Synthesis of penta- and hexasaccharide fragments corresponding to the O-antigen of Escherichia coli O150

Tetrahedron: Asymmetry 21 (2010) 2142–2152

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Synthesis of penta- and hexasaccharide fragments corresponding to the O-antigen of Escherichia coli O150 Rajib Panchadhayee, Anup Kumar Misra * Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII-M, Kolkata 700 054, India

a r t i c l e

i n f o

Article history: Received 2 June 2010 Accepted 17 June 2010 Available online 17 August 2010

a b s t r a c t The chemical synthesis of a pentasaccharide and a hexasaccharide corresponding to the O-antigen of Escherichia coli O150 has been achieved using sequential glycosylation and [3+3] block glycosylation strategies. Suitably protected monosaccharide synthons have been prepared from the commercially available reducing sugars and then stereoselectively coupled to give the pentasaccharide and a hexasaccharide in excellent yields. 4-Methoxyphenyl and 2-(4-methoxyphenoxy) ethyl groups have been used as the anomeric-protecting groups in the target pentasaccharide and a hexasaccharide, respectively. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Escherichia coli (E. coli) is a Gram-negative, nonmotile, facultatively anaerobic bacterium capable of both respiratory and fermentative metabolisms.1 E. coli serves a useful function in the human body by suppressing the growth of harmful bacteria and by synthesizing considerable amounts of vitamins.2 It colonizes the lower gut of animals and survives when released into the natural environment, allowing widespread distribution to the new hosts. Despite the usefulness of E. coli, a number of virulent E. coli strains are responsible for severe infection of the enteric/diarrheal, urinary tract infection, pulmonary, and nervous systems.3 Based on their nature of infections, the enteropathogenic strains of E. coli have been classified into several classes,4 which include (a) enteropathogenic E. coli (EPEC); (b) enteroinvasive E. coli (EIEC); (c) enterotoxigenic E. coli (ETEC); (d) enteroaggregative E. coli (EAEC); (e) diffusely adherent E. coli (DAEC); and (f) enterohemorrhagic E. coli (EHEC). Because of their capability to produce a bacteriophage-mediated Shiga-like toxin, EHEC strains are also termed as ‘Shiga toxin-producing E. coli’ (STEC).5 A well-studied and frequently found Shiga toxin-producing EHEC strain is E. coli O157:H7, which has been found to be associated with several lethal intestinal infections and outbreaks in developed countries.6 Besides E. coli O157:H7, several other E. coli serotypes associated with diarrheal diseases have been included in the STEC category, which include E. coli O4, O26, O55, O103, O111, O145, O150 etc.7 Recently, Perepelov et al. reported the structure of the oligosaccharide repeating unit of the O-antigen of E. coli O150,8 which is a member of STEC family and is associated with several outbreaks of gastrointestinal diseases in Europe (Fig. 1).9

* Corresponding author. Tel.: +91 33 25693240; fax: +91 33 2355 3886. E-mail address: [email protected] (A.K. Misra). 0957-4166/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetasy.2010.06.022

Being an integral part of the endotoxin, the O-antigen plays an important role in the pathogenicity of E. coli. In the past, glycoconjugates derived from the O-antigenic oligosaccharides from the bacterial cell wall have been used to develop antibacterial vaccine candidates.10 Several biological experiments are necessary for a detailed understanding of the relationship between the O-antigen with the pathogenicity of a particular bacterial strain, which in turn demands substantial quantities of the oligosaccharides in hand. The oligosaccharides isolated from the natural source cannot meet such a requirement. Therefore, the development of a concise chemical synthetic strategy is essential to provide the large quantity of a particular oligosaccharide. In this direction,11 we report herein a convenient synthesis of a pentasaccharide as its 4methoxyphenyl glycoside 1 together with a hexasaccharide as its 2-(4-methoxyphenoxy) ethyl glycoside 2 corresponding to the Oantigen of E. coli O150 using sequential and [3+3] block glycosylation strategies (Figs. 2 and 3). The 4-methoxyphenyl (PMP) group can be easily removed under oxidative conditions to provide the pentasaccharide hemiacetal and hexasaccharide derivative linked to an ethylene glycol linker useful for the a preparation of glycoconjugate derivatives. 2. Results and discussion This report comprises the synthesis of a pentasaccharide 1 and a hexasaccharide 2 using sequential glycosylation and block synthetic strategies, respectively. A [3+3] block synthetic strategy has been developed for the convenient synthesis of the target hexasaccharide as its 2-(4-methoxyphenoxy) ethyl glycoside 2 (Fig. 3). The synthetic strategy comprises a number of noteworthy features, which are (a) the stereoselective [3+3] block glycosylation; (b) the use of readily available suitably protected monosaccharide intermediates; (c) the exploitation of the orthogonal

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β-D-Glcp 1 ↓ 2 →3)-β-D-GlcpNAc4(Slac)-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1→ A OR β-D-Glcp 1 ↓ 2 →3)-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1→3)-β-D-GlcpNAc4(Slac)-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→ B Figure 1. Structure of the hexasaccharide repeating unit corresponding to the O-antigen of Escherichia coli O150.

OH O

HO O B

HO

HO HO

HO

B

OH OH O HO D O NHAc

HO O

NHAc E

O

O HO

OH OH

HO

E

HO

HO O

O OPMP NPhth

AcO

3

BnO BnO

Ph

BnO

O OAc 5 AcO AcO

O SEt NPhth 6

OAc O

Ph

OPMP

SEt

O O HO

OH 17

OPMP

SEt AcO 7

PMP: 4-Methoxyphenyl

O BnO BnO OAc 5 Ph O O O O AcO SEt SEt NPhth NPhth 6 18

BnO

OMBn

O O AcO

O

O

4 SEt

O

2

O

O

BnO

O

NHAc

OH OH

F

O HO

SEt

O O HO

C

HO OH HO O

OPMP

O

O

PMP: 4-Methoxyphenyl

1

Ph

HO

O

O

A

HO

O

C

D

O

OPMP NHAc

O

O HO

A

O

BnO BnO OH 19

AcO AcO

OAc O SEt AcO 7

Figure 2. Structure of the synthesized pentasaccharide 1 corresponding to the Oantigen of Escherichia coli O150 as its 4-methoxyphenyl glycoside. Figure 3. Structure of the synthesized hexasaccharide 2 corresponding to the Oantigen of Escherichia coli O150 as its 2-(4-methoxyphenoxy) ethyl glycoside.

property of thioglycoside 18 in the glycosylation of glycosyl trichloroacetimidate donor; (d) the use of PMP disaccharide 26 as a source of disaccharide trichloroacetimidate donor 27 to couple with the thioglycoside 18; and (e) the preparation of 2-(4methoxyphenoxy) ethyl glycoside, which offers the access to an ethylene glycol-linked hexasaccharide derivative for its further use. Initially, the synthesis of a hexasaccharide as its 4-methoxyphenyl glycoside corresponding to the structure A (Fig. 1) was attempted. For this purpose, a series of suitably protected monosaccharide intermediates 3,12 4, 5,13 6,14 and 715 have been prepared following the literature (Fig. 2). Starting from ethyl 4-O-benzyl-1-

thio-a-L-rhamnopyranoside 8,16 compound 4 was prepared by the selective 4-methoxybenzylation under phase transfer condition followed by acetylation of the remaining hydroxyl group in 70% yield. The stereoselective glycosylation of D-glucosamine hydrochloridederived compound 3 with L-rhamnopyranosyl thioglycoside derivative 4 in the presence of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) combination17 furnished disaccharide derivative 9 in 81% yield, which was deacetylated to give disaccharide acceptor 10 almost quantitatively. The NMR spectroscopic analysis of compound 9 confirmed its formation [d 5.69 (d,

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J = 8.4 Hz, H-1A), 5.49 (s, PhCH), 4.56 (br s, H-1B) in 1H NMR and d 101.8 (PhCH), 98.0 (C-1A), 97.7 (C-1B) in 13C NMR spectra). The NIS–TMSOTf17-mediated stereoselective glycosylation of compound 10 with the thioglycoside derivative 5 afforded trisaccharide derivative 11 in 80% yield, which on deacetylation gave trisaccharide acceptor 12 in 98% yield. The appearance of characteristic signals [d 102.0 (PhCH), 99.1 (C-1C), 97.9 (C-1A), 97.7 (C-1B)] in the 13C NMR spectra confirmed the formation of compound 11. Glycosylation of compound 12 with compound 5 in the presence of NIS–TMSOTf furnished tetrasaccharide derivative 13 in 81% yield, which was deacetylated to give tetrasaccharide acceptor 14 in 90% yield. The appearance of characteristic signals in the NMR spectra [d 5.84 (d, J = 8.4 Hz, H-1A), 5.55 (s, 1H, PhCH), 4.92 (br s, H-1C), 4.82 (br s, H1D), 4.58 (br s, H-1B) in 1H NMR and d 101.8 (PhCH), 100.2 (C-1C), 98.9 (C-1D), 97.9 (C-1A), 97.4 (C-1B) in 13C NMR) confirmed the formation of compound 13. The stereoselective glycosylation of compound 14 with the thioglycoside derivative 6 in the presence of NIS–TMSOTf furnished pentasaccharide derivative 15 in 71% yield, which was characterized by the appearance of signals in the NMR spectra [d 5.76 (d, J = 8.4 Hz, H-1A), 5.54 (s, PhCH), 5.42 (s, PhCH), 5.40 (d, J = 8.3 Hz, H-1E), 4.82–4.80 (m, 2H, H-1C, H-1D), 5.45 (br s, H-1B) in 1H NMR and d 101.8 (PhCH), 101.5 (PhCH), 100.8 (2C, C1C, C-1D), 100.4 (C-1E), 98.0 (C-1A), 97.6 (C-1B) in 13C NMR]. The DDQ-mediated oxidative removal18 of the 4-methoxybenzyl group from compound 15 afforded pentasaccharide acceptor 16 in 70% yield. However, after the successful preparation of pentasaccharide derivative 16, further NIS–TMSOTf-mediated b-selective glycosylation of the compound 16 with the thioglycoside 7 did not furnish any expected hexasaccharide except the formation of the hemiacetal derivative from compound 7. In order to overcome this difficulty, compound 7 was replaced with its per-O-benzoylated derivative to act as a glycosyl donor and a series of thioglycoside activators e.g. NIS–TfOH, methyl triflate, DMTST, IDCP etc. were used in different reaction solvents such as, CH2Cl2, CH3CN, toluene, and 1,2-(CH2Cl)2. However, all glycosylation conditions failed to produce the glycosylation product. The use of a per-O-acetylated glucosyl trichloroacetimidate derivative in place of thioglycoside derivative in the presence TMSOTf as a glycosylation activator did not lead to the formation of the desired glycosylation product. This finding may be explained by considering the severe steric crowding around the hydroxyl group of the acceptor. At this point, complete removal of the functional groups of the compound 16 by hydrogenolysis19 followed by deacetylation furnished the pentasaccharide fragment 1 as its 4-methoxyphenyl glycoside in 62% yield (Structure A, Figs. 1 and 2, Scheme 1). The structure of compound 1 was confirmed from its NMR spectroscopic data [d 5.20–5.11 (m, H-1A, H-1E), 5.07 (br s, H-1D), 4.71 (br s, H-1C), 4.52 (br s, H-1B) in the 1H NMR and d 102.9 (C-1A), 101.7 (C-1E), 101.8 (C-1D), 101.4 (C-1C), 96.5 (C-1B) in the 13 C NMR spectra]. It is worth mentioning that in the immunological studies the smaller oligosaccharide fragments are used to discover the immunodominant fragment of the repeating unit. Therefore, the structure of the repeating unit has been slightly rearranged (Structure B, Fig. 1) to minimize such steric crowding and a hexasaccharide repeating unit has been successfully synthesized as its 2-(4methoxyphenoxy) ethyl glycoside 2 (Fig. 3). The target hexasaccharide as its 2-(4-methoxyphenoxy) ethyl glycoside 2 was synthesized from a series of suitably protected monosaccharide synthons 5,13 6,14 7,15 17, 18,14 and 1920 (Fig. 3). 2-(4-Methoxyphenoxy) ethyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranoside 20 was prepared from L-rhamnose in 87% yield in a twostep sequence involving per-O-acetylation using acetic anhydride and HClO4–SiO221 followed by a reaction of the acetate with 2(4-methoxyphenoxy) ethanol in the presence of boron trifluoride diethyl etherate.22 Saponification of compound 20 followed by acetal formation using 2,2-dimethoxypropane and p-toluenesulfonic acid furnished 2-(4-methoxyphenoxy) ethyl 2,3-O-isopropyli-

