Synthesis of the trisaccharide repeating unit of the O-antigen related to the enterohemorrhagic Escherichia coli type O26:H

Article abstract of DOI:10.1081/CAR-120020480

L-Fucose was converted to the 2-azido-2-deoxy-L-fucose derivative, which together with the monosaccharide synthons prepared from L-rhamnose and D-glucosamine hydrochloride were utilized for the synthesis of the p-ethoxyphenyl glycoside of the trisaccharid

Full text of DOI:10.1081/CAR-120020480

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Synthesis of the Trisaccharide Repeating Unit of
the O‐Antigen Related to the Enterohemorrhagic
Escherichia coli Type O26:H
Kakali Sarkar a , Indrani Mukherjee a & Nirmolendu Roy a
a Department of Biological Chemistry , Indian Association for the Cultivation of Science ,
Jadavpur, Calcutta, 700 032, India
Published online: 16 Aug 2006.
To cite this article: Kakali Sarkar , Indrani Mukherjee & Nirmolendu Roy (2003) Synthesis of the Trisaccharide Repeating Unit
of the O‐Antigen Related to the Enterohemorrhagic Escherichia coli Type O26:H, Journal of Carbohydrate Chemistry, 22:2,
95-107, DOI: 10.1081/CAR-120020480
To link to this article: http://dx.doi.org/10.1081/CAR-120020480
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 22, No. 2, pp. 95–107, 2003
Synthesis of the Trisaccharide Repeating Unit of
the O-Antigen Related to the Enterohemorrhagic
Escherichia coli Type O26:H
*
Kakali Sarkar, Indrani Mukherjee, and Nirmolendu Roy
Department of Biological Chemistry, Indian Association for the
Cultivation of Science, Jadavpur, Calcutta, India
ABSTRACT
L-Fucose was converted to the 2-azido-2-deoxy-L-fucose derivative, which together
with the monosaccharide synthons prepared from L-rhamnose and D-glucosamine
hydrochloride were utilized for the synthesis of the p-ethoxyphenyl glycoside of the
trisaccharide repeating unit of the antigen from enterohemorrhagic Escherichia coli
type O26:H.
Key Words: Synthesis; Escherichia coli type O26:H; Trisaccharide.
INTRODUCTION
Escherichia coli play an important role in maintaining intestinal physiology.
Within this species,[1] however, there are fully pathogenic strains that cause distinct
syndromes of diarrheal disease. Of the four major categories of diarrheagenic E. coli,
enterohemorrhagic E. coli is one of the most virulent types of pathogen. The
*
Correspondence: Nirmolendu Roy, Department of Biological Chemistry, Indian Association
for the Cultivation of Science, Jadavpur, Calcutta 700 032, India; Fax: + 91-33-2473-2805;
E-mail: bcnr@mahendra.iacs.res.in.
95
DOI: 10.1081/CAR-120020480
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

