Carbohydrate Research 399 (2014) 21–25

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Efficient synthesis of the tetrasaccharide repeating unit of the O-antigen of Escherichia coli O174 strain Ishani Bhaumik, Tamashree Ghosh, Anup Kumar Misra ⇑ Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII-M, Kolkata 700054, India

a r t i c l e

i n f o

Article history: Received 12 June 2014 Received in revised form 21 July 2014 Accepted 4 August 2014 Available online 12 August 2014

a b s t r a c t The tetrasaccharide repeating unit of the O-antigen of Escherichia coli O174 strain was synthesized applying sequential glycosylations of suitably functionalized monosaccharide intermediates. Activation of glycosyl trichloroacetimidate derivatives using nitrosyl tetrafluoroborate (NOBF4) has been used during the synthesis. The glycosylation steps were high yielding with satisfactory stereo outcome. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Glycosylation Trichloroacetimidate Nitrosyl tetrafluoroborate Escherichia coli Tetrasaccharide Thioglycoside

1. Introduction Most of the gastroenteric infections in humans are the result of the intake of contaminated food and water as well as lack of adequate sanitations.1 The major causes for the diarrhoeal infections recorded till date are due to the infections of pathogenic Escherichia coli (E. coli), Shigella and Salmonella strains.2–4 E. coli strains associated with gastrointestinal infections, are classified into several pathotypes,5 among which Verotoxin producing E. coli (VTEC) strains are considered as the most important human pathogens, which cause diarrhoea as well as life threatening haemorrhagic colitis and haemolytic uraemic syndrome.6 E. coli O174 strain belongs to the VTEC in general and causes diarrhoea and other gastric complications in humans.7 Since, the cell wall O-antigen is associated with the virulence factor of the pathogenic bacteria, the structure of the tetrasaccharide repeating unit of the O-antigenic polysaccharide of E. coli O174 has been reported by Fontana et al. (Fig. 1).8 Because of the important roles of the cell wall O-antigens in the initial stage of bacterial infections, it is quite pertinent to develop therapeutics based on the glycoconjugate derivatives related to them. However, it is quite difficult to isolate the sufficient quantity of the oligosaccharides from the natural sources (bacterial cell wall) with appropriate purity for their comprehensive biological studies. Therefore, development of efficient synthetic strategies for the chemical synthesis of the oligosaccharides is highly essential for getting access ⇑ Corresponding author. Fax: +91 33 2355 3886. E-mail address: [email protected] (A.K. Misra). http://dx.doi.org/10.1016/j.carres.2014.08.003 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

to the significant quantity of materials. Hence, an efficient synthesis of the tetrasaccharide repeating unit of the O-antigenic polysaccharide of E. coli O174 is reported herein (Fig. 1). 2. Results and discussion The synthesis of the target tetrasaccharide (1), the 2-(pmethoxyphenoxy)ethyl glycoside was achieved using sequential glycosylations of suitably protected monosaccharide intermediates (Fig. 2) applying a number of recently developed reaction methodologies. Commercially available reducing sugars were judiciously protected using a number of literature reported reaction conditions to get protected glycosyl donors and acceptors 2,9 3,10 411 and 512 in excellent yield. D-Galactosamine 2-(p-methoxyphenoxy)ethyl glycoside derivative (2) was prepared from tri-O-acetyl-D-galactal using a recently reported reaction condition.9 Stereoselective preparation of glycosidic linkages was achieved using nitrosyl tetrafluoroborate (NOBF4) mediated glycosylation of glycosyl trichloroacetimidate derivatives.13 Late stage oxidation of the protected tetrasaccharide derivative using a combination of TEMPO and iodobenzene diacetate (BAIB) furnished D-glucuronic acid containing tetrasaccharide derivative.14 The presence of a 2-(p-methoxyphenoxy)ethyl group as the anomeric protecting group could provide the scope for the further glycoconjugate formation with relevant aglycons after oxidative removal of the PMP group.15 Stereoselective glycosylation of D-galactosamine derivative (2) with earlier reported D-galactose trichloroacetimidate derivative (3)10 in the presence of NOBF413 furnished disaccharide derivative

22

I. Bhaumik et al. / Carbohydrate Research 399 (2014) 21–25

→4)-[β-D-GlcpNAc-(1→2)]-β-D-GlcpA-(1→3)-β-D-Galp-(1→3)-β-D-GalpNAc-(1→ Figure 1. Structure of the repeating unit of the cell wall O-antigen of Escherichia coli O174.

