL) and filtered through a Celite® bed and washed with CH₂Cl₂. The combined organic layer was successively washed with 5% Na₂S₂O₃, satd. NaHCO₃ and water, dried (Na₂SO₄) and concentrated. The crude product was purified over SiO₂ using hexane–EtOAc (5 : 1) as eluent to give pure compound 15 (1 g, 66%). White solid; m.p. 95–97 °C [EtOH]; [α]²⁵_D +12 (c 1.2, CHCH₃); IR (KBr): 3477, 2932, 2869, 1775, 1714, 1507, 1454, 1392, 1243, 1213, 1100, 1045, 996 cm⁻¹; ¹H NMR (500 MHz, CDCl₃): δ 7.94–6.52 (m, 72H, Ar–H), 5.78 (t, J = 9.5 Hz each, 1H, H-3_F), 5.63 (t, J = 10.0 Hz each, 1H, H-4_F), 5.61 (t, J = 9.5 Hz each, 1H, H-2_F), 5.54 (s, 1H, PhCH), 5.48 (d, J = 8.5 Hz, 1H, H-1_B), 5.46 (d, J = 8.5 Hz, 1H, H-1_D), 5.42 (t, J = 9.0 Hz each, 1H, H-3_D), 5.30 (s, 1H, PhCH), 5.05 (d, J = 3.0 Hz, 1H, H-1_A), 4.97 (d, J = 8.0 Hz, 1H, H-1_F), 4.84 (d, J = 12.0 Hz, 1H, PhCH₂), 4.78–4.72 (m, 3H, H-3_B, PhCH₂), 4.71 (d, J = 3.0 Hz, 1H, H-1_C), 4.68–4.62 (m, 2H, H-5_B, PhCH₂), 4.60–4.52 (m, 2H, PhCH₂), 4.51 (d, J = 3.0 Hz, 1H, H-1_E), 4.48–4.44 (m, 1H, H-6_aF), 4.37 (t, J = 8.5 Hz each, 1H, H-2_D), 4.34 (d, J = 3.5 Hz, 1H, H-4_A), 4.30–4.20 (m, 5H, H-5_E, H-6_aA, H-6_aF, PhCH₂), 4.18–4.12 (m, 2H, H-6_aD, PhCH₂), 4.08–3.97 (m, 4H, H-2_B, H-6_aB, H-6_bD, PhCH₂), 3.94–3.79 (m, 8H, H-2_A, H-3_C, H-4_C, H-4_E, H-5_C, H-5_F, PhCH₂), 3.78–3.70 (m, 2H, H-3_A, H-5_D), 3.73 (s, 3H, OCH₃), 3.69–3.62 (m, 4H, H-3_E, H-4_B, H-6_bA, H-6_bB), 3.52–3.45 (m, 2H, H-2_C, H-2_E), 3.22–3.20 (m, 1H, H-5_A), 3.14 (t, J = 9.5 Hz each, 1H, H-4_D), 1.55 (s, 3H, COCH₃), 0.88–0.84 (m, 6H, 2 CCH₃); ¹³C NMR (125 MHz, CDCl₃): δ 170.1 (COCH₃), 167.7 (2C) (Phth), 167.4 (2C) (Phth), 165.8, 165.7, 165.0, 164.6 (4 COPh), 154.8–114.4 (Ar–C), 101.9 (PhCH), 101.8 (C-1_F), 100.5 (PhCH), 100.4 (C-1_B), 100.3 (C-1_C), 99.2 (C-1_E), 96.8 (C-1_A), 95.0 (C-1_D), 80.4 (C-4_D), 78.8 (C-5_A), 78.3 (C-2_C), 78.2 (C-3_E), 77.1 (2C, C-2_E, C-4_A), 76.6 (C-5_D), 75.9 (C-4_E), 75.8 (2C, C-3_B, C-4_B), 75.1 (C-3_A), 74.8 (PhCH₂), 74.2 (PhCH₂), 74.0 (PhCH₂), 73.6 (C-3_C), 73.2 (2C, C-3_D, PhCH₂), 73.1 (C-3_F), 72.5 (PhCH₂), 72.3 (2C, C-2_F, C-5_F), 72.0 (PhCH₂), 71.7 (C-5_C), 69.7 (2C, C-4_F, C-6_A), 69.1 (C-6_D), 68.6 (C-6_B), 67.1 (C-2_A), 66.6 (C-5_B), 66.2 (C-5_E), 63.2 (C-4_C), 63.1 (C-6_F), 56.2 (C-2_D), 55.5 (OCH₃), 55.3 (C-2_B), 20.4 (COCH₃), 15.9, 15.7 (2 CCH₃); MALDI-MS: 2519.8 [M + Na]⁺; anal. calcd for C₁₄₅H₁₃₆N₂O₃₇ (2496.88): C, 69.70; H, 5.49; found: C, 69.54; H, 5.68.

p-Methoxyphenyl (sodium β-D-glucopyranosyluronate)-(1→4)-(α-L-fucopyranosyl)-(1→4)-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→3)-(α-L-fucopyranosyl)-(1→3)-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→3)-α-D-galactopyranoside (1)