96
Sarkar, Mukherjee, and Roy
structures (I) of the O-antigen of enterohemorrhagic E. coli 026:H has already been
reported.[2]
!4ÞÀaÀL ÀFucpNAcÀð1!3ÞÀbÀD ÀGlcpNAcÀð1!3ÞÀaÀL ÀRhapÀð1!
I
The sugar moieties a-L-Rhap, a-L-FucpNAc and b-D-GlcpNAc may be respon-
sible[3] individually or collectively for the immunogenicity of the native antigen. It is
therefore pertinent to synthesize the complex oligosaccharides related to the repeating
unit of this antigen. They can act as inhibitors and can also be attached to a solid
support or a protein carrier for utilization as immunoadsorbent and artificial antigens
(vaccines), respectively. Carbohydrate-based antibacterial vaccines are among the most
successful carbohydrate pharmaceuticals.[4] As a part of our programme to determine
the relationship between the structure and the immunological specificity of the
carbohydrate moieties, our primary aim in this communication is to synthesize the
trisaccharide repeating unit of the antigen from E. coli O26.
RESULTS AND DISCUSSION
Our strategy is to synthesize the target trisaccharide from the three monosaccharide
synthons, namely ethyl 4,6-O-benzylidene-3-O-chloroacetyl-2-phthalimido-1-thio-b-D-
glucopyranoside (5), 4-methoxyphenyl 2,4-di-O-benzyl-a-L-rhamnopyranoside (11) and
2-azido-3,4-di-O-acetyl-2-deoxy-b-galactopyranosyl trichloroacetimidate (15).
For the synthesis of 5, 1,3,4,5-tetra-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopy-
ranose (1)[5] was converted into the ethyl 1-thioglycoside (2).[6] Removal of the
O-acetyl groups of 2 followed by treatment of the product with a,a-dimethoxytoluene[7]
in the presence of p-toluenesulfonic acid (p-TsOH) in acetonitrile gave the benzylidine
derivative 4. Chloroacetylation[8] of 4 with chloroacetic anhydride and triethylamine
gave 5 (Scheme 1) in the form of fine crystals.
Scheme 1. (a) EtSH, BF3ꢀOEt2, CH2Cl2; (b) NaOMe, MeOH; (c) a,a-dimethoxytoluene, p-TsOH,
CH3CN; (d) (ClCH2CO)2O, Et3N, CH2CH2.

Escherichia coli Type O26:H
97
Scheme 2. (a) NaOMe, MeOH; (b) 2,2-Dimethoxypropane, p-TsOH, DMF; (c) Benzoyl chloride,
pyr; (d) 80% AcOH, 80°C; (e) i. Trimethylorthobenzoate, camphor-10-sulfonic acid, CH2Cl2; ii.
80% AcOH, rt.
In another experiment, 4-methoxyphenyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranoside
(6) was prepared according to the known method[9] starting from L-rhamnose. The
product was conventionally deacetylated and then treated with 2,2-dimethoxypro-
pane[10] in the presence of p-TsOH. The resulting 2,3-O-isopropylidine derivative (8)
was benzoylated to give the 4-O-benzoyl derivative 9. Opening of the isopropylidine
ring with 80% acetic acid, treatment of the product 10 with trimethyl orthobenzoate[11]
and camphor-10-sulfonic acid followed by mild hydrolysis afforded the acceptor 11
(Scheme 2).
In a separate experiment, 3,4-di-O-acetyl-L-fucal (12) was prepared according to
the standard method[12] from L-fucose. Azidonitration[13] of 12 followed by treat-
ment[14] of the product (13) with sodium nitrite in dioxane-water (20:1) at 80°C gave
the reducing 2-azido compound 14 which on treatment[15,16] with trichloroacetonitrile
and potassium carbonate in dichloroethane afforded the b-trichloroacetimidate 15
(Scheme 3).
Scheme 3. (a) CAN, NaN3; (b) NaNO2, 1:20 H2O-dioxane, 80°C; (c) Cl3CCN, K2CO3.