6, which was immediately treated with benzyl bromide in the presence of solid sodium hydroxide16 to give compound 7 in 79% over all yield. Spectral analysis of compound 7 confirmed its formation [signals at d 4.81 (d, J = 7.5 Hz, H-1A), 4.48 (d, J = 8.0 Hz, H-1B) in the 1H NMR and at d 103.9 (C-1B), 103.0 (C-1A) in the 13 C NMR spectra (please see Section 3)]. Oxidative removal of p-methoxybenzyl (PMB)17 group from compound 7 was achieved by the treatment with 2,3-dicholoro-5,6-dicyano-1,4-benzoquinone (DDQ) to give disaccharide acceptor 8 in 79% yield. NOBF4 mediated stereoselective glycosylation of compound 8 with D-glucose trichloroacetimidate derivative (4) furnished trisaccharide derivative 9 in 76% yield. Formation of compound 9 was unambiguously confirmed from its spectral analysis [signals at d 4.84 (d, J = 10.0 Hz, H-1C), 4.81 (d, J = 8.0 Hz, H-1B) and 4.47 (d, J = 7.5 Hz, H-1A) in the 1H NMR and at d 103.5 (C-1B), 103.0 (C-1A) and 101.4 (C-1C) in the 13C NMR spectra (please see Section 3)]. DeO-acetylation of compound 9 using sodium methoxide18 resulted in the formation of trisaccharide acceptor 10 in 90% yield. Stereoselective 1,2-trans glycosylation of compound 10 with D-glucosamine thioglycoside derivative (5) in the presence of a combination of N-iodosuccinimide (NIS) and triflic acid (TfOH)19,20 produced tetrasaccharide derivative 11 in 69% yield. Spectral analysis of compound 11 supported its formation having required stereochemistry at the glycosyl linkages [signals at d 5.90 (d, J = 8.0 Hz, H-1D), 5.13 (d, J = 7.0 Hz, H-1C), 4.86 (d, J = 8.0 Hz, H-1B) and 4.56 (d, J = 7.5 Hz, H-1A) in the 1H NMR and d 103.4 (C-1A), 103.0 (C-1B), 101.0 (C-1C) and 99.0 (C-1D) in the 13C NMR spectra (please see Section 3)]. Finally, compound 11 was subjected to a sequence of consecutive functional group transformations, which include (a) treatment with thioacetic acid in pyridine for the direct conversion of azido group into acetamido group;21 (b) treatment with hydrazine monohydrate to remove phthaloyl group22 followed by acetylation using acetic anhydride and pyridine; (c) acidic hydrolysis of benzylidene acetals by the treatment with 80% aq acetic acid at elevated temperature followed by acetylation of the free hydroxyl groups using acetic anhydride and pyridine; (d) removal of benzyl groups using a catalytic transfer hydrogenation condition in the presence of a combination of 20% Pd(OH)2–C and triethylsilane;23 (e) selective oxidation of the primary hydroxyl group19 into carboxylic functionality using a combination of TEMPO and BAIB, without affecting the secondary hydroxyl groups and (f) saponification of the oxidized tetrasaccharide derivative using sodium methoxide to furnish the tetrasaccharide as its 2-(p-methoxyphenoxy)ethyl glycoside (1). The deprotected tetrasaccharide was passed through a column of

HO HO HO

OH O

D

C

3. Experimental 3.1. 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 in hot plate. Silica gel 230–400 mesh was used for column chromatography. NMR spectra were recorded on Brucker Avance 500 MHz using CDCl3 as solvent and TMS as internal reference unless stated otherwise. Chemical shift value is expressed in d ppm. The complete assignment of proton and carbon spectra was carried out by using a standard set of NMR experiments, for example, 1H NMR, 13C NMR, 13C DEPT 135, 2D COSY and 2D HSQC. MS were recorded on a Micromass and Brucker mass spectrometer. Optical rotations were recorded on a Jasco P-2000 spectrometer. Commercially available grades of organic solvents of adequate purity are used in all reactions. 3.2. 2-(p-Methoxyphenoxy)ethyl O-[2-O-benzyl-4,6-Obenzylidene-3-O-(p-methoxy)benzyl-b-D-galactopyranosyl](1 ? 3)-2-azido-4,6-O-benzylidene-2-deoxy-b-Dgalactopyranoside (7) A solution of compound 2 (1.5 g, 3.38 mmol) and compound 3 (2.6 g, 4.08 mmol) in anhydrous CH2Cl2 (20 mL) was cooled to – 20 °C under argon. To the cooled reaction mixture was added NOBF4 (480 mg, 4.11 mmol) and the reaction mixture was allowed

HO HO OH O B O O OH

O

A

OH O O NHAc

OPMP

1

NHAc Ph

Ph

O O

O O HO

O HO HO O

Sephadex LH-20 column to give pure compound 1 in 49% over all yield. Spectral analysis of compound 1 unambiguously confirmed its formation with required stereochemistry at the glycosyl linkages present in it [signals at d 4.66 (d, J = 8.5 Hz, H-1D), 4.53 (d, J = 7.0 Hz, H-1C), 4.46 (d, J = 9.0 Hz, H-1A) and 4.35 (d, J = 7.0 Hz, H-1B) in the 1H NMR and at d 104.1 (C-1B), 102.4 (C-1C), 101.9 (C-1D) and 101.4 (C-1A) in the 13C NMR spectra (please see Section 3)] (Scheme 1). In summary, a straightforward synthetic strategy has been developed for the synthesis of the tetrasaccharide repeating unit corresponding to the cell wall O-antigen of Escherichia coli O174 strain. A linear approach using sequential glycosylations has been applied to achieve the target tetrasaccharide as its 2-(p-methoxyphenoxy)ethyl glycoside. Glycosyl trichloroacetimidate and thioglycoside derivatives have been used as glycosyl donors during the synthesis. The yield of glycosylation steps was very good.