To a solution of compound 15 (800 mg, 0.32 mmol) in C₂H₅OH (15 mL) was added hydrazine monohydrate (0.3 mL) and the reaction was allowed to stir at 80 °C for 10 h. The solvents were removed under reduced pressure and the crude product was dissolved in acetic anhydride and pyridine (4 mL, 1 : 1 v/v) and kept at room temperature for 3 h. Solvents were removed under reduced pressure and the acetylated product was dissolved in 0.1 M CH₃ONa in CH₃OH (15 mL) and the reaction mixture was allowed to stir at room temperature for 2 h. The reaction mixture was neutralized using Dowex 50W-X8 (H⁺) resin, filtered and concentrated under reduced pressure. To a solution of the hexasaccharide pentahydroxy derivative in CH₂Cl₂ (25 mL) and H₂O (5 mL) were successively added 1 M aq. NaBr (3 mL), 1 M aq. TBAB (5 mL), TEMPO (150 mg, 0.96 mmol), satd. NaHCO₃ (20 mL) and 4% aq. NaOCl (15 mL) and the reaction mixture was allowed to stir at 5 °C for 3 h and neutralized with 1 N HCl. To the reaction mixture were added tert-butanol (20 mL), 2-methyl-but-2-ene (20 mL; 2 M solution in THF), aq. NaClO₂ (2 g per 10 mL) and aq. NaH₂PO₄ (2 g per 10 mL) in succession and the reaction mixture was allowed to stir at room temperature for 3 h. The reaction mixture was diluted with satd. aq. NaH₂PO₄ and extracted with CH₂Cl₂ (100 mL). The organic layer was washed with water, dried (Na₂SO₄) and concentrated to dryness to give the crude product, which was successively passed through a short pad of SiO₂ and a short column of Dowex 50W-X8 (Na⁺) to give the sodium salt of glucuronic acid containing hexasaccharide derivative. To a solution of the oxidized product and 10% Pd–C (150 mg) in CH₃OH (10 mL) was added Et₃SiH (0.7 mL, 4.38 mmol) dropwise over 30 min and the reaction mixture was allowed to stir at room temperature for 10 h. The reaction mixture was filtered through a Celite® bed, washed with CH₃OH–H₂O (2 : 1, v/v) and concentrated under reduced pressure to give compound 1, which was passed through a column of Sephadex LH-20 gel using CH₃OH–H₂O (3 : 1) as eluent to give pure compound 1 as p-methoxyphenyl glycoside and sodium salt (200 mg, 53%). Glass; [α]²⁵_D +10 (c 1.0, CH₃OH); IR (KBr): 3478, 2933, 2867, 1777, 1714, 1507, 1457, 1392, 1243, 1213, 1100, 1045, 996 cm⁻¹; ¹H NMR (500 MHz, D₂O): δ 7.02–6.86 (2 d, J = 9.0 Hz each, 4H, Ar–H), 5.34 (br s, 1H, H-1_A), 4.99, 4.95 (2 d, J = 8.5 Hz each, 2H, H-1_B, H-1_D), 4.62 (d, J = 8.0 Hz, 1H, H-1_F), 4.35, 4.34 (2 d, J = 3.0 Hz each, 2H, H-1_C, H-1_E), 4.20–4.02 (m, 2H, H-5_C, H-5_E), 4.0–3.86 (m, 5H, H-2_B, H-2_C, H-3_F, H-4_A, H-4_E), 3.85–3.75 (m, 10H, H-2_D, H-2_E, H-3_C, H-3_D, H-4_B, H-5_D, H-6_abA, H-6_abD), 3.69 (s, 3H, OCH₃), 3.68–3.50 (m, 7H, H-3_A, H-3_E, H-4_D, H-4_F, H-5_A, H-6_abB), 3.46–3.25 (m, 6H, H-2_A, H-2_F, H-3_B, H-4_C, H-5_B, H-5_F), 2.05, 1.98 (2 s, 6H, 2 COCH₃), 1.18, 1.12 (2 d, J = 6.0 Hz each, 6H, 2 CCH₃); ¹³C NMR (125 MHz, D₂O): δ 175.9, 175.7 (2 COCH₃), 173.0 (COONa), 154.6–115.0 (Ar–C), 103.1 (3C, C-1_C, C-1_E, C-1_F), 100.6 (C-1_B), 100.1 (C-1_D), 98.5 (C-1_A), 80.6 (2C, C-3_D, C-4_B), 75.9 (4C, C-3_A, C-4_C, C-5_B, C-5_F), 75.5 (2C, C-3_B, C-4_A), 74.7 (C-3_E), 73.5 (2C, C-4_D, C-5_A), 72.1 (C-3_F), 71.2 (C-3_C), 69.4 (2C, C-4_F, C-5_D), 69.0 (2C, C-2_F, C-4_E), 68.7 (2C, C-2_C, C-5_C), 68.3 (C-2_A), 67.8 (C-5_E), 66.9 (C-2_E), 60.9 (C-6_A), 60.5 (2C, C-6_B, C-6_D), 55.7 (OCH₃), 55.0 (C-2_B), 54.9 (C-2_D), 23.7, 22.4 (2 COCH₃), 15.1 (2C, 2 CCH₃); MALDI-MS: 1183.3 [M + 1]⁺; anal. calcd for C₄₇H₇₁N₂NaO₃₁ (1182.39): C, 47.72; H, 6.05; found: C, 47.51; H, 6.28.

Acknowledgements

T. G. and A. S. thank CSIR, New Delhi for providing Junior and Senior Research Fellowship, respectively. This work was supported by DST, New Delhi [Project no. SR/S1/OC-83/2010].

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Copies of 1D and 2D NMR spectra of compounds 1 and 8–15. See DOI: 10.1039/c3ra45493b

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