SEt

SEt a

O

BnO HO

O

BnO AcO

OH

OMBn 4

8 Ph

O O O

b

3+4

BnO

O

A

OPMP NPhth

O

B

RO

OMBn 9: R = Ac c 10: R = H 5 b

Ph BnO BnO

C

BnO RO

B

O O O

O

A

OPMP NPhth

O

O

O

OMBn 11: c 12 R = Ac R=H 5 b Ph BnO

BnO

C

B

D

13: R = Ac 14: R = H

c

6 b Ph BnO

BnO

C

O

B

BnO E

O

D

BnO

O

O

O

O O O

A

O

O OPMP NPhth

O OR

BnO O O AcO

OPMP NPhth

OMBn

BnO RO

Ph

O

O

O

O

O

A

O

BnO BnO

O O O

d

15: R = MBn 16: R = H

e

1

7 b

NPhth PMP: 4-Methoxyphenyl

No glycosylation

Scheme 1. Reagents and conditions: (a) (i) 4-methoxybenzyl chloride, 5% aq NaOH, TBAB, CH2Cl2, room temperature, 5 h; (ii) acetic anhydride, pyridine, room temperature, 2 h, 70%; (b) N-iodosuccinimide (NIS), TMSOTf, CH2Cl2, MS 4 Å, 40 °C, 45 min, 81% for 9; 80% for 11; 81% for 13 and 71% for 15; (c) 0.01 M CH3ONa, CH3OH, 30 min, room temperature, 97% for 10, 98% for 12 and 90% for 14; (d) DDQ, CH2Cl2–H2O (6:1), room temperature, 2 h, 70%; (e) (i) ethylenediamine, n-butanol, 90 °C, 5 h; (ii) acetic anhydride, pyridine, room temperature, 2 h; (iii) H2, 20% Pd(OH)2–C, CH3OH, room temperature, 24 h; (iv) 0.1 M CH3ONa, CH3OH, room temperature, 5 h, 62% in four steps.

dene-a-L-rhamnopyranoside 21 in 90% yield. Compound 21 was subjected to a sequence of reactions involving benzylation using benzyl bromide and sodium hydroxide,23 removal of the acetal ring using 80% aqueous acetic acid, and selective benzylation of the equatorial hydroxy group via stanylidene acetal formation24 to give 2-(4-methoxyphenoxy) ethyl 3,4-di-O-benzyl-a-L-rhamnopyranoside 17 in 79% yield (Scheme 2). The stereoselective glycosylation of compound 17 with L-rhamnose-derived thioglycoside 5 in the presence of NIS–TMSOTf17 fur-

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O L-Rhamnose

a

OPMP

O

AcO AcO

OAc 20 b

O BnO BnO

O

OPMP

O

c, d

OPMP

O

HO O

OH 17

O 21

Scheme 2. Reagents and conditions: (a) (i) acetic anhydride, HClO4–SiO2, room temperature, 10 min; (ii) 2-(4-methoxyphenoxy) ethanol, BF3OEt2, CH2Cl2, room temperature, 18 h, 87%; (b) (i) 0.1 M CH3ONa, CH3OH, room temperature, 3 h; (ii) 2,2-dimethoxypropane, p-TsOH, DMF, room temperature, 10 h, 90%; (c) (i) benzyl bromide, NaOH, THF, TBAB, room temperature, 1 h; (ii) 80% aq AcOH, 80 °C, 1.5 h; (d) (i) dibutyltin oxide, CH3OH, 80 °C, 2 h; (ii) benzyl bromide, TBAB, DMF, 60 °C, 20 h, 79%.

nished 2-(4-methoxyphenoxy) ethyl (2-O-acetyl-3,4-di-O-benzyla-L-rhamnopyranosyl)-(1?2)-3,4-di-O-benzyl-a-L-rhamnopyranoside 22 in 88% yield, which on deacetylation using sodium methoxide gave compound 23 almost quantitatively. The formation of disaccharide derivative 22 was confirmed from its NMR spectra [d 4.89 (br s, H-1A), 4.70 (br s, H-1B) in 1H NMR and d 99.6 (C-1A), 99.3 (C-1B) in 13C NMR]. Iodonium ion-mediated stereoselective glycosylation of compound 23 with the thioglycoside donor 6 in the presence of NIS–TMSOTf17 furnished trisaccharide derivative 24 in 84% yield, which on deacetylation afforded trisaccharide acceptor 25 in 94% yield (Scheme 3). The presence of signals at d 5.43 (s, PhCH), 5.38 (d, J = 8.3 Hz, H-1C), 4.87 (br s, H-1A), 4.66 (br s, H-1B) in the 1H NMR and d 101.9 (PhCH), 101.4 (C-1C), 100.8 (C-1A), 99.3 (C-1B) in the 13C NMR spectra confirmed the formation of compound 24.

In another experiment, compound 7 was allowed to couple stereoselectively with compound 19 in the presence of NIS–TMSOTf17 to furnish disaccharide derivative 26 in 78% yield. The presence of signals in the NMR spectra confirmed its formation [d 5.46 (br s, H1E), 4.60 (d, J = 8.0 Hz, H-1F) in 1H NMR and d 103.1 (C-1F), 98.0 (C1E) in 13C NMR]. Oxidative removal of the anomeric PMP group from compound 26 using ceric ammonium nitrate (CAN)25 resulted in the disaccharide hemiacetal derivative, which upon treatment with trichloroacetonitrile in the presence of DBU26 furnished disaccharide trichloroacetimidate derivative 27 in 73% yield, which was used immediately for the next step. The stereoselective glycosylation of disaccharide trichloroacetimidate derivative 27 with thioglycoside glycosyl acceptor 18 under the Schmidt’s glycosylation condition26 furnished exclusively the trisaccharide thioglycoside derivative 28 in 75% yield. The appearance of the signals in the NMR spectra [d 5.45 (s, PhCH), 5.28 (d, J = 10.6 Hz, H-1D), 4.66 (br s, H-1E), 4.21 (d, J = 7.8 Hz, H-1F) in 1H NMR and d 102.5 (PhCH), 102.1 (C-1F), 99.8 (C-1E), 82.1 (C-1D) in 13C NMR] confirmed the stereoselective formation of compound 28 (Scheme 4). Finally, the stereoselective [3+3] glycosylation of compound 25 with compound 28 in the presence of NIS–TfOH17 furnished hexasaccharide derivative 29 in 76% yield. Spectroscopic analysis of compound 29 confirmed its formation [d 102.4 (C-1F), 102.0 (PhCH), 101.4 (PhCH), 101.3 (C-1D), 101.1 (C-1E), 100.0 (C-1B), 99.3 (C-1A), 97.8 (C-1C) in the 13C NMR]. Conversion of N-phthalimido group to acetamido group27 followed by hydrogenolysis19 and saponification of compound 29 furnished the target hexasaccharide 2 as its 2-(4methoxyphenoxy) ethyl glycoside in 66% yield. 1D and 2D NMR spectroscopic analyses confirmed the structure of compound 2 [d 5.18 (d, J = 8.3 Hz, H-1C), 4.95 (d, J = 8.5 Hz, H-1D), 4.78 (br s, H1E), 4.62 (br s, H-1A), 4.56 (br s, H-1B), 4.48 (d, J = 7.6 Hz, H-1F) in 1 H NMR and d 104.9 (C-1F), 101.3 (C-1E), 100.2 (C-1B), 99.9 (C1A), 99.0 (C-1D), 98.2 (C-1C) in 13C NMR spectra] (Scheme 5).

7 + 19 a

O BnO 5 + 17

BnO

a

OPMP

OR

O

A

E O AcO BnO OAc F BnO O OAc O AcO 26: R = PMP b 27: R = C(NH)CCl 3

O

B O BnO BnO OR

b 22: R = Ac 23: R = H

18 c

6 a O BnO

A

BnO

Ph

Ph

O O O

OPMP

O E

BnO BnO O

O

B O BnO BnO O O O C O RO NPhth

b 24: R = Ac 25: R = H Scheme 3. Reagents and conditions: (a) N-iodosuccinimide (NIS), TMSOTf, CH2Cl2, MS 4 Å, 30 °C, 1 h, 88% for 22 and 84% for 24; (b) 0.01 M CH3ONa, CH3OH, 30 min, room temperature, 95% for 23 and 94% for 25.

O

28

D

O SEt NPhth

AcO F

O AcO

OAc OAc

Scheme 4. Reagents and conditions: (a) NIS, TMSOTf, CH2Cl2, MS 4 Å, 30 °C, 45 min, 78%; (b) (i) CAN, CH3CN–H2O (9:1, v/v), room temperature, 2 h; (ii) CCl3CN, DBU, CH2Cl2, 10 °C, 1 h, 73%; (c) TMSOTf, CH2Cl2, 20 °C, 1 h, 75%.

3. Conclusion In conclusion, a convenient synthesis of a pentasaccharide as its 4-methoxyphenyl glycoside and a hexasaccharide as its 2-(4-

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(C-1), 79.4 (C-4), 77.2 (PhCH2), 76.5 (PhCH2), 73.9 (C-2), 72.1 (C-3), 68.0 (C-5), 55.1 (OCH3), 25.1 (SCH2CH3), 20.9 (COCH3), 18.0 (CCH3), 14.9 (SCH2CH3); ESI-MS: m/z 483.2 [M+Na]+; Anal. Calcd for C25H32O6S (460.19): C, 65.19; H, 7.00. Found: C, 65.0; H, 7.26.

25 + 28 a O A

BnO

OPMP

O

BnO O O BnO BnO O O C B

Ph

E

O O O O

BnO BnO O

D

O

Ph

O O O

NPhth

NPhth AcO F

O AcO

OAc OAc PMP: 4-methoxyphenyl 29 b

2 Scheme 5. Reagents and conditions: (a) NIS, TMSOTf, CH2Cl2, MS 4 Å, 30 °C, 1 h, 76%; (b) (i) ethylenediamine, n-butanol, 90 °C, 5 h; (ii) acetic anhydride, pyridine, room temperature, 2 h; (iii) H2, 20% Pd(OH)2–C, CH3OH, room temperature, 24 h; (iv) 0.1 M CH3ONa, CH3OH, room temperature, 5 h, 66% in four steps.

methoxyphenoxy) ethyl glycoside corresponding to the O-antigen of E. coli O150 has been achieved using sequential and [3+3] block glycosylation strategies, respectively. All glycosylation reactions were highly stereoselective and high yielding. A thioglycoside derivative has been successfully used as a glycosyl acceptor exploiting its orthogonal behavior. The use of a [3+3] glycosylation made it possible to achieve the target hexasaccharide in a minimum number of steps. 2-(4-Methoxyphenoxy) ethyl glycoside provides the option to obtain the final hexasaccharide linked with an ethylene glycol linkage, which is useful for its further transformation. 4. Experimental 4.1. General methods General methods are the same as reported earlier.11 4.1.1. Ethyl 3-O-acetyl-4-O-benzyl-2-O-(4-methoxybenzyl)-1thio-a-L-rhamnopyranoside 4 To a solution of compound 8 (3.0 g, 10.05 mmol) in CH2Cl2 (20 mL) were added 4-methoxybenzyl chloride (1.5 mL, 11.06 mmol), 5% aq NaOH (10 mL), TBAB (200 mg) and the biphasic reaction mixture was allowed to stir at room temperature for 5 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and washed with water, dried (Na2SO4), and concentrated under reduced pressure. A solution of the crude product in acetic anhydride–pyridine (10 mL; 1:1 v/v) was kept at room temperature for 2 h and concentrated under reduced pressure. The crude product was purified over SiO2 using hexane–EtOAc (5:1) as an eluant to give pure 4 (3.2 g, 70%). Colorless oil; ½a25 D ¼ 89 (c 1.0, CHCl3); mmax (neat): 3020, 1734, 1514, 1370, 1216, 1092, 761 cm1; 1H NMR (300 MHz, CDCl3): d 7.30–7.22 (m, 7H, Ar-H), 6.85 (d, J = 8.6 Hz, 2H, Ar-H), 5.16 (d, J = 1.5 Hz, 1H, H-1), 5.07 (dd, J = 9.4, 3.3 Hz, 1H, H-3), 4.69 (d, J = 11.4 Hz, 1H, PhCH2), 4.62 (d, J = 11.4 Hz, 1H, PhCH2) 4.57 (d, J = 11.8 Hz, 1H, PhCH2), 4.43 (d, J = 11.8 Hz, 1H, PhCH2), 4.09–4.05 (m, 1H, H-5), 3.91–3.89 (m, 1H, H-2), 3.80 (s, 3H, OCH3), 3.58 (t, J = 9.4 Hz, 1H, H-4), 2.64–2.49 (m, 2H, SCH2CH3), 1.90 (s, 3H, COCH3), 1.31 (d, J = 6.2 Hz, 3H, CCH3), 1.27 (t, J = 7.4 Hz, 3H, SCH2CH3); 13C NMR (75 MHz, CDCl3): d 169.7 (COCH3), 159.4–113.8 (Ar-C), 81.6