A

O O

OPMP PMBO

B

O NH

BnO BnO

N3 BzO O CCl 3 2 3 PMB: p-methoxybenzyl; PMP: p-methoxyphenyl

C

Ph

OBn O NH

AcO O 4

CCl3

O O AcO

D

O

O N

SEt O

5

Figure 2. Structure of the synthesized tetrasaccharide 1, the 2-(p-methoxyphenoxy)ethyl glycoside corresponding to the repeating unit of the O-antigen of Escherichia coli O174.

I. Bhaumik et al. / Carbohydrate Research 399 (2014) 21–25

Ph

Ph

O O

O O 2+3

a

B

PMBO b

O O OR 6: R = Bz 7: R = Bn

O

A

O

OPMP

N3

c Ph

Ph

O O

O O O

B

HO

O

A

O

O

OPMP

N3

OBn 8

Ph

BnO BnO

O O

O O

OBn O

C

B

O OR e

O

A

O OBn 9: R = Ac 10: R = H

O O

OPMP

N3

5 f Ph

Ph

Ph

O O AcO

BnO BnO O D

C

N O

OBn O

O O

O O B

O

O O OBn

O O

H-1A), 4.76–4.65 (3 d, J = 11.0 Hz each, 3H, PhCH2), 4.48 (d, J = 8.0 Hz, 1H, H-1B), 4.34 (d, J = 3.5 Hz, 1H, H-4B), 4.27–4.19 (m, 3H, H-6abA, OCH2), 4.15–4.12 (m, 2H, H-6abB), 4.03 (d, J = 3.0 Hz, 1H, H-4A), 4.01–3.95 (m, 3H, OCH2), 3.92 (dd, J = 7.5 Hz each, 1H, H-2A), 3.88 (dd, J = 8.0 Hz each, 1H, H-2B), 3.79 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.62 (dd, J = 10.5, 3.0 Hz, 1H, H-3B), 3.55 (dd, J = 10.5, 3.0 Hz, 1H, H-3A), 3.39–3.38 (m, 1H, H-5A), 3.31–3.30 (m, 1H, H-5B); 13C NMR (125 MHz, CDCl3): d 159.2–113.7 (Ar-C), 103.9 (C-1B), 103.0 (C-1A), 101.2 (PhCH), 100.7 (PhCH), 78.5 (C-3A), 78.4 (C-2B), 75.9 (C-3B), 75.3 (C-4B), 75.1 (PhCH2), 74.1 (C-4A), 71.9 (PhCH2), 69.2 (C-6A), 68.9 C-6B), 68.1 (OCH2), 68.0 (OCH2), 66.8 (C-5B), 66.5 (C-5A), 62.4 (C-2A), 55.6 (OCH3), 55.2 (OCH3); ESI-MS: 926.3 [M+Na]+; Anal. Calcd for C50H53N3O13 (903.36): C, 66.43; H, 5.91. Found: C, 66.26; H, 6.05. 3.3. 2-(p-Methoxyphenoxy)ethyl O-(2-O-benzyl-4,6-Obenzylidene-b-D-galactopyranosyl)-(1 ? 3)-2-azido-4,6-Obenzylidene-2-deoxy-b-D-galactopyranoside (8)

4 d Ph

23

A

O O

OPMP

N3

11 g, h, i, j, k, l, m 1

Scheme 1. Reagents and conditions: (a) NOBF4, CH2Cl2, 20 °C, 30 min; (b) benzyl bromide, NaOH, THF, TBAB, 50 °C, 3 h, 79% in two steps; (c) DDQ, CH2Cl2–H2O (3:1), 5 °C, 4 h, 79%; (d) NOBF4, CH2Cl2, 30 °C, 20 min, 76%; (e) 0.1 M CH3ONa, CH3OH, room temperature, 2 h, 90%; (f) NIS, TMSOTf, MS 4 Å, CH2Cl2, 10 °C, 45 min, 69%; (g) CH3COSH, pyridine, room temperature, 18 h; (h) (1) NH2NH2
H2O, EtOH, 90 °C, 10 h, (2) acetic anhydride, pyridine, room temperature, 3 h; (i) 80% aq AcOH, 80 °C, 2 h; (j) acetic anhydride, pyridine, room temperature, 3 h; (k) 20% Pd(OH)2–C, Et3SiH, CH3OH–CHCl3 (1:1), room temperature, 10 h; (l) TEMPO, BAIB, CH2Cl2, H2O, room temperature, 1 h; (m) 0.1 M CH3ONa, CH3OH, room temperature, 4 h, 49% over all yield.

to stir at same temperature for 30 min. The reaction mixture was diluted with CH2Cl2 (100 mL) and successively washed with satd. aq NaHCO3 and water, dried (Na2SO4) and concentrated. To a solution of the crude product in THF (20 mL) were added benzyl bromide (0.5 mL, 4.20 mmol), powdered NaOH (300 mg, 7.5 mmol) and tetrabutylammonium bromide (TBAB; 50 mg) and the reaction mixture was stirred briskly at 50 °C for 3 h. The reaction mixture was poured into water and extracted with CH2Cl2 (100 mL). The organic layer was washed with satd. aq NaHCO3, water, dried (Na2SO4) and concentrated. The crude product was purified over SiO2 using hexane-EtOAc (5:1) as eluent to give pure compound 7 (2.4 g, 79%). Yellow oil; [a]25 D +34.5 (c 1.0, CHCl3); IR (neat): 3100, 3078, 2965, 2870, 2125, 1515, 1508, 1455, 1365, 1286, 1253, 1217, 1100, 1092, 1058, 822, 755, 699 cm 1; 1H NMR (500 MHz, CDCl3): d 7.58–6.78 (m, 23H, Ar-H), 5.57 (s, 1H, PhCH), 5.47 (s, 1H, PhCH), 4.99 (d, J = 11.0 Hz, 1H, PhCH2), 4.81 (d, J = 7.5 Hz, 1H,

To a solution of compound 7 (2.2 g, 2.43 mmol) in CH2Cl2–H2O (20 mL; 3:1 v/v) was added DDQ (750 mg, 3.30 mmol) and the biphasic reaction mixture was stirred at 5 °C for 4 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and the organic layer was successively washed with satd. aq 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 8 (1.5 g, 79%). White solid; mp 73–74 °C [EtOH]; [a]25 D +20 (c 1.0, CHCl3); IR (KBr): 3420, 2999, 2920, 2846, 2180, 1501, 1492, 1360, 1273, 1207, 1006, 995, 813, 696 cm 1; 1H NMR (500 MHz, CDCl3): d 7.56–6.79 (m, 19 H, Ar-H), 5.58 (s, 1H, PhCH), 5.53 (s, 1H, PhCH), 5.03 (d, J = 11.0 Hz, 1H, PhCH2), 4.84 (d, J = 7.5 Hz, 1H, H-1B), 4.73 (d, J = 11.0 Hz, 1H, PhCH2), 4.50 (d, J = 8.0 Hz, 1H, H1A), 4.34 (d, J = 3.0 Hz, 1H, H-4B), 4.29–4.20 (m, 3H, H-6aA, OCH2), 4.18 (d, J = 3.0 Hz, 1H, H-4A), 4.17–4.13 (m, 2H, H-6abB), 4.07– 3.96 (m, 3H, H-6bA, OCH2), 3.91 (dd, J = 8.0 Hz each, 1H, H-2A), 3.75 (s, 3H, OCH3), 3.74–3.71 (m, 1H, H-2B), 3.66–3.62 (m, 2H, H3A, H-3B), 3.43–3.42 (m, 1H, H-5A), 3.41–3.40 (m, 1H, H-5B); 13C NMR (125 MHz, CDCl3): d 155.0–114.6 (Ar-C), 103.8 (C-1B), 102.9 (C-1A), 101.3 (PhCH), 100.8 (PhCH), 78.7 (C-3A), 76.0 (C-3B), 75.3 (2 C, C-4A, C-4B), 74.7 (PhCH2), 72.1 (C-2B), 69.2 (C-6A), 68.9 (C6B), 68.1 (OCH2), 68.0 (OCH2), 66.8 (C-5A), 66.5 (C-5B), 62.5 (C2A), 55.6 (OCH3); ESI-MS: 806.3 [M+Na]+; Anal. Calcd for C42H45N3O12 (783.30): C, 64.36; H, 5.79. Found: C, 64.20; H, 6.00. 3.4. 2-(p-Methoxyphenoxy)ethyl O-(2-O-acetyl-3,4,6-tri-Obenzyl-b-D-glucopyranosyl)-(1 ? 3)-O-(2-O-benzyl-4,6-Obenzylidene-b-D-galactopyranosyl)-(1 ? 3)-2-azido-4,6-Obenzylidene-2-deoxy-b-D-galactopyranoside (9) A solution of compound 8 (1.4 g, 1.78 mmol) and compound 4 (1.5 g, 2.35 mmol) in anhydrous CH2Cl2 (15 mL) was cooled to 30 °C under argon. To the cooled reaction mixture was added NOBF4 (300 mg, 2.56 mmol) and the reaction mixture was allowed to stir at same temperature for 20 min. The reaction mixture was diluted with CH2Cl2 (100 mL) and successively washed with satd. aq 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 9 (1.7 g, 76%). Yellow oil; [a]25 D +29 (c 1.0, CHCl3); IR (neat): 3089, 3075, 3020, 2850, 1744, 1496, 1460, 1385, 1215, 1045, 752, 699, 667 cm 1; 1H NMR (500 MHz, CDCl3): d 7.51–6.78 (m, 34H, Ar-H), 5.53 (s, 1H, PhCH), 5.48 (s, 1H, PhCH), 5.07 (t, J = 8.0 Hz, 1H, H-2C), 4.92 (d, J = 11.0 Hz, 1H, PhCH2), 4.84 (d, J = 10.0 Hz, 1H, H-1C), 4.81 (d, J = 8.0 Hz, 1H, H-1B), 4.77–4.74 (m, 2H, PhCH2), 4.66 (d, J = 11.0 Hz, 1H, PhCH2), 4.58–4.48 (4d, J = 11.0 Hz each, 4H, PhCH2), 4.47 (d, J = 7.5 Hz, 1H, H-1A), 4.29 (d, J = 3.0 Hz, 1H, H-4B), 4.25 (br s, 1H, H-4A), 4.22–4.12 (m, 5H,