4.1.2. 4-Methoxyphenyl [2-O-acetyl-4-O-benzyl-2-O-(4methoxybenzyl)-a-L-rhamnopyranosyl]-(1?3)-4,6-Obenzylidene-2-deoxy-2-N-phthalimido-b-D-glucopyranoside 9 To a solution of compound 3 (2.0 g, 3.97 mmol) and compound 4 (2.2 g, 4.78 mmol) in anhydrous CH2Cl2 (15 mL) was added MS 4 Å (3 g) and the reaction mixture was allowed to stir at room temperature under argon for 30 min. The reaction mixture was cooled to 40 °C and N-iodosuccinimide (NIS; 1.3 g, 5.77 mmol) followed by TMSOTf (10 lL) was added to it. After stirring at same temperature for 45 min, the reaction mixture was filtered through a CeliteÒ bed and washed with CH2Cl2 (100 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 (6:1) as the eluant to give pure 9 (2.9 g, 81%). Colorless oil; ½a25 D ¼ þ22 (c 1.0, CHCl3); mmax (neat): 3020, 2361, 1718, 1508, 1385, 1216, 1099, 758 cm1; 1H NMR (300 MHz, CDCl3): d 7.80– 7.07 (m, 14H, Ar-H), 6.78–6.62 (m, 8H, Ar-H), 5.69 (d, J = 8.4 Hz, 1H, H-1A), 5.49 (s, 1H, PhCH), 5.01 (dd, J = 9.4, 3.3 Hz, 1H, H-3B), 4.61 (t, J = 8.2 Hz each, 1H, H-3A), 4.50–4.35 (m, 6H, PhCH2, H-1B, H-2A), 3.85–3.83 (m, 1H, H-5B), 3.84–3.62 (m, 4H, H-4A, H-5A, H6abA), 3.72 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 3.46–3.45 (m, 1H, H2B), 3.35 (t, J = 9.4 Hz each, H-4B), 1.74 (s, 3H, COCH3), 0.71 (d, J = 6.2 Hz, 3H, CCH3); 13C NMR (75 MHz, CDCl3): d 169.5 (COCH3), 167.6, 167.5 (2CO, Phth), 159.1–113.5 (Ar-C), 101.8 (PhCH), 98.0 (C-1A), 97.7 (C-1B), 80.1 (C-4A), 78.9 (C-4B), 76.3 (C-2B), 74.1 (PhCH2), 73.2 (C-3B), 72.6 (C-3A), 72.1 (PhCH2), 68.6 (C-6A), 67.8 (C-5B), 66.6 (C-5A), 56.5 (C-2A), 55.3 (OCH3), 55.0 (OCH3), 20.8 (COCH3), 17.1 (CCH3); ESI-MS: m/z 924.3 [M+Na]+; Anal. Calcd for C51H51NO14 (901.33): C, 67.91; H, 5.70. Found: C, 67.70; H, 6.00. 4.1.3. 4-Methoxyphenyl [4-O-benzyl-2-O-(4-methoxybenzyl)-aL-rhamnopyranosyl]-(1?3)-4,6-O-benzylidene-2-deoxy-2-Nphthalimido-b-D-glucopyranoside 10 A solution of compound 9 (2.8 g, 3.10 mmol) in 0.01 M CH3ONa in CH3OH (30 mL) was allowed to stir at room temperature for 30 min. The reaction mixture was neutralized with Dowex 50 W X8 (H+), filtered, and evaporated to dryness. The crude product was passed through a short pad of SiO2 using hexane–EtOAc (3:1) as the eluant to give pure compound 10 (2.6 g, 97%). Colorless oil; ½a25 D ¼ 11 (c 1.0, CHCl3); mmax (neat): 3020, 2361, 1720, 1598, 1513, 1426, 1216, 1046, 928, 761 cm1; 1H NMR (300 MHz, CDCl3): d 7.89–7.21 (m, 14H, Ar-H), 6.85–6.70 (m, 8H, Ar-H), 5.78 (d, J = 8.4 Hz, 1H, H-1A), 5.53 (s, 1H, PhCH), 4.73–4.63 (m, 2H, H-3A, H-1B), 4.53–4.38 (m, 5H, PhCH2, H-2A), 3.86–3.61 (m, 6H, H-4A, H-5A, H-6abA, H-3B, H-5B), 3.76 (s, 3H, OCH3), 3.68 (s, 3H, OCH3), 3.36 (br s, 1H, H-2B), 3.02 (t, J = 9.4 Hz each, 1H, H-4B), 0.73 (d, J = 6.2 Hz, 3H, CCH3); 13C NMR (75 MHz, CDCl3): d 167.6, 167.5 (2CO, Phth), 159.3–113.7 (Ar-C), 102.0 (PhCH), 98.0 (C-1A), 97.3 (C-1B), 81.9 (C-4A), 80.4 (C-4B), 79.0 (C-2B), 74.4 (PhCH2), 73.7 (C3B), 72.0 (PhCH2), 70.8 (C-3A), 68.6 (C-6A), 67.5 (C-5B), 66.7 (C5A), 56.6 (C-2A), 55.4 (OCH3), 55.0 (OCH3), 17.2 (CCH3); ESI-MS: m/z 882.3 [M+Na]+; Anal. Calcd for C49H49NO13 (859.32): C, 68.44; H, 5.74. Found: C, 68.22; H, 5.92. 4.1.4. 4-Methoxyphenyl (2-O-acetyl-3,4-di-O-benzyl-a-Lrhamnopyranosyl)-(1?3)-[4-O-benzyl-2-O-(4-methoxybenzyl)a-L-rhamnopyranosyl]-(1?3)-4,6-O-benzylidene-2-deoxy-2-Nphthalimido-b-D-glucopyranoside 11 To a solution of compound 10 (2.2 g, 2.56 mmol) and compound 5 (1.3 g, 3.02 mmol) in anhydrous CH2Cl2 (15 mL) was added MS

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4 Å (3 g) and the reaction mixture was allowed to stir at room temperature under argon for 30 min. The reaction mixture was cooled to 40 °C and NIS (0.8 g, 3.55 mmol) followed by TMSOTf (10 lL) was added to it. After stirring at the same temperature for 45 min, the reaction mixture was filtered through a CeliteÒ bed and washed with CH2Cl2 (100 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 (6:1) as the eluant to give pure 11 (2.5 g, 80%). Colorless oil; ½a25 D ¼ 9 (c 1.0, CHCl3); mmax (neat): 3020, 2362, 1723, 1593, 1216, 1042, 762, 670 cm1; 1H NMR (300 MHz, CDCl3): d 7.83–7.45 (m, 24H, ArH), 7.30–7.21 (m, 8H, Ar-H), 6.60 (d, J = 8.4 Hz, 1H, H-1A), 5.54 (s, 1H, PhCH), 5.39 (br s, 1H, H-2C), 4.98 (br s, 1H, H-1C), 4.88 (d, J = 11.0 Hz, 1H, PhCH2), 4.62–4.36 (m, 10H, H-1B, H-2A, PhCH2), 3.96–3.92 (m, 1H, H-5C), 3.84–3.65 (m, 8H, H-3A, H-3B, H-3C, H4A, H-5A, H-5B, H-6abA), 3.71 (s, 3H, OCH3), 3.65 (s, 3H, OCH3), 3.43–3.33 (m, 2H, H-4B, H-4C), 3.30 (br s, 1H, H-2B), 2.07 (s, 3H, COCH3), 1.35 (d, J = 6.1 Hz, 3H, CCH3), 0.72 (d, J = 6.2 Hz, 3H, CCH3); 13C NMR (75 MHz, CDCl3): d 169.6 (COCH3), 167.7, 167.6 (2CO, Phth), 158.9–113.3 (Ar-C), 102.0 (PhCH), 99.1 (C-1C), 97.9 (C-1A), 97.7 (C-1B), 80.6 (C-4A), 80.2 (C-4B), 80.0 (C-4C), 78.9 (C2C), 78.0 (C-2B), 76.7 (C-3C), 75.1 (PhCH2), 74.8 (PhCH2), 74.4 (C3B), 71.9 (PhCH2), 71.6 (PhCH2), 68.8 (C-3A), 68.6 (C-6A), 68.3 (C5A), 68.2 (C-5B), 66.7 (C-5C), 56.5 (C-2A), 55.3 (OCH3), 54.9 (OCH3), 20.8 (COCH3), 17.9 (CCH3), 17.1 (CCH3); ESI-MS: m/z 1250.4 [M+Na]+; Anal. Calcd for C71H73NO18 (1227.48): C, 69.42; H, 5.99. Found: C, 69.18; H, 6.25. 4.1.5. 4-Methoxyphenyl (3,4-di-O-benzyl-a-L-rhamnopyranosyl)(1?3)-[4-O-benzyl-2-O-(4-methoxybenzyl)-a-L-rhamnopyranosyl](1?3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-b-D-glucopyranoside 12 A solution of compound 11 (2.0 g, 1.63 mmol) in 0.01 M CH3ONa in CH3OH (25 mL) was allowed to stir at room temperature for 30 min. The reaction mixture was neutralized with Dowex 50 W X8 (H+), filtered, and evaporated to dryness. The crude product was passed through a short pad of SiO2 using hexane–EtOAc (3:1) as the eluant to give pure compound 12 (1.9 g, 98%). Colorless oil; ½a25 D ¼ þ29 (c 1.0, CHCl3); mmax (neat): 3020, 2359, 1719, 1512, 1425, 1216, 1403, 761 cm1; 1H NMR (300 MHz, CDCl3): d 7.83– 7.18 (m, 24H, Ar-H), 6.83–6.62 (m, 8H, Ar-H), 5.78 (d, J = 8.2 Hz, 1H, H-1A), 5.55 (s, 1H, PhCH), 5.01 (s, 1H, H-1C). 4.84 (d, J = 11.1 Hz, 1H, PhCH2), 4.64–4.60 (m, 10H, H-1B, H-2A, PhCH2), 3.94–3.90 (m, 1H, H-5C), 3.85–3.65 (m, 9H, H-2C, H-3A, H-3B, H3C, H-4A, H-5A, H-5B, H-6abA), 3.70 (s, 3H, OCH3), 3.66 (s, 3H, OCH3), 3.43–3.25 (m, 3H, H-2B, H-4B, H-4C), 1.29 (d, J = 6.2 Hz, 3H, CCH3), 0.72 (d, J = 6.1 Hz, 3H, CCH3); 13C NMR (75 MHz, CDCl3): d 167.7, 167.6 (2CO, Phth), 158.9–113.5 (Ar-C), 101.8 (PhCH), 100.7 (C-1C), 97.9 (C-1A), 97.7 (C-1B), 80.6 (C-4A), 80.2 (C-4B), 80.0 (C-4C), 79.9 (C-2B), 78.8 (C-2C), 77.0(C-3C), 75.0 (PhCH2), 74.8 (PhCH2), 74.4 (C-3B), 71.8 (2C, 2PhCH2), 68.7 (C-3A), 68.5 (C-6A), 68.3 (C5A), 67.9 (C-5B), 66.6 (C-5C), 56.5 (C-2A), 55.3 (OCH3), 54.9 (OCH3), 17.8 (CCH3), 17.1 (CCH3); ESI-MS: m/z 1208.4 [M+Na]+; Anal. Calcd for C69H71NO17 (1185.47): C, 69.86; H, 6.03. Found: C, 69.67; H, 6.30. 4.1.6. 4-Methoxyphenyl (2-O-acetyl-3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1?2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1?3)[4-O-benzyl-2-O-(4-methoxybenzyl)-a-L-rhamnopyranosyl]-(1?3)4,6-O-benzylidene-2-deoxy-2-N-phthalimido-b-D-glucopyranoside 13 To a solution of compound 12 (1.7 g, 1.43 mmol) and compound 5 (750 mg, 1.74 mmol) in anhydrous CH2Cl2 (10 mL) was added MS