24

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H-6aA, H-6abB, OCH2), 3.98–3.93 (m, 2H, OCH2), 3.90–3.87 (m, 1H, H-6bA), 3.85 (t, J = 8.0 Hz, 1H, H-2A), 3.83–3.80 (m, 2H, H-2B, H-4C), 3.75 (s, 3H, OCH3), 3.71–3.58 (m, 5H, H-3A, H-3B, H-3C, H6abc), 3.50–3.45 (m, 1H, H-5C), 3.37–3.36 (m, 1H, H-5A), 3.31–3.30 (m, 1H, H-5 B), 1.78 (s, 3H, COCH3); 13C NMR (125 MHz, CDCl3): d 169.2 (COCH3), 155.0–114.6 (Ar-C), 103.5 (C-1B), 103.0 (C-1A), 101.4 (C-1C), 100.7 (PhCH), 100.3 (PhCH), 83.1 (C-3C), 78.2 (C-3B), 78.0 (C-2B), 77.7 (C-4C), 75.7 (C-5C), 75.1 (2 C, C-4A, C-4B), 74.9 (PhCH2), 74.8 (PhCH2), 74.7 (PhCH2), 74.6 (C-3A), 73.4 (PhCH2), 73.0 (C-2C), 69.2 (C-6C), 68.9 (C-6A), 68.8 (C-6B), 68.1 (OCH2), 67.9 (OCH2), 66.8 (C-5A), 66.6 (C-5B), 62.3 (C-2A), 55.6 (OCH3), 20.8 (COCH3); ESI-MS: 1280.5 [M+Na]+; Anal. Calcd for C71H75N3O18 (1257.50): C, 67.77; H, 6.01. Found: C, 67.60; H, 6.15. 3.5. 2-(p-Methoxyphenoxy)ethyl O-(3,4,6-tri-O-benzyl-b-Dglucopyranosyl)-(1 ? 3)-O-(2-O-benzyl-4,6-O-benzylidene-b-Dgalactopyranosyl)-(1 ? 3)-2-azido-4,6-O-benzylidene-2-deoxyb-D-galactopyranoside (10) A solution of compound 9 (1.5 g, 1.19 mmol) in CH3ONa (0.1 M solution in CH3OH; 30 mL) was allowed to stir at room temperature for 2 h. The reaction mixture was neutralized with Dowex 50 W X8 (H+) resin, filtered and concentrated. The crude product was passed through a short pad of SiO2 using hexane–EtOAc (2:1) as eluent to give pure compound 10 (1.3 g, 90%). White solid; mp 150–152 °C; [a]25 D +19 (c 1.0, CHCl3); IR (KBr): 3314, 3013, 2929, 1605, 1496, 1372, 1218, 1051, 760, 746 cm 1; 1H NMR (500 MHz, CDCl3): d 7.54–6.79 (m, 34H, Ar-H), 5.58 (s, 1H, PhCH), 5.48 (s, 1H, PhCH), 4.98–4.94 (2 d, J = 11.0 Hz each, 2H, PhCH2), 4.84 (d, J = 9.5 Hz, 1H, H-1C), 4.83–4.65 (3 d, J = 11.0 Hz each, 3H, PhCH2), 4.59 (d, J = 7.5 Hz, 1H, H-1B), 4.57–4.47 (3 d, J = 11.0 Hz each, 3H, PhCH2), 4.46 (d, J = 8.0 Hz, 1H, H-1A), 4.35 (d, J = 3.5 Hz, 1H, H-4B), 4.30–4.13 (m, 6H, H-4A, H-6aA, OCH2, H-6abB), 4.04– 3.85 (m, 5H, H-2A, H-3A, H-6bA, OCH2), 3.79 (dd, J = 10.0, 3.0 Hz, 1H, H-3B), 3.75 (s, 3H, OCH3), 3.67–3.60 (m, 3H, H-2C, H-6abC), 3.58–3.48 (m, 3H, H-2B, H-3C, H-4C), 3.46–3.34 (m, 3H, H-5A, H-5B, H-5C); 13C NMR (125 MHz, CDCl3): d 155.0–114.3 (Ar-C), 104.3 (C-1B), 103.9 (C-1C), 103.0 (C-1A), 101.0 (PhCH), 100.8 (PhCH), 84.2 (C-3C), 78.5 (C-3B), 77.8 (C-2B), 77.3 (C-4C), 76.7 (C2C), 75.9 (C-5C), 75.6 (C-4A), 75.3 (C-4B), 75.1 (C-3A), 75.0 (PhCH2), 74.9 (2 C, 2 PhCH2), 73.4 (PhCH2), 69.3 (C-6C), 69.0 (C-6A), 68.9 (C6B), 68.1 (OCH2), 68.0 (OCH2), 66.8 (C-5A), 66.5 (C-5B), 62.3 (C-2A), 55.6 (OCH3); ESI-MS: 1238.4 [M+Na]+; Anal. Calcd for C69H73N3O17 (1215.49): C, 68.13; H, 6.05. Found: C, 67.95; H, 6.25. 3.6. 2-(p-Methoxyphenoxy)ethyl O-(3-O-acetyl-4,6-Obenzylidene-2-deoxy-2-phthalimido-b-D-glucopyranosyl)(1 ? 2)-O-(3,4,6-tri-O-benzyl-b-D-glucopyranosyl)-(1 ? 3)-O-(2O-benzyl-4,6-O-benzylidene-b-D-galactopyranosyl)-(1 ? 3)-2azido-4,6-O-benzylidene-2-deoxy-b-D-galactopyranoside (11) To a solution of compound 10 (1.2 g, 0.98 mmol) and compound 5 (525 mg, 1.08 mmol) in anhydrous CH2Cl2 (10 mL) was added MS 4 Å (1 g) and it was cooled to 10 °C under argon. To the cooled reaction mixture were added NIS (275 mg, 1.22 mmol) and TfOH (5 lL) and the reaction mixture was allowed to stir at same temperature for 45 min. The reaction mixture was filtered and washed with CH2Cl2 (100 mL). The organic layer was successively washed with 5% Na2S2O3, satd. aq 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 11 (1.1 g, 69%). White solid; mp 110–112 °C; [a]25 D +15 (c 1.0, CHCl3); IR (KBr): 3063, 3032, 2923, 2860, 2114, 1776, 1743, 1717, 1611, 1508, 1454, 1387, 1230, 1092, 1055, 999, 821, 721, 697 cm 1; 1H NMR (500 MHz, CDCl3): d 7.72–6.75 (m, 43H, Ar-H), 5.90 (d, J = 8.0 Hz, 1H, H-1D), 5.60 (s, 1H, PhCH), 5.57 (s, 1H, PhCH), 5.46