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4 Å (2 g) and the reaction mixture was allowed to stir at room temperature under argon for 30 min. The reaction mixture was cooled to 40 °C and NIS (470 mg, 2.08 mmol) followed by TMSOTf (5 lL) was added to it. After stirring at the same temperature for 45 min, the reaction mixture was filtered through a CeliteÒ bed and washed with CH2Cl2 (100 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 (6:1) as the eluant to give pure 13 (1.8 g, 81%). Colorless oil; ½a25 D ¼ 3 (c 1.0, CHCl3); mmax (neat): 3021, 2360, 1722, 1598, 1514, 1423, 1216, 1046, 762 cm1; 1H NMR (300 MHz, CDCl3): d 7.81–7.16 (m, 34H, ArH), 6.85–6.80 (m, 4H, Ar-H), 6.70 (d, J = 9.1 Hz, 2H, Ar-H), 6.62 (d, J = 9.2 Hz, 2H, Ar-H), 5.84 (d, J = 8.4 Hz, 1H, H-1A), 5.55 (s, 1H, PhCH), 5.44 (s, 1H, H-2D), 4.92 (br s, 1H, H-1C), 4.89 (d, J = 11.0 Hz, 1H, PhCH2), 4.84 (d, J = 11.2 Hz, 1H, PhCH2), 4.82 (br s, 1H, H-1D), 4.71 (d, J = 11.1 Hz, 1H, PhCH2), 4.65–4.32 (m, 12H, H1B, H-2A, H-3A, PhCH2), 3.92–3.60 (m, 11H, H-2C, H-3B, H-3C, H3D, H-4A, H-5A, H-5B, H-5C, H-5D, H-6abA), 3.75 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 3.43–3.21 (m, 4H, H-2B, H-4B, H-4C, H-4D), 2.11 (s, 3H, COCH3), 1.25 (d, J = 6.0 Hz, 3H, CCH3), 1.13 (d, J = 6.2 Hz, 3H, CCH3), 0.64 (d, J = 6.1 Hz, 3H, CCH3); 13C NMR (75 MHz, CDCl3): d 169.6 (COCH3), 167.8, 167.7 (2CO, Phth), 158.9–113.4 (Ar-C), 101.8 (PhCH), 100.2 (C-1C), 98.9 (C-1D), 97.9 (C-1A), 97.4 (C-1B), 80.2 (2C, C-4A, C-4B), 80.0 (2C, C-2B, C-4C), 79.6 (C-2D), 79.1 (C2C), 77.4 (2C, C-3C, C-4D), 75.1 (PhCH2), 74.9 (PhCH2), 74.8 (PhCH2), 74.6 (C-3D), 74.4 (C-3B), 71.9 (PhCH2), 71.6 (PhCH2), 71.5 (PhCH2), 68.8 (C-3A), 68.5 (C-6A), 68.3 (2C, C-5A, C-5B), 68.2 (C-5D), 66.6 (C-5C), 56.4 (C-2A), 55.3 (OCH3), 54.8 (OCH3), 20.9 (COCH3), 17.9 (CCH3), 17.8 (CCH3), 17.1 (CCH3); ESI-MS: m/z 1576.6 [M+Na]+; Anal. Calcd for C91H95NO22 (1553.63): C, 70.30; H, 6.16. Found: C, 70.10; H, 6.40. 4.1.7. 4-Methoxyphenyl (3,4-di-O-benzyl-a-L-rhamnopyranosyl)(1?2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1?3)-[4-O-benzyl-2-O-(4-methoxybenzyl)-a-L-rhamnopyranosyl]-(1?3)-4,6-Obenzylidene-2-deoxy-2-N-phthalimido-b-D-glucopyranoside 14 A solution of compound 13 (1.5 g, 0.96 mmol) in 0.01 M CH3ONa in CH3OH (20 mL) was allowed to stir at room temperature for 30 min. The reaction mixture was neutralized with Dowex 50 W X8 (H+), filtered, and evaporated to dryness. The crude product was passed through a short pad of SiO2 using hexane–EtOAc (3:1) as the eluant to give pure compound 14 (1.3 g, 90%). Colorless oil; ½a25 D ¼ þ4 (c 1.0, CHCl3); mmax (neat): 3020, 2361, 1717, 1610, 1509, 1216, 1098, 1404, 761 cm1; 1H NMR (300 MHz, CDCl3): d 7.81–7.16 (m, 34H, Ar-H), 6.85–6.80 (m, 4H, Ar-H), 6.70 (d, J = 9.1 Hz, 2H, Ar-H), 6.61 (d, J = 9.1 Hz, 2H, Ar-H), 5.79 (d, J = 8.4 Hz, 1H, H-1A), 5.55 (s, 1H, PhCH), 4.98–4.94 (m, 4H, H-1C, H-1D, PhCH2), 4.85 (2 d, J = 10.9 Hz each, 2H, PhCH2), 4.69 (br s, 2H, PhCH2), 4.61 (br s, 1H, H-1B), 4.58–4.30 (m, 10H, H-2A, H3A, PhCH2), 4.08–4.05 (m, 1H, H-2D), 3.87–3.61 (m, 11H, H-2C, H-3B, H-3C, H-3D, H-4A, H-5A, H-5B, H-5C, H-5D, H-6abA), 3.72 (s, 3H, OCH3), 3.66 (s, 3H, OCH3), 3.43–3.23 (m, 4H, H-2B, H-4B, H4C, H-4D), 1.29 (d, J = 6.1 Hz, 3H, CCH3), 1.11 (d, J = 6.1 Hz, 3H, CCH3), 0.63 (d, J = 6.1 Hz, 3H, CCH3); 13C NMR (75 MHz, CDCl3): 167.8, 167.7 (2CO, Phth), 158.9–113.4 (Ar-C), 101.9 (PhCH), 100.5 (2C, C-1C, C-1D), 98.0 (C-1A), 97.6 (C-1B), 80.3 (C-4A), 80.2 (2C, C-4C, C-4B), 80.0 (C-2B), 79.7 (C-2D), 79.4 (2C, C-2C, C-3C), 79.1 (2C, C-4D), 78.8 (PhCH2), 75.2 (PhCH2), 74.8 (PhCH2), 74.7 (C-3D), 74.4 (C-3B), 72.2 (PhCH2), 72.0 (PhCH2), 71.7 (PhCH2), 68.6 (2C, C-3A, C-5A), 68.4 (2C, C-5B, C-6A), 67.9 (C-5D), 66.7 (C5C), 56.5 (C-2A), 55.4 (OCH3), 54.9 (OCH3), 18.0 (CCH3), 17.8 (CCH3), 17.1 (CCH3); ESI-MS: m/z 1534.6 [M+Na]+; Anal. Calcd for C89H93NO21 (1511.62): C, 70.67; H, 6.20. Found: C, 70.84; H, 6.42.

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4.1.8. 4-Methoxyphenyl (3-O-acetyl-4,6-O-benzylidene-2deoxy-2-N-phthalimido-b-D-glucopyranosyl-(1?2)-(3,4-di-Obenzyl-a-L-rhamnopyranosyl)-(1?2)-(3,4-di-O-benzyl-a-Lrhamnopyranosyl)-(1?3)-[4-O-benzyl-2-O-(4-methoxybenzyl)a-L-rhamnopyranosyl]-(1?3)-4,6-O-benzylidene-2-deoxy-2-Nphthalimido-b-D-glucopyranoside 15 To a solution of compound 14 (1.0 g, 0.66 mmol) and compound 6 (380 mg, 0.78 mmol) in anhydrous CH2Cl2 (5 mL) was added MS 4 Å (1 g) and the reaction mixture was allowed to stir at room temperature under argon for 30 min. The reaction mixture was cooled to 40 °C and NIS (200 mg, 8.44 mmol) followed by TMSOTf (3 lL) was added to it. After stirring at the same temperature for 45 min, the reaction mixture was filtered through a CeliteÒ 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 (6:1) as the eluant to give pure 15 (910 mg, 71%). Colorless oil; ½a25 D ¼ 5 (c 1.0, CHCl3); mmax (neat): 3021, 2359, 1520, 1425, 1216, 1403, 928, 764 cm1; 1H NMR (300 MHz, CDCl3): d 7.76–7.07 (m, 43H, Ar-H), 7.06–6.77 (m, 4H, Ar-H), 6.80 (d, J = 9.1 Hz, 2H, Ar-H), 6.78 (d, J = 9.0 Hz, 2H, Ar-H), 6.02 (t, J = 9.9 Hz, 1H, H-3E), 5.76 (d, J = 8.4 Hz, 1H, H-1A), 5.54 (s, 1H, PhCH), 5.42 (s, 1H, PhCH), 5.40 (d, J = 8.3 Hz, 1H, H1E), 4.82–4.80 (m, 2H, H-1C, H-1D), 4.74 (d, J = 11.1 Hz, PhCH2), 5.44–4.25 (m, 12H, H-1B, H-2A, H-2E, PhCH2), 4.25–4.01 (m, 2H, PhCH2), 4.15–3.44 (m, 17H, H-2C, H-2D, H-3A, H-3B, H-3C, H-3D, H-4A, H-4E, H-5A, H-5B, H-5C, H-5D, H-5E, H-6abA, H-6abE), 3.70 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 3.27–2.94 (m, 4H, H-2B, H-4B, H4C, H-4D), 1.93 (s, 3H, COCH3), 1.20 (d, J = 6.0 Hz, 3H, CCH3), 1.02 (d, J = 6.1 Hz, 3H, CCH3), 0.61 (d, J = 6.0 Hz, 3H, CCH3); 13C NMR (75 MHz, CDCl3): d 169.5 (COCH3), 167.8, 167.7 (4CO, 2Phth), 158.9–113.4 (Ar-C), 101.8 (PhCH), 101.5 (PhCH), 100.8 (2C, C-1C, C-1D), 100.4 (C-1E), 98.0 (C-1A), 97.6 (C-1B), 80.5 (2C, C-4A, C-4E), 80.2 (2C, C-4B, C-4C), 79.1 (2C, C-2B, C-2D), 78.9 (C-4D), 78.7 (C2C), 77.6 (C-3C), 77.4 (C-3B), 75.8 (C-3D), 74.9 (PhCH2), 74.7 (PhCH2), 74.4 (C-3E), 72.5 (PhCH2), 71.8 (PhCH2), 71.7 (PhCH2), 69.3 (C-3A), 68.5 (2C, C-5A, C-6A), 68.3 (2C, C-5E, C-6E), 68.2 (C-5B), 66.7 (C5D), 65.6 (C-5C), 56.5 (C-2A), 55.3 (OCH3), 55.2 (C-2E), 54.9 (OCH3), 20.5 (COCH3), 18.0 (CCH3), 17.5 (CCH3), 17.3 (CCH3); MALDI-MS: m/z 1955.7 [M+Na]+; Anal. Calcd for C112H112N2O28 (1932.74): C, 69.55; H, 5.84. Found: C, 69.36; H, 6.10. 4.1.9. 4-Methoxyphenyl (3-O-acetyl-4,6-O-benzylidene-2deoxy-2-N-phthalimido-b-D-glucopyranosyl-(1?2)-(3,4-di-Obenzyl-a-L-rhamnopyranosyl)-(1?2)-(3,4-di-O-benzyl-a-Lrhamnopyranosyl)-(1?3)-(4-O-benzyl-a-L-rhamnopyranosyl)(1?3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-b-Dglucopyranoside 16 To a solution of compound 15 (900 mg, 0.46 mmol) in CH2Cl2 (10 mL) was added DDQ (200 mg, 0.88 mmol) in H2O (5 mL) and the reaction mixture was allowed to stir at room temperature for 2 h. The reaction mixture was diluted with CH2Cl2 (50 mL) and washed with satd. NaHCO3, and water, dried (Na2SO4), and concentrated. The crude product was purified over SiO2 using hexane– EtOAc (4:1) as the eluant to give pure 16 (580 mg, 70%). Colorless oil; ½a25 D ¼ 18 (c 1.0, CHCl3); mmax (neat): 2928, 2361, 1718, 1630, 1386, 1225, 1098, 768 cm1; 1H NMR (300 MHz, CDCl3): d 7.80– 7.05 (m, 43H, Ar-H), 6.77 (d, J = 9.0 Hz, 2H, Ar-H), 6.69 (d, J = 9.2 Hz, 2H, Ar-H), 6.01 (t, J = 9.9 Hz, 1H, H-3E), 5.72 (d, J = 8.2 Hz, H-1A), 5.52 (s, 1H, PhCH), 5.43 (s, 1H, PhCH), 5.41 (d, J = 8.2 Hz, 1H, H-1E), 4.83 (br s, 1H, H-1C), 4.80 (br s, 1H, H-1D), 4.74 (d, J = 10.8 Hz, 1H, PhCH2), 4.61–4.26 (m, 12H, H-1B, H-2A, H-2E, H-3A, PhCH2), 4.09–4.02 (m, 2H, H-5C, PhCH2), 3.93–3.46 (m, 16H, H-2B, H-2C, H-2D, H-3B, H-3C, H-3D, H-4A, H-4E, H-5A, H5B, H-5D, H-5E, H-6abA, H-6abE), 3.69 (s, 3H, OCH3), 3.21–2.94 (m, 3H, H-4B, H-4C, H-4D), 1.94 (s, 3H, COCH3), 1.16 (d, J = 6.1 Hz, 3H,

CCH3), 1.06 (d, J = 6.1 Hz, 3H, CCH3), 0.65 (d, J = 6.2 Hz, 3H, CCH3); C NMR (75 MHz, CDCl3): d 169.7 (COCH3), 167.8, 167.7 (4CO, 2Phth), 155.5–114.4 (Ar-C), 101.8 (PhCH), 101.5 (PhCH), 100.8 (C1C), 100.5 (C-1D), 100.4 (C-1E), 99.5 (C-1B), 98.0 (C-1A), 80.5 (C4A), 80.4 (C-4E), 80.3 (C-4B), 79.8 (C-4C), 79.1 (2C, C-2B, C-3C), 79.0 (C-2D), 78.9 (C-4D), 75.8 (C-3D), 75.4 (PhCH2), 74.8 (PhCH2), 74.7 (PhCH2), 74.2 (C-3B), 72.5 (PhCH2), 71.8 (PhCH2), 70.9 (C-3E), 69.3 (C-3A), 68.6 (2C, C-5A, C-5E), 68.4 (C-6A), 68.3 (C-6E), 67.9 (C5B), 66.5 (C-5D), 65.7 (C-5C), 56.2 (C-2A), 55.3 (OCH3), 55.2 (C-2E), 20.5 (COCH3), 17.8 (CCH3), 17.6 (CCH3), 17.1 (CCH3); MALDI-MS: m/z 1835.6 [M+Na]+; Anal. Calcd for C104H104N2O27 (1812.68): C, 68.86; H, 5.78. Found: C, 68.64; H, 6.05. 13

4.1.10. 2-(4-Methoxyphenoxy) ethyl 2,3,4-tri-O-acetyl-a-Lrhamnopyranoside 20 To a well-stirred solution of L-rhamnose monohydrate (5.0 g, 27.45 mmol) in acetic anhydride (30 mL, 317.65 mmol) was added HClO4-SiO2 (500 mg) and the reaction mixture was allowed to stir at room temperature for 10 min. The reaction mixture was poured into ice-water and extracted with CH2Cl2 (200 mL). The organic layer was washed with satd. NaHCO3 and water, dried (Na2SO4), and concentrated under reduced pressure to give 1,2,3,4-tetra-Oacetyl-a/b-L-rhamnopyranose (9.0 g), which was used directly for the next step. To a solution of 1,2,3,4-tetra-O-acetyl-a/b-L-rhamnopyranose (8.0 g, 24.07 mmol) in anhydrous CH2Cl2 (80 mL) were added 2-(4-methoxyphenoxy) ethanol (8.0 g, 47.57 mmol) and BF3OEt2 (6.0 mL, 48.61 mmol) and the reaction mixture was allowed to stir at room temperature for 18 h. The reaction mixture was poured into ice-water and extracted with CH2Cl2 (150 mL). The organic layer was washed with satd. NaHCO3 and water, dried (Na2SO4), and concentrated under reduced pressure. The crude product was purified over SiO2 using hexane–EtOAc (5:1) as the eluant to give pure 20 (9.2 g, 87%). Colorless oil; ½a25 D ¼ 53 (c 1.0, CHCl3); mmax (neat): 2937, 1747, 1508, 1371, 1228, 1088, 1050, 827, 757 cm1; 1H NMR (500 MHz, CDCl3): d 6.86–6.82 (m, 4H, Ar-H), 5.31–5.27 (m, 2H, H-2, H-3), 5.06 (t, J = 9.8 H33z, 1H, H-4), 4.83 (d, J = 1.5 Hz, 1H, H-1), 4.11–4.09 (m, 2H, OCH2a), 3.99–3.93 (m, 2H, OCH2b), 3.85–3.81 (m, 1H, H-5), 3.76 (s, 3H, OCH3), 2.15 (s, 3H, COCH3), 2.04 (s, 3H, COCH3), 1.98 (s, 3H, COCH3), 1.21 (d, J = 6.3 Hz, 3H, CCH3); 13C NMR (125 MHz, CDCl3): d 170.5 (COCH3), 170.4 (COCH3), 170.3 (COCH3), 154.5–115.0 (Ar-C), 98.0 (C-1), 71.5 (C-4), 70.2 (C-3), 69.4 (C-2), 67.9 (C-5), 66.9 (OCH2), 66.8 (OCH2), 56.1 (OCH3), 21.3 (COCH3), 21.2 (COCH3), 21.1 (COCH3), 17.7 (CCH3); ESI-MS: m/z 463.1 [M+Na]+; Anal. Calcd for C21H28O10 (440.17): C, 57.27; H, 6.41. Found: C, 57.04; H, 6.60. 4.1.11. 2-(4-Methoxyphenoxy) ethyl 2,3-O-isopropylidene-a-Lrhamnopyranoside 21 A solution of compound 20 (8.0 g, 18.16 mmol) in 0.1 M CH3ONa in CH3OH (100 mL) was allowed to stir at room temperature for 3 h. The reaction mixture was neutralized with Dowex 50 W X8 (H+) resin, filtered, and concentrated. To a solution of the deacetylated product (5.7 g, 18.13 mmol) in dry DMF (15 mL) were added 2,2-dimethoxypropane (6.0 mL, 48.8 mmol) and p-toluenesulfonic acid (200 mg) and the reaction mixture was allowed to stir at room temperature for 10 h. The reaction was quenched with Et3N (1.0 mL) and the solvents were removed under reduced pressure. The crude product was purified over SiO2 using hexane–EtOAc (3:1) as the eluant to give pure 21 (5.8 g, 90%). Colorless oil; ½a25 D ¼ 27 (c 1.0, CHCl3); mmax (neat): 2936, 1639, 1509, 1456, 1383, 1230, 1096, 1053, 924, 858 cm1; 1H NMR (500 MHz, CDCl3): d 6.86–6.81 (m, 4H, Ar-H), 5.04 (br s, 1H, H-1), 4.19–4.17 (m, 1H, OCH2a), 4.11–4.06 (m, 3H, H-2, H-3, OCH2a), 4.02–3.98 (m, 1H, OCH2b), 3.83–3.79 (m, 1H, OCH2b), 3.76 (s, 3H, OCH3), 3.75–3.70 (m, 1H, H-4), 3.42–3.38 (m, 1H, H-5), 1.52 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.28 (d, J = 6.2 Hz, 3H, CCH3); 13C NMR (125 MHz, CDCl3):

R. Panchadhayee, A. K. Misra / Tetrahedron: Asymmetry 21 (2010) 2142–2152

d 154.4–115.0 (Ar-C), 109.8 [C(OCH3)2], 97.7 (C-1), 78.6 (C-4), 76.0 (C-3), 74.6 (C-2), 68.0 (OCH2), 66.5 (C-5), 66.3 (OCH2), 56.1 (OCH3), 28.3 (CH3), 26.4 (CH3), 17.9 (CCH3); ESI-MS: m/z 377.1 [M+Na]+; Anal. Calcd for C18H26O7 (354.17): C, 61.00; H, 7.39. Found: C, 60.78; H, 7.60. 4.1.12. 2-(4-Methoxyphenoxy) ethyl 3,4-di-O-benzyl-a-Lrhamnopyranoside 17 To a solution of compound 21 (5.0 g, 14.11 mmol) in THF (15 mL) were added powdered solid NaOH (1.8 g, 45.0 mmol), tetrabutylammonium bromide (TBAB, 200 mg, 0.62 mmol), and benzyl bromide (3.5 mL, 29.42 mmol) and the reaction mixture was allowed to stir at room temperature for 1 h. The reaction mixture was poured into satd. NH4Cl solution and extracted with CH2Cl2 (100 mL). The organic layer was washed with water, dried (Na2SO4), and concentrated. A solution of the benzylated product in 80% aq AcOH (100 mL) was allowed to stir at 80 °C for 1.5 h and the solvents were removed under reduced pressure. To a solution of the crude product (5.0 g, 12.36 mmol) in anhydrous CH3OH (100 mL) was added dibutyltin oxide (4.5 g, 18.07 mmol) and the reaction mixture was allowed to stir at 80 °C for 2 h. The solvents were removed under reduced pressure and the crude product was dissolved in dry DMF (15 mL). To the reaction mixture were added benzyl bromide (3.0 mL, 25.22 mmol) and TBAB (500 mg, 1.55 mmol) and the reaction mixture was allowed to stir at 60 °C for 10 h. The reaction mixture was poured into satd. NH4Cl and extracted with EtOAc (mL). The organic layer was washed with water, dried (Na2SO4), and evaporated to dryness. The crude product was purified over SiO2 using hexane–EtOAc (4:1) as the eluant to give pure 17 (5.5 g, 79%). Colorless oil; ½a25 D ¼ 35 (c 1.0, CHCl3); mmax (neat): 2931, 2876, 1508, 1454, 1364, 1232, 1109, 1075, 1042, 985, 827, 746 cm1; 1H NMR (500 MHz, CDCl3): d 7.25–7.20 (m, 10H, Ar-H), 6.77–6.72 (m, 4H, Ar-H), 4.82 (d, J = 1.2 Hz, 1H, H-1), 4.78 (d, J = 10.9 Hz, 1H, PhCH2), 4.58 (br s, 2H, 2PhCH2), 4.54 (d, J = 11.0 Hz, 1H, PhCH2), 3.99–3.96 (m, 3H, H-2, H-3, H-5), 3.87– 3.83 (m, 1H, OCH2a), 3.78–3.75 (m, 1H, OCH2a), 3.74–3.66 (m, 2H, 2OCH2b), 3.66 (s, 3H, OCH3), 3.37 (t, J = 9.4 Hz, 1H, H-4), 1.22 (d, J = 6.2 Hz, 3H, CCH3); 13C NMR (125 MHz, CDCl3): d 154.4–115.0 (Ar-C), 99.6 (C-1), 80.4 (C-4), 80.3 (C-3), 75.7 (PhCH2), 72.4 (PhCH2), 68.8 (C-2), 68.1 (OCH2), 67.8 (C-5), 66.4 (OCH2), 56.1 (OCH3), 18.3 (CCH3); ESI-MS: m/z 517.2 [M+Na]+; Anal. Calcd for C29H34O7 (494.23): C, 70.43; H, 6.93. Found: C, 70.25; H, 7.18. 4.1.13. 2-(4-Methoxyphenoxy) ethyl (2-O-acetyl-3,4-di-Obenzyl-a-L-rhamnopyranosyl)-(1?2)-3,4-di-O-benzyl-a-Lrhamnopyranoside 22 To a solution of compound 17 (3.0 g, 6.06 mmol) and compound 5 (3.1 g, 7.2 mmol) in anhydrous CH2Cl2 (25 mL) was added MS 4 Å (5 g) and the reaction mixture was allowed to stir at room temperature under argon for 30 min. The reaction mixture was cooled to 30 °C and N-iodosuccinimide (NIS; 1.9 g, 8.44 mmol) followed by TMSOTf (20 lL) was added to it. After stirring at the same temperature for 1 h, the reaction mixture was filtered through a CeliteÒ bed and washed with CH2Cl2 (100 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 (6:1) as the eluant to give pure 22 (4.6 g, 88%). Colorless oil; ½a25 D ¼ 34 (c 1.0, CHCl3); mmax (neat): 2921, 1744, 1508, 1454, 1369, 1233, 1106, 1055, 983, 826, 739 cm1; 1H NMR (500 MHz, CDCl3): d 7.25–7.17 (m, 20H, Ar-H), 6.74 (d, J = 9.2 Hz, 2H, Ar-H), 6.71 (d, J = 9.2 Hz, 2H, Ar-H), 5.44–5.43 (m, 1H, H-2B), 4.89 (br s, 1H, H1A), 4.82 (d, J = 10.9 Hz, 1H, PhCH2), 4.80 (d, J = 11.0 Hz, 1H, PhCH2), 4.70 (br s, 1H, H-1B), 4.66 (d, J = 11.0 Hz, 1H, PhCH2), 4.57 (br s, 2H, PhCH2), 4.55 (d, J = 11.0 Hz, 1H, PhCH2), 4.51 (d, J = 10.9 Hz, 1H, PhCH2), 4.46 (d, J = 11.0 Hz, 1H, PhCH2), 3.97–3.93 (m, 3H, H-2A,