(t, J = 8.0 Hz, 1H, H-3D), 5.13 (d, J = 7.0 Hz, 1H, H-1C), 5.02 (s, 1H, PhCH), 4.98 (d, J = 11.0 Hz, 1H, PhCH2), 4.86 (d, J = 8.0 Hz, 1H, H-1B), 4.77 (d, J = 11.0 Hz, 1H, PhCH2), 4.68 (d, J = 11.0 Hz, 1H, PhCH2), 4.56 (d, J = 7.5 Hz, 1H, H-1A), 4.55–4.50 (m, 2H, PhCH2), 4.43–4.36 (m, 4H, H-4B, PhCH2), 4.30 (br s, 1H, H-4A), 4.29–4.14 (m, 8H, H-2D, H-4A, H-6abA, H-6abB, OCH2), 4.03–3.93 (m, 6H, H-2A, H-2B, H-6abD, OCH2), 3.75 (s, 3H, OCH3), 3.73–3.71 (m, 1H, H-3A), 3.56–3.50 (m, 4H, H-2C, H-4C, H-6abC), 3.48 (t, J = 9.5 Hz, 1H, H-4D), 3.44–3.37 (m, 3H, H-3B, H-5A, H-5B), 3.31 (t, J = 8.5 Hz, 1H, H-3C), 3.30–3.26 (m, 1H, H-5D), 3.12–3.10 (m, 1H, H-5C), 1.78 (s, 3H, COCH3); 13C NMR (125 MHz, CDCl3): d 170.5 (COCH3), 167.7, 167.4 (PhthCO), 155.0–114.6 (Ar-C), 103.4 (C-1A), 103.0 (C1B), 101.0 (C-1C), 100.9 (PhCH), 100.7 (PhCH), 100.5 (PhCH), 99.0 (C-1D), 83.6 (C-3C), 81.8 (C-4C), 79.6 (C-2B), 78.7 (C-4D), 77.9 (C-3B), 75.7 (C-4A), 75.2 (C-4B), 74.6 (2 C, 2 PhCH2), 74.5 (C-3A), 74.4 (PhCH2), 73.9 (C-2C), 73.7 (C-5C), 73.4 (PhCH2), 70.2 (C-3D), 68.9 (C-6C), 68.8 (C-6D), 68.5 (2 C, C-6A, C-6B), 68.1 (OCH2), 67.9 (OCH2), 66.9 (C-5A), 66.6 (C-5B), 65.7 (C-5D), 62.5 (C-2A), 56.4 (C-2D), 55.6 (OCH3), 20.5 (COCH3); MALDI-MS: 1659.6 [M+Na]+; Anal. Calcd for C92H92N4O24 (1636.61): C, 67.47; H, 5.66. Found: C, 67.30; H, 5.85. 3.7. 2-(p-Methoxyphenoxy)ethyl O-(2-acetamido-2-deoxy-b-Dglucopyranosyl)-(1 ? 2)-O-(b-D-glucopyranosiduronic acid)(1 ? 3)-O-(b-D-galactopyranosyl)-(1 ? 3)-2-acetamido-2-deoxyb-D-galactopyranoside (1) To a solution of compound 11 (1 g, 0.61 mmol) in pyridine (5 mL) was added CH3COSH (0.5 mL, 7.0 mmol) and the reaction mixture was stirred at room temperature for 18 h. The solvents were removed under reduced pressure to give the crude product, which was used directly in the next step. To a solution of the product in EtOH (20 mL) was added NH2NH2H2O (0.5 mL) and the reaction mixture was stirred at 90 °C for 10 h. The solvents were removed under reduced pressure and a solution of the crude product in acetic anhydride and pyridine (5 mL; 1:1 v/v) was kept at room temperature for 3 h. The solvents were removed under reduced pressure and a solution of the crude product in 80% aq AcOH (15 mL) was stirred at 80 °C for 2 h. The solvents were removed under reduced pressure and a solution of the crude product in acetic anhydride and pyridine (5 mL; 1:1 v/v) was kept at room temperature for 3 h. The solvents were removed under reduced pressure and the crude product was passed through a short pad of SiO2 using EtOAc as eluent. To a solution of the acetylated product in CH3OH–CHCl3 (10 mL; 1:1, v/v) were added 20% Pd(OH)2–C (0.1 g) and Et3SiH (2 mL, 12.52 mmol) and the reaction mixture was allowed to stir at room temperature for 10 h. The reaction mixture was filtered through a CeliteÒbed and the filtering bed was washed with CH3OH–CH2Cl2 (50 mL; 1:1 v/v). The combined filtrate was concentrated to give the crude product, which was dissolved in CH2Cl2–H2O (15 mL; 2:1, v/v). To the solution of the hydrogenolized product were added TEMPO (25 mg, 0.16 mmol) and BAIB (0.5 g, 1.55 mmol) at 5 °C and the reaction mixture was allowed to stir vigorously at room temperature for 1 h. The reaction was quenched by addition of satd. aq Na2S2O3 (5 mL) and diluted with CH2Cl2 (50 mL). The organic layer was successively washed with satd. NaCl and water, dried (Na2SO4) and concentrated to give the oxidized product, which was passed through a short pad of SiO2. Finally, a solution of the oxidized product in CH3ONa (0.1 M solution in CH3OH; 20 mL) was stirred at room temperature for 4 h. The reaction mixture was neutralized with Dowex 50W X8 (H+) resin, filtered and concentrated. The product was passed through a column of Sephadex LH-20 using CH3OH–H2O (3:1) as eluent to give pure compound 1 (275 mg, 49%). White powder; [a]25 D +8 (c 1.0, H2O); IR (KBr): 3420, 2929, 2856, 1706, 1640, 1595, 1556, 1510, 1435, 1221, 1149, 1080,