2149

2OCH2a), 3.86 (dd, J = 9.4 Hz, 3.3 Hz, 1H, H-3A), 3.85–3.80 (m, 1H, OCH2b), 3.76 (dd, J = 9.4 Hz, 3.0 Hz, 1H, H-3A), 3.74–3.69 (m, 1H, H-5A), 3.68–3.61 (m, 2H, H-5B, OCH2b), 3.66 (s, 3H, OCH3), 3.31 (2 t, J = 9.5 Hz each, 2H, H-4A, H-4B), 2.04 (s, 3H, COCH3), 1.22 (d, J = 6.2 Hz, 3H, CCH3), 1.21 (d, J = 6.2 Hz, 3H, CCH3); 13C NMR (125 MHz, CDCl3): d 170.2 (COCH3), 154.4–115.0 (Ar-C), 99.6 (C1A), 99.3 (C-1B), 80.4 (C-4A), 80.3 (C-4B), 80.2 (C-3B), 78.0 (C-3A), 75.8 (PhCH2), 75.6 (PhCH2), 74.6 (C-2A), 72.4 (PhCH2), 72.1 (PhCH2), 69.2 (C-2B), 68.6 (C-5A), 68.4 (C-5B), 68.1 (OCH2), 66.2 (OCH2), 56.0 (OCH3), 21.4 (COCH3), 18.4 (2C, CCH3); ESI-MS: m/z 885.3 [M+Na]+; Anal. Calcd for C51H58O12 (862.39): C, 70.98; H, 6.77. Found: C, 70.75; H, 7.00. 4.1.14. 2-(4-Methoxyphenoxy) ethyl (3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1?2)-3,4-di-O-benzyl-a-L-rhamnopyranoside 23 A solution of compound 22 (4.0 g, 4.63 mmol) in 0.01 M CH3ONa in CH3OH (20 mL) was allowed to stir at room temperature for 30 min. The reaction mixture was neutralized with Dowex 50 W X8 (H+), filtered, and evaporated to dryness. The crude product was passed through a short pad of SiO2 using hexane–EtOAc (3:1) as the eluant to give pure compound 23 (3.6 g, 95%). Colorless oil; ½a25 D ¼ 26 (c 1.0, CHCl3); mmax (neat): 3466, 3011, 2911, 1509, 1454, 1360, 1235, 1055, 981, 920, 828, 753 cm1; 1H NMR (500 MHz, CDCl3): d 7.34–7.26 (m, 20H, Ar-H), 6.81 (d, J = 9.2 Hz, 2H, Ar-H), 6.78 (d, J = 9.1 Hz, 2H, Ar-H), 5.05 (br s, 1H, H-1A), 4.87–4.84 (m, 2H, PhCH2), 4.80 (br s, 1H, H-1B), 4.69–4.57 (m, 6H, PhCH2), 4.10–4.09 (m, 1H, H-2B), 4.04–4.01 (m, 3H, H-2A, 2OCH2a), 3.92–3.88 (m, 1H, OCH2b), 3.87–3.82 (m, 2H, H-3A, H3B), 3.80–3.78 (m, 1H, H-5A), 3.76–3.70 (m, 2H, H-5B, OCH2b), 3.72 (s, 3H, OCH3), 3.45 (t, J = 9.3 Hz, 1H, H-4A), 3.37 (t, J = 9.3 Hz, 1H, H-4B), 1.28, 1.27 (2 d, J = 6.2 Hz each, 6H, 2CCH3); 13C NMR (125 MHz, CDCl3): d 154.4–115.0 (Ar-C), 101.1 (C-1A), 99.5 (C-1B), 80.7 (C-4A), 80.4 (C-4B), 80.2 (C-3A), 80.0 (C-3B), 75.7 (PhCH2), 75.6 (PhCH2), 74.8 (C-2A), 72.7 (PhCH2), 72.5 (PhCH2), 69.1 (C-2B), 68.4 (C-5A), 68.3 (C-5B), 68.2 (OCH2), 66.3 (OCH2), 56.0 (OCH3), 18.5 (CCH3), 18.3 (CCH3); ESI-MS: m/z 843.3 [M+Na]+; Anal. Calcd for C49H56O11 (820.38): C, 71.69; H, 6.88. Found: C, 71.46; H, 7.10. 4.1.15. 2-(4-Methoxyphenoxy) ethyl (3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-b-D-glucopyranosyl-(1?2)-(3,4di-O-benzyl-a-L-rhamnopyranosyl)-(1?2)-3,4-di-O-benzyl-a-Lrhamnopyranoside 24 To a solution of compound 23 (3.0 g, 3.65 mmol) and compound 6 (2.1 g, 4.34 mmol) in anhydrous CH2Cl2 (20 mL) was added MS 4 Å (3 g) and the reaction mixture was allowed to stir at room temperature under argon for 30 min. The reaction mixture was cooled to 30 °C and NIS (1.2 g, 5.33 mmol) followed by TMSOTf (20 lL) was added to it. After stirring at the same temperature for 1 h, the reaction mixture was filtered through a CeliteÒ bed and washed with CH2Cl2 (100 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 the eluant to give pure 24 (3.8 g, 84%). Colorless oil; ½a25 D ¼ 11 (c 1.0, CHCl3); mmax (neat): 2932, 1776, 1746, 1718, 1508, 1388, 1229, 1104, 1081, 1050, 1029, 998, 827, 738, 722 cm1; 1H NMR (500 MHz, CDCl3): d 7.42–7.08 (m, 29H, Ar-H), 6.79–6.78 (m, 4H, Ar-H), 6.03 (t, J = 9.3 Hz each, 1H, H-3C), 5.43 (s, 1H, PhCH), 5.38 (d, J = 8.3 Hz, 1H, H-1C), 4.87 (br s, 1H, H-1A), 4.73 (d, J = 10.8 Hz, 1H, PhCH2), 4.66 (br s, 1H, H-1B), 4.58 (br s, 2H, PhCH2), 4.50 (d, J = 10.8 Hz, 1H, PhCH2)3333, 4.38 (dd, J = 8.4 Hz each, 1H, H-2C), 4.29 (d, J = 11.2 Hz, 1H, PhCH2), 4.08–4.03 (m, 2H, PhCH2), 3.99–3.94 (m, 3H, H-2A, H-2B, PhCH2), 3.85–3.83 (m, 3H, H-6abC, OCH2a), 3.73– 3.58 (m, 7H, H-3A, H-3B, H-5A, H-5B, OCH2a, 2OCH2b), 3.72 (s, 3H, OCH3), 3.52 (t, J = 10.2 Hz each, 1H, H-4C), 3.49–3.47 (m, 1H, H5C), 3.11 (t, J = 9.3 Hz each, 1H, H-4A), 3.06 (t, J = 9.4 Hz each, 1H,

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H-4B), 1.93 (s, 3H, COCH3), 1.20 (d, J = 6.2 Hz, 3H, CCH3), 1.15 (d, J = 6.1 Hz, 3H, CCH3); 13C NMR (125 MHz, CDCl3): d 170.3 (COCH3), 167.7, 167.6 (2CO, Phth), 154.4–115.0 (Ar-C), 101.9 (PhCH), 101.4 (C-1C), 100.8 (C-1A), 99.3 (C-1B), 81.0 (C-4C), 80.9 (C-4B), 79.6 (3C, C-3A, C-3B, C-4A), 78.1 (C-5B), 76.0 (C-2A), 75.6 (PhCH2), 75.3 (PhCH2), 73.0 (PhCH2), 72.1 (PhCH2), 69.7 (C-3C), 68.9 (C-2B), 68.8 (C-6C), 68.1 (2C, C-5A, OCH2), 66.2 (OCH2), 66.1 (C-5C), 56.0 (OCH3), 55.7 (C-2C), 21.1 (COCH3), 18.4 (CCH3), 18.1 (CCH3); ESIMS: m/z 1264.5 [M+Na]+; Anal. Calcd for C72H75NO18 (1241.49): C, 69.61; H, 6.08. Found: C, 69.40; H, 6.32. 4.1.16. 2-(4-Methoxyphenoxy) ethyl (4,6-O-benzylidene-2deoxy-2-N-phthalimido-b-D-glucopyranosyl)-(1?2)-(3,4-di-Obenzyl-a-L-rhamnopyranosyl)-(1?2)-3,4-di-O-benzyl-a-Lrhamnopyranoside 25 A solution of compound 24 (3.0 g, 2.41 mmol) in 0.01 M CH3ONa in CH3OH (30 mL) was allowed to stir at room temperature for 30 min. The reaction mixture was neutralized with Dowex 50 W X8 (H+), filtered, and evaporated to dryness. The crude product was passed through a short pad of SiO2 using hexane–EtOAc (3:1) as the eluant to give pure compound 25 (2.7 g, 94%). Colorless oil; ½a25 D ¼ 10 (c 1.0, CHCl3); mmax (neat): 2926, 1715, 1508, 1390, 1232, 1100, 1050, 994, 827, 739 cm1; 1H NMR (500 MHz, CDCl3): d 7.47–7.08 (m, 29H, Ar-H), 6.78–6.77 (m, 4H, Ar-H), 5.46 (s, 1H, PHCH), 5.19 (d, J = 8.4 Hz, 1H, H-1C), 4.88 (br s, 1H, H-1A), 4.77 (t, J = 8.4 Hz each, 1H, H-3C), 4.73 (d, J = 10.8 Hz, 1H, PhCH2), 4.67 (br s, 1H, H-1B), 4.57 (2 d, J = 10.8 Hz each, 2H, PhCH2), 4.48 (d, J = 10.8 Hz, 1H, PhCH2), 4.31 (dd, J = 8.4 Hz each, 1H, H-2C), 4.25 (d, J = 12.2 Hz, 1H, PhCH2), 4.08 (d, J = 10.8 Hz, 1H, PhCH2), 4.02 (d, J = 11.2 Hz, 1H, PhCH2), 3.99–3.95 (m, 3H, H-2A, H-2B, PhCH2), 3.85–3.82 (m, 3H, H-6abC, OCH2a), 3.73–3.57 (m, 6H, H-3A, H-3B, H-5A, 2OCH2b, OCH2a), 3.72 (s, 3H, OCH3), 3.52–3.49 (m, 2H, H-4C, H-5B), 3.33–3.29 (m, 1H, H-5C), 3.09 (2 t, J = 9.7 Hz each, 2H, H4A, H-4B), 1.20 (d, J = 6.2 Hz, 3H, CCH3), 1.18 (d, J = 6.2 Hz, 3H, CCH3); 13C NMR (125 MHz, CDCl3): d 168.7, 168.6 (2CO, Phth), 154.4–115.0 (Ar-C), 102.2 (PhCH), 101.5 (C-1C), 101.2 (C-1A), 99.4 (C-1B), 82.4 (C-4C), 81.0 (C-4A), 80.9 (C-4B), 79.6 (C-3A), 79.5 (C3B), 78.1 (C-5B), 76.0 (C-2A), 75.6 (PhCH2), 75.4 (PhCH2), 72.9 (PhCH2), 72.0 (PhCH2), 68.9 (C-3C), 68.8 (C-6C), 68.5 (C-2B), 68.1 (2C, C-5A, OCH2), 66.2 (OCH2), 66.1 (C-5C), 57.0 (C-2C), 56.0 (OCH3), 18.4 (CCH3), 18.1 (CCH3); ESI-MS: m/z 1222.4 [M+Na]+; Anal. Calcd for C70H73NO17 (1199.48): C, 70.04; H, 6.13. Found: C, 70.25; H, 6.38. 4.1.17. 4-Methoxyphenyl (2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-(1?2)-3,4-di-O-benzyl-a-L-rhamnopyranoside 26 To a solution of compound 19 (2.0 g, 4.44 mmol) and compound 7 (2.1 g, 5.35 mmol) in anhydrous CH2Cl2 (15 mL) was added MS 4 Å (2 g) and the reaction mixture was allowed to stir at room temperature under argon for 30 min. The reaction mixture was cooled to 30 °C and NIS (1.4 g, 6.22 mmol) followed by TMSOTf (25 lL) was added to it. After stirring at the same temperature for 45 min, the reaction mixture was filtered through a CeliteÒ bed and washed with CH2Cl2 (100 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 (5:1) as the eluant to give pure 26 (2.7 g, 78%). Colorless oil; ½a25 D ¼ 31 (c 1.0, CHCl3); mmax (neat): 2937, 1754, 1508, 1366, 1223, 1089, 1039, 978, 829, 753 cm1; 1H NMR (500 MHz, CDCl3): d 7.37– 7.25 (m, 10H, Ar-H), 6.92 (d, J = 9.1 Hz, 2H, Ar-H), 6.79 (d, J = 9.1 Hz, 2H, Ar-H), 5.46 (br s, 1H, H-1E), 5.22 (t, J = 9.6 Hz, 1H, H-3F), 5.09 (dd, J = 7.9 Hz each, 1H, H-2F), 5.01 (t, J = 9.6 Hz, 1H, H-4F), 4.81 (d, J = 10.8 Hz, 1H, PhCH2), 4.74 (d, J = 11.4 Hz, 1H,

PhCH2), 4.66 (d, J = 11.4 Hz, 1H, PhCH2), 4.61 (d, J = 11.2 Hz, 1H, PhCH2), 4.60 (d, J = 8.0 Hz, 1H, H-1F), 4.20 (dd, J = 12.2, 5.5 Hz, 1H, H-6aF), 4.04 (dd, J = 12.2, 2.4 Hz, 1H, H-6bF), 4.01–3.99 (m, 2H, H2E, H-3E), 3.77–3.73 (m, 1H, H-5E), 3.75 (s, 3H, OCH3), 3.64–3.60 (m, 1H, H-5F), 3.42 (t, J = 9.4 Hz each, 1H, H-4E), 2.02 (br s, 6H, 2COCH3), 1.90 (s, 3H, COCH3), 1.85 (s, 3H, COCH3), 1.24 (d, J = 6.2 Hz, 3H, CCH3); 13C NMR (125 MHz, CDCl3): d 170.7 (COCH3), 170.5 (COCH3), 169.6 (2C, 2COCH3), 155.2–114.9 (Ar-C), 103.1 (C1F), 98.0 (C-1E), 80.6 (C-3E), 79.8 (C-4E), 78.3 (C-5E), 75.8 (PhCH2), 73.3 (PhCH2), 72.7 (C-3F), 72.1 (C-5F), 71.5 (C-2F), 69.0 (2C, C-2E, C-4F), 62.3 (C-6F), 55.9 (OCH3), 21.0 (COCH3), 20.9 (2C, 2COCH3), 20.8 (COCH3), 18.2 (CCH3); ESI-MS: m/z 803.3 [M+Na]+; Anal. Calcd for C41H48O15 (780.29): C, 63.07; H, 6.20. Found: C, 63.26; H, 6.46. 4.1.18. Ethyl (2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-(1?2)(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1?3)-(4,6-O-benzylidene-2-deoxy-2-N-phthalimido-1-thio-b-D-glucopyranoside 28 To a solution of compound 26 (2.5 g, 3.20 mmol) in CH3CN–H2O (20 mL; 4:1, v/v) was added ammonium cerium nitrate (CAN; 2.6 g, 4.74 mmol) and the reaction mixture was allowed to stir at room temperature for 2 h. The reaction mixture was diluted with CH2Cl2 (80 mL) and the organic layer was washed with satd. NaHCO3 and water, dried (Na2SO4), and evaporated to dryness to give disaccharide hemiacetal. To a solution of the hemiacetal in anhydrous CH2Cl2 (15 mL) was added trichloroacetonitrile (2.8 mL, 27.92 mmol) and the reaction mixture was cooled to 10 °C. To the cooled reaction mixture was added DBU (0.1 mL, 0.65 mmol) and it was allowed to stir at 10 °C for 1 h. The reaction mixture was evaporated to dryness and the crude product was passed through a short pad of SiO2 to furnish (2,3,4,6-tetra-O-acetyl-b-Dglucopyranosyl)-(1?2)-3,4-di-O-benzyl-a-L-rhamnopyranosyl trichloroacetimidate (27; 1.9 g, 73%), which was used immediately for the next step. A solution of compound 18 (0.7 g, 1.58 mmol) and compound 27 (1.9 g, 2.32 mmol) in anhydrous CH2Cl2 (15 mL) was cooled to 20 °C. To the cooled reaction mixture was added TMSOTf (20 lL) and it was allowed to stir at 20 °C for 1 h. The reaction mixture was diluted with CH2Cl2 (50 mL) and the organic layer was washed with satd. NaHCO3 and water in succession, dried (Na2SO4), and evaporated to dryness. The crude product was purified over SiO2 using hexane–EtOAc (5:1) as the eluant to give pure 28 (1.3 g, 75%). Colorless oil; ½a25 D ¼ 15 (c 1.0, CHCl3); mmax (neat): 2931, 1757, 1716, 1429, 1382, 1225, 1096, 1041, 994, 751, 722 cm1; 1H NMR (500 MHz, CDCl3): d 7.82–7.12 (m, 19H, Ar-H), 5.45 (s, 1H, PhCH), 5.28 (d, J = 10.6 Hz, 1H, H-1D), 4.91 (t, J = 9.5 Hz each, 1H, H-3F), 4.78 (t, J = 9.6 Hz each, 1H, H-4F), 4.75 (dd, J = 7.8 Hz each, 1H, H-2F), 4.66 (br s, 1H, H-1E), 4.62 (d, J = 10.9 Hz, 1H, PhCH2), 4.56 (t, J = 9.2 Hz each, 1H, H-3D), 4.45 (d, J = 11.5 Hz, 1H, PhCH2), 4.38 (d, J = 10.9 Hz, 1H, PhCH2), 4.35–4.32 (m, 2H, H-4D, PhCH2), 4.25 (t, J = 10.2 Hz each, 1H, H2D), 4.21 (d, J = 7.8 Hz, 1H, H-1F), 4.01 (dd, J = 12.2, 4.2 Hz, 1H, H6aF), 3.73–3.64 (m, 4H, H-2E, H-5D, H-6abD), 3.63–3.51 (m, 2H, H-3E, H-6bF), 3.47–3.45 (m, 1H, H-5E), 3.14–3.08 (m, 2H, H-4E, H-5F), 2.63–2.52 (m, 2H, SCH2CH3), 2.02 (s, 3H, COCH3), 1.92 (s, 3H, COCH3), 1.88 (s, 3H, COCH3), 1.65 (s, 3H, COCH3), 1.11 (t, J = 7.4 Hz each, 3H, SCH2CH3), 0.77 (d, J = 6.2 Hz, 3H, CCH3); 13C NMR (125 MHz, CDCl3): d 171.0 (COCH3), 170.5 (COCH3), 169.5 (COCH3), 169.3 (COCH3), 167.7, 167.6 (2CO, Phth), 138.9–124.1 (Ar-C), 102.5 (PhCH), 102.1 (C-1F), 99.8 (C-1E), 82.1 (C-1D), 81.2 (C-3E), 80.5 (C-4E), 79.7 (C-5D), 78.0 (C-5E), 75.6 (C-3D), 75.5 (PhCH2), 72.8 (PhCH2), 72.7 (C-3F), 71.8 (C-5F), 71.3 (C-4D), 71.1 (C-2F), 69.0 (2C, C-4F, C-6D), 68.6 (C-2E), 62.1 (C-6F), 56.0 (C-2D), 24.6 (SCH2CH3), 21.1 (COCH3), 21.0 (COCH3), 20.9 (COCH3), 20.8 (COCH3), 17.7 (SCH2CH3), 15.3 (CCH3); ESI-MS: m/z 1120.3 [M+Na]+; Anal. Calcd for C57H63NO19S (1097.37): C, 62.34; H, 5.78. Found: C, 62.15; H, 6.00.

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4.1.19. 2-(4-Methoxyphenoxy) ethyl (2,3,4,6-tetra-O-acetyl-b-Dglucopyranosyl)-(1?2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)(1?3)-(4,6-O-benzylidene-2-deoxy-2-N-phthalimido-b-D-glucopyranosyl)-(1?3)-(4,6-O-benzylidene-2-deoxy-2-N-phthalimidob-D-glucopyranosyl)-(1?2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1?2)-3,4-di-O-benzyl-a-L-rhamnopyranoside 29 To a solution of compound 25 (1.0 g, 0.83 mmol) and compound 28 (1.2 g, 1.09 mmol) in anhydrous CH2Cl2 (10 mL) was added MS 4 Å (2 g) and the reaction mixture was allowed to stir at room temperature under argon for 30 min. The reaction mixture was cooled to 30 °C and NIS (300 mg, 1.33 mmol) followed by TMSOTf (5 lL) was added to it. After stirring at the same temperature for 1 h, the reaction mixture was filtered through a CeliteÒ 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 (3:1) as the eluant to give pure 29 (1.4 g, 76%). Colorless oil; ½a25 D ¼ 9 (c 1.0, CHCl3); mmax (neat): 2931, 1745, 1717, 1507, 1381, 1225, 1104, 1096, 1050, 994, 827, 722 cm1; 1H NMR (500 MHz, CDCl3): d 7.30–7.12 (m, 48H, ArH), 6.77–6.76 (m, 4H, Ar-H), 5.42 (s, 1H, PhCH), 5.41 (s, 1H, PhCH), 5.29 (d, J = 8.3 Hz, 1H, H-1C), 4.99 (d, J = 8.4 Hz, 1H, H-1D), 4.94 (t, J = 9.5 Hz each, 1H, H-2F), 4.90 (t, J = 9.5 Hz each, 1H, H-3F), 4.80 (br s, 1H, H-1E), 4.79 (t, J = 9.3 Hz each, 1H, H-4F), 4.73 (t, J = 8.4 Hz each, 1H, H-3C), 4.71 (d, J = 11.0 Hz, 1H, PhCH2), 4.63 (br s, 1H, H-1A), 4.62 (d, J = 11.8 Hz, 1H, PhCH2), 4.52 (br s, 2H, PhCH2), 4.50 (br s, 1H, H-1B), 4.45 (d, J = 10.9 Hz, 1H, PhCH2), 4.40–4.37 (m, 2H, PhCH2), 4.09 (m, 6H, H-2C, H-2D, H-3D, PhCH2), 4.08–4.0 (m, 1H, H-6aF), 3.98–3.95 (m, 5H, H-1F, H-6abC, PhCH2), 3.84–3.76 (m, 4H, H-6abD, 2OCH2a), 3.71 (s, 3H, OCH3), 3.71–3.39 (m, 14H, H-2A, H-2B, H-2E, H-3A, H-3B, H-3E, H-4C, H-4D, H-5A, H5B, H-5D, H-6bF, 2OCH2b), 3.48–3.45 (m, 1H, H-5C), 3.30 (br s, 1H, H-5E), 3.10 (2 t, J = 9.3 Hz each, 2H, H-4A, H-4B), 3.08–3.06 (m, 1H, H-5F), 2.88 (t, J = 9.5 Hz each, 1H, H-4E), 2.04 (s, 3H, COCH3), 1.98 (s, 3H, COCH3), 1.92 (s, 3H, COCH3), 1.64 (s, 3H, COCH3), 1.17 (d, J = 6.2 Hz, 3H, CCH3), 1.09 (d, J = 6.2 Hz, 3H, CCH3), 0.72 (d, J = 6.2 Hz, 3H, CCH3); 13C NMR (125 MHz, CDCl3): d 171.0 (COCH3), 170.5 (COCH3), 169.5 (COCH3), 169.3 (COCH3), 167.7, 167.6 (4CO, 2Phth), 154.0–115.0 (Ar-C), 102.4 (C-1F), 102.0 (PhCH), 101.4 (PhCH), 101.3 (C-1D), 101.1 (C-1E), 100.0 (C-1B), 99.3 (C-1A), 97.8 (C-1C), 81.2 (C-4E), 80.9 (C-4A), 80.8 (C-4B), 80.4 (C-3A), 80.0 (C3B), 79.7 (C-5B), 79.5 (C-3E), 79.0 (C-5D), 78.1 (C-5E), 78.0 (C-4F), 75.8 (2C, C-3D, C-4C), 75.6 (PhCH2), 75.4 (PhCH2), 75.2 (PhCH2), 74.6 (C-2A), 73.0 (PhCH2), 72.6 (C-3F), 72.5 (PhCH2), 72.0 (PhCH2), 71.7 (C-3C), 71.2 (C-2B), 69.1 (C-6C), 68.9 (C-6D), 68.7 (C-5C), 68.5 (C-4D), 68.1 (3C, C-2F, C-5F, OCH2), 66.5 (C-2E), 66.3 (C-5A), 66.2 (OCH2), 62.0 (C-6F), 57.0 (C-2D), 56.1 (C-2C), 56.0 (OCH3), 21.1 (COCH3), 20.9 (COCH3), 20.7 (COCH3), 18.4 (COCH3), 18.4 (CCH3), 18.0 (CCH3), 17.6 (CCH3); MALDI-MS: m/z 2257.8 [M+Na]+; Anal. Calcd for C125H130N2O36 (2234.84): C, 67.13; H, 5.86. Found: C, 66.92; H, 6.15. 4.1.20. 4-Methoxyphenyl (2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?2)-(a-L-rhamnopyranosyl)-(1?2)-(a-L-rhamnopyranosyl)-(1?3)-(a-L-rhamnopyranosyl)-(1?3)-2-acetamido-2-deoxyb-D-glucopyranoside 1 To a solution of compound 16 (400 mg, 0.22 mmol) in n-butanol (10 mL) was added ethylenendiamine (0.5 mL, 7.48 mmol) and the reaction mixture was allowed to stir at 90 °C for 5 h. The solvents were removed under reduced pressure and a solution of the crude product in acetic anhydride–pyridine (2 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 passed through a short pad of SiO2. To a solution of the N-acetylated product in CH3OH (10 mL) was added 20% Pd(OH)2-C (100 mg) and the reaction mixture was allowed to stir at room temperature under a positive