I. Bhaumik et al. / Carbohydrate Research 399 (2014) 21–25

1030, 969 cm 1; 1H NMR (500 MHz, D2O): d 6.83–6.81 (m, 4H, ArH), 4.66 (d, J = 8.5 Hz, 1H, H-1D), 4.53 (d, J = 7.0 Hz, 1H, H-1C), 4.46 (d, J = 9.0 Hz, 1H, H-1A), 4.35 (d, J = 7.0 Hz, 1H, H-1B), 4.03 (d, J = 3.5 Hz, 1H, H-4A), 4.02–3.96 (m, 3H, OCH2), 3.95 (d, J = 3.0 Hz, 1H, H-4B), 3.85 (t, J = 8.0 Hz, 1H, H-2A), 3.82–3.77 (m, 2H, H-3A, OCH2), 3.76–3.68 (m, 2H, H-4C, H-5A), 3.63 (s, 3H, OCH3), 3.61– 3.48 (m, 11H, H-2B, H-2D, H-3B, H-6abA, H-6abB, H-6abC, H-6abD), 3.42–3.32 (m, 4H, H-2C, H-3C, H-5B, H-5C), 3.28–3.18 (m, 3H, H3D, H-4D, H-5D), 1.92, 1.76 (2 s, 6H, 2 COCH3); 13C NMR (125 MHz, D2O): d 175.2 (COOH), 174.8, 174.4 (2 COCH3), 153.4– 115.1 (Ar-C), 104.1 (C-1B), 102.4 (C-1C), 101.9 (C-1D), 101.4 (C-1A), 83.4 (C-3B), 80.8 (C-2C), 80.4 (C-3A), 76.2 (C-4C), 76.1 (C-5C), 75.5 (C-5B), 74.7 (C-5D), 74.6 (C-3C), 73.5 (C-3D), 70.1 (C-5A), 69.6 (2 C, C-2B, C-4D), 68.3 (OCH2), 68.1 (OCH2), 67.8 (2 C, C-4A, C-4B), 61.0 (C-6D), 60.9 (C-6A), 60.4 (C-6B), 56.0 (C-2D), 55.9 (OCH3), 51.0 (C-2A), 22.2 (2 C, 2 COCH3); ESI-MS: 935.3 [M+Na]+; Anal. Calcd for C37H56N2O24 (912.32): C, 48.68; H, 6.18. Found: C, 48.50; H, 6.38. Acknowledgments I.B. and T.G. thank CSIR, India for providing Junior and Senior Research Fellowships respectively. This work was supported by the Department of Science and Technology, India (Project No. SR/S1/OC-83/2010). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carres.2014. 08.003.

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References 1. Scott, E. Can. J. Infect. Dis. 2003, 14, 277–280. 2. DuPont, H. L.; Formal, S. B.; Hornick, R. B.; Snyder, M. J.; Libonati, J. P.; Sheahan, D. G.; LaBrec, E. H.; Kalas, J. P. N. Engl. J. Med. 1971, 285, 1–9. 3. Hodges, K.; Gill, R. Gut Microbes 2010, 1, 4–21. 4. Leclerc, H.; Schwartbrod, L.; Dei-Cas, E. Crit. Rev. Microbiol. 2002, 28, 371–409. 5. Nataro, J. P.; Kaper, J. B. Clin. Microbiol. Rev. 1998, 11, 142–201. 6. Piérard, D.; De Greve, H.; Haesebrouck, F.; Mainil, J. Vet. Res. 2012, 43, 13. 7. Scheutz, F.; Cheasty, T.; Woodward, D.; Smith, H. R. APMIS 2004, 112, 569–584. 8. Fontana, C.; Lundborg, M.; Weintraub, A.; Widmalm, G. Carbohydr. Res. 2012, 354, 102–105. 9. Santra, A.; Misra, A. K. Tetrahedron: Asymmetry 2010, 21, 2612–2618. 10. Hsu, C.-H.; Chu, K.-C.; Lin, Y.-S.; Han, J.-L.; Peng, Y.-S.; Ren, C.-T.; Wu, C.-Y.; Wong, C.-H. Chem. Eur. J. 2010, 16, 1754–1760. 11. Schmidt, R. R.; Effenberger, G. Liebigs Ann. Chem. 1987, 825–831. 12. Kihlberg, J. O.; Leigh, D. A.; Bundle, D. R. J. Org. Chem. 1990, 55, 2860–2863. 13. Sau, A.; Santra, A.; Misra, A. K. Synlett 2012, 2341–2348. 14. van den Bos, L. J.; Codée, J. D. C.; van der Toorn, J. C.; Boltje, T. J.; van Boom, J. H.; Overkleeft, H. S.; van der Marel, G. A. Org. Lett. 2004, 6, 2165–2168. 15. Tanikawa, T.; Fridman, M.; Zhu, W.; Faulk, B.; Joseph, I. C.; Kahne, D.; Wagner, B. K.; Clemons, P. A. J. Am. Chem. Soc. 2009, 131, 5075–5083. 16. Madhusudan, S. K.; Agnihotri, G.; Negi, D. S.; Misra, A. K. Carbohydr. Res. 2005, 340, 1373–1377. 17. Okiawa, Y.; Yoshioko, T.; Yonemitsu, O. Tetrahedron Lett. 1982, 23, 885–888. 18. Zemplén, G. Ber. Dtsch. Chem. Ges. 1926, 59, 1254–1266. 19. Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H. Tetrahedron Lett. 1990, 31, 1331–1334. 20. Konradsson, P.; Udodong, U. E.; Fraser-Reid, B. Tetrahedron Lett. 1990, 31, 4313–4316. 21. Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1996, 292, 71–81. 22. Lee, H.-H.; Schwartz, D. A.; Harris, J. F.; Carver, J. P.; Krepinsky, J. J. Can. J. Chem. 1986, 64, 1912–1918. 23. Santra, A.; Ghosh, T.; Misra, A. K. Beilstein J. Org. Chem. 2013, 9, 74–78.