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pressure of hydrogen for 24 h. The reaction mixture was filtered through a CeliteÒ bed, washed with CH3OH (50 mL), and concentrated. A solution of the crude product in 0.1 M CH3ONa in CH3OH (10 mL) was allowed to stir at room temperature for 5 h. The reaction mixture was neutralized with Dowex 50 W X8 (H+), filtered, and evaporated to dryness to give compound 1, which was purified through a Sephadex LH-20 column using CH3OH–H2O (2:1) as the eluant to give pure compound 1 (130 mg, 62%). White powder; ½a25 D ¼ 8 (c 1.0, CH3OH); mmax (KBr): 3018, 2929, 1744, 1376, 1220, 1069, 764 cm1; 1H NMR (500 MHz, CD3OD): d 6.85 (d, J = 9.1 Hz, 2H, Ar-H), 6.76 (d, J = 9.1 Hz, 2H, Ar-H), 5.20–5.11 (m, 2H, H-1A, H-1E), 5.07 (br s, 1H, H-1D), 4.71 (br s, 1H, H-1C), 4.52 (br s, 1H, H-1B), 4.13–4.03 (m, 2H, H-2A, H-2E), 3.92–3.74 (m, 7H, H-2B, H-2C, H-2D, H-3B, H-3C, H-3D, H-5B), 3.73–3.52 (m, 10H, H3A, H-3E, H-4A, H-5A, H-5C, H-5D, H-6abA, H-6abE), 3.68 (s, 3H, OCH3), 3.50–3.35 (m, 5H, H-4B, H-4C, H-4D, H-4E, H-5E), 2.12 (s, 3H, COCH3), 2.10 (s, 3H, COCH3), 1.27–1.17 (m, 9H, 3CCH3); 13C NMR (125 MHz, CDCl3): d 169.9, 169.8 (2COCH3), 154.4–115.0 (Ar-C), 102.9 (C-1A), 101.7 (C-1E), 101.8 (C-1D), 101.4 (C-1C), 96.5 (C-1B), 80.7 (C-3A), 80.0 (C-2B), 78.8 (2C, C-2C, C-2D), 78.0 (C-2E), 77.1 (C-5A), 77.0 (2C, C-3B, C-5E), 73.2 (C-4A), 73.0 (C-4C), 71.9 (C4B), 71.3 (C-4D), 71.0 (2C, C-3C, C-3D), 70.7 (C-4E), 69.9 (C-5D), 69.3 (C-5C), 68.9 (C-5B), 61.8 (C-6A), 61.5 (C-6E), 57.2 (C-2A), 56.4 (C-2E), 55.1 (OCH3), 23.9, 21.7 (2COCH3), 16.8 (3C, 3CH3); ESI-MS: m/z 991.3 [M+Na]+; Anal. Calcd for C41H64N2O24 (968.38): C, 50.82; H, 6.66. Found: C, 50.60; H, 6.94. 4.1.21. 2-(4-Methoxyphenoxy) ethyl (b-D-glucopyranosyl)-(1?2)(a-L-rhamnopyranosyl)-(1?3)-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1?3)-(2-acetamido-2-deoxy-b-D-glucopyranosyl)(1?2)-(a-L-rhamnopyranosyl)-(1?2)-a-L-rhamnopyranoside 2 To a solution of compound 29 (1.0 g, 0.45 mmol) in n-butanol (20 mL) was added ethylenendiamine (0.5 mL, 7.48 mmol) and the reaction mixture was allowed to stir at 90 °C for 5 h. The solvents were removed under reduced pressure and a solution of the crude product in acetic anhydride–pyridine (3 mL, 1:1 v/v) was kept at the room temperature for 2 h. The solvents were removed under reduced pressure and the crude product was passed through a short pad of SiO2. To a solution of the N-acetylated product in CH3OH (10 mL) was added 20% Pd(OH)2-C (150 mg) and the reaction mixture was allowed to stir at room temperature under a positive pressure of hydrogen for 24 h. The reaction mixture was filtered through a CeliteÒ bed, washed with CH3OH (50 mL), and concentrated. A solution of the crude product in 0.1 M CH3ONa in CH3OH (20 mL) was allowed to stir at room temperature for 5 h. The reaction mixture was neutralized with Dowex 50 W X8 (H+), filtered, and evaporated to dryness to give compound 1, which was purified through a Sephadex LH-20 column using CH3OH– H2O (2:1) as the eluant to give pure compound 2 (350 mg, 66%). White powder; ½a25 D ¼ 14 (c 1.0, H2O); mmax (KBr): 3448, 3066, 3033, 2926, 2370, 1368, 1057, 696 cm1; 1H NMR (500 MHz, D2O): d 6.76–6.74 (m, 4H, Ar-H), 5.18 (d, J = 8.3 Hz, 1H, H-1C), 4.95 (d, J = 8.5 Hz, 1H, H-1D), 4.78 (br s, 1H, H-1E), 4.62 (br s, 1H, H-1A), 4.56 (br s, 1H, H-1B), 4.48 (d, J = 7.6 Hz, 1H, H-1F), 3.93– 3.91 (m, 2H, H-2B, H-2C), 3.87–3.71 (m, 6H, H-2A, H-2D, H-2E, H6aC, H-6abF), 3.66–3.53 (m, 7H, H-2F, H-3A, H-3B, H-3E, H-6bC, 2OCH2a), 3.58 (s, 3H, OCH3), 3.51–3.57 (m, 9H, H-2F, H-3C, H-3D, H-3F, H-4C, H-4F, H-5A, H-6abD), 3.35–3.25 (m, 3H, H-4D, 2OCH2b), 3.23–3.06 (m, 3H, H-5B, H-5C, H-5D), 2.94 (br s, 1H, H-5E), 2.87 (t, J = 9.4 Hz each, 1H, H-4E), 2.80 (t, J = 9.2 Hz each, 1H, H-4B), 2.43– 2.41 (m, 1H, H-4A), 2.15–2.13 (m, 1H, H-5F), 1.86 (br s, 6H, 2COCH3), 1.00 (d, J = 6.2 Hz, 3H, CCH3), 0.94 (d, J = 6.2 Hz, 3H, CCH3), 0.81 (d, J = 6.2 Hz, 3H, CCH3); 13C NMR (125 MHz, D2O): d 170.5, 170.2 (2COCH3), 153.9–115.5 (Ar-C), 104.9 (C-1F), 101.3 (C-1E), 100.2 (C-1B), 99.9 (C-1A), 99.0 (C-1D), 98.2 (C-1C), 82.1 (C5E), 80.9 (2C, C-2A, C-2E), 78.8 (2C, C-4E, C-5B,), 76.6 (C-5F), 75.8

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(C-2F), 75.5 (C-3F), 75.3 (C-3C), 73.2 (C-5C), 72.6 (C-5D), 72.4 (2C, C4A, C-4B), 70.3 (C-3A), 70.0 (C-3B), 69.8 (C-3E), 69.7 (C-4F), 69.5 (C3D), 69.3 (C-4C), 69.1 (C-4D), 69.0 (C-5A), 68.7 (C-2B), 68.3 (C-6D), 66.5 (C-6C), 61.0 (2C, C-6F, OCH2), 60.2 (OCH2), 56.4 (C-2D), 56.2 (OCH3), 55.7 (C-2C), 24.4 (COCH3), 24.3 (COCH3), 17.0 (CCH3), 16.8 (2C, CCH3); ESI-MS: m/z 1197.4 [M+Na]+; Anal. Calcd for C49H78N2O30 (1174.46): C, 50.08; H, 6.69. Found: C, 50.25; H, 6.96.

8. 9. 10.

Acknowledgments R.P. thanks CSIR, New Delhi for providing a Senior Research Fellowship. This work was supported by the Ramanna Fellowship (A.K.M.),the Department of Science and Technology, New Delhi (SR/S1/RFPC-06/2006) and the Bose Institute, Kolkata. References 1. (a) Jann, B.; Jann, K. Curr. Top. Microbiol. Immunol. 1990, 150, 19–42; (b) Ørskov, F.; Ørskov, I. Can. J. Microbiol. 1992, 38, 699–704. 2. (a) Abbott, S. L.; O’Conner, J.; Robin, T.; Zimmer, B. L.; Janda, J. M. J. Clin. Microbiol. 2003, 41, 4852–4854; (b) Kaper, J. B.; Nataro, J. P.; Mobley, H. L. Nat. Rev. Microbiol. 2004, 2, 123–140. 3. (a) Gerber, A.; Karch, H.; Allerberger, F.; Verweyen, H. M.; Zimmerhackl, L. B. J. Infect. Dis. 2002, 186, 493–500; (b) Ørskov, I.; Ørskov, F.; Jann, B.; Jann, K. Bacteriol. Rev. 1977, 41, 667–710. 4. (a) Nataro, J. P.; Kaper, J. B. Clin. Microbiol. Rev. 1998, 11, 142–201; (b) Russmann, H.; Kothe, E.; Schmidth, H.; Franke, S.; Harmsen, D.; Caprioli, A.; Karch, H. J. Med. Microbiol. 1995, 42, 404–410; (c) Beutin, L.; Aleksic, S.; Zimmermann, S.; Gleier, K. Med. Microbiol. Immunol. 1994, 183, 13–21. 5. (a) Ludwig, K.; Sarkim, V.; Bitzan, M.; Karmali, M. A.; Bobrowski, C.; Ruder, H.; Laufs, R.; Sobottka, I.; Petric, M.; Karch, H.; Müller-Wiefel, D. E. J. Clin. Microbiol. 2002, 40, 1773–1782; (b) Bower, J. R. Pediatr. Infect. Dis. J. 1999, 18, 909–910. 6. (a) Boyce, T. G.; Swerdlow, D. L.; Griffin, P. M. N. Eng. J. Med. 1995, 333, 364– 368; (b) Kaper, J. B. Curr. Opin. Microbiol. 1998, 1, 103–108; (c) Grimm, L. M.; Goldoft, M.; Kobayashi, J.; Lewis, J. H.; Alfi, D.; Perdichizzi, A. M.; Tarr, P. I.; Ongerth, J. E.; Moseley, S. L.; Samadpour, M. J. Clin. Microbiol. 1995, 33, 2155– 2158; (d) Ezawa, A.; Gocho, F.; Saitoh, M.; Tamura, T.; Kawata, K.; Takahashi, T.; Kikuchi, N. J. Vet. Med. Sci. 2004, 66, 779–784. 7. (a) Beutin, L.; Zimmermann, S.; Gleier, K. Emerg. Infect. Dis. 1998, 4, 635–639; (b) Stenutz, R.; Weintraub, A.; Widmalm, G. FEMS Microbiol. Rev. 2006, 30, 382–

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