98
Sarkar, Mukherjee, and Roy
The thioglycoside donor 5 was allowed to react with the acceptor 11 in the
presence of N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH)[17,18] in
dichloromethane to afford the disaccharide derivative 16 in 55% yield (Scheme 4). The
compound 16 was characterized by its signals at d 3.70 (ClCH2CO), 1.02 (CCH3) in the
1H NMR spectrum and at d165.6, 165.1 (2COPh), 163.9 (COCH2Cl), 100.6 (CHPh),
97.9 (C-1II), 95.3 (C-1I), 54.3 (OCH3), 53.9 (C-2II), 39.2 (COCH2Cl), and 16.5 (CCH3)
in its 13C NMR spectrum. The chloroacetyl group of 16 was removed[19] with thiourea
in the presence of excess sodium bicarbonate to give the acceptor 17 (Scheme 4) which
was characterized from its NMR signals for benzylidene, CHCH3, OMe and two
anomeric protons and carbons.
The disaccharide acceptor 17 was then allowed to react[20] with the trichlor-
oacetimidate donor 15 in the presence of triethysilyl trifluoromethanesulfonate to give
the trisaccharide derivative 18 as crystals in 62% isolated yield (Scheme 5). The
formation of a-glycoside results from employing the nonparticipating azido group in
the 2-position of 15. The compound 18 was characterized from the signals for ben-
zylidene, OMe, two acetyl groups, two CHCH3 and three anomeric protons and carbons
1
in its H and 13C NMR spectra.
Compound 18 was hydrogenolyzed with hydrogen and 10% Pd/C in the presence
of acetic anhydride, conditions under which the azido group was converted[21] into an
acetamido, with simultaneous removal of the benzylidene moiety to give 19. Treatment
of 19 with ethylenediamine in 1-butanol[22] followed by acetic anhydride/pyridine
afforded the peracetate derivative 20 which could be purified by column chromato-
graphy. Compound 20 was characterized by the presence of OMe, three anomeric
protons and carbons, two CHCH3 and the appearance of two NHCOCH3 groups in its
NMR spectra. Conventional deacetylation of the acetate 20 gave the desired target
trisaccharide repeating unit 21 of the antigen from E. coli O26. The final compound 21
was characterized from its NMR signals for two CHCH3, OMe, two NHCOCH3 and
three anomeric protons and carbons.
Scheme 4. (a) NIS-TfOH; (b) Thiourea, NaHCO3, 2:1 MeOH-CH2Cl2.

Escherichia coli Type O26:H
99
Scheme 5. (a) TESOTf, dichloroethane, À 30°C, 30 min; (b) 10% Pd on charcoal, Ac2O, EtOH;
(c) i. Ethylenediamine, 1-butanol, 90°C, 20 h; (d) Ac2O, Pyr; (e) 0.5 N NaOMe, MeOH.
EXPERIMENTAL
General. All reactions were monitored by TLC on silica gel G (E. Merck).
Column chromatography was performed on 100–200 mesh silica gel (SRL, India). All
solvents were distilled and/or dried before use and all evaporations were conducted
below 50°C under reduced pressure unless stated otherwise. Optical rotations were
measured with a Perkin Elmer model 241 MC polarimeter. The 1H and 13C NMR
spectra were recorded on a Bruker DPX 300 Spectrometer using CDCl3 as solvent and
tetramethylsilane as internal standard unless otherwise mentioned. Melting points were
determined on a paraffin oil bath and are uncorrected.
Ethyl 3,4,5-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio- -D-glucopyranoside (2).
Ethane thiol (5 mL, 67.6 mmol) and BF3.OEt2 (10 mL, 78.9 mmol) were added to a
solution of 1,3,4,5-tetra-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranose (1)5
(10.6 g, 22.2 mmol) in CH2Cl2 (86 mL) with stirring. The reaction was monitored
with TLC and after 22 h the solution was washed with water, saturated NaHCO3
and water in succession, dried (Na2SO4) and concentrated to give 2 (6.2 g, 62.5%) in
25
the form of fine crystals: mp 116°C (EtOAc-hexane); [a]D + 43.07 (c 1.7, CHCl3).
1H NMR: d 7.80, 7.68 (2m, 4H, phthalimido protons), 5.76 (t, 1H, J = 9.9 Hz, H-3),
5.41 (d,1H, J1,2 = 10.5 Hz, H-1), 5.11 (t,1H, J = 9.6 Hz, H-4), 4.33 (t, 1H, J = 10.5

100
Sarkar, Mukherjee, and Roy
Hz, H-2), 4.25, 4.11 (2m, 2H, H-6); 3.82 (m, 1H, H-5), 2.61 (m, 2H, SCH2CH3) 2.04,
1.96, 1.79 (3s, 9H, 3OCOCH3), 1.15 (t, 2H, J = 7.2 Hz, SCH2CH3).
Ethyl 2-deoxy-2-phthalimido-1-thio- -D-glucopyranoside (3). Compound 2 (3 g,
6.7 mmol) was deacetylated in the usual way with 0.05 N sodium methoxide in
methanol (25 mL) for 3 h. Column chromatography (EtOAc) of the product gave pure
3 (1.49 g, 69.5%) as an amorphous solid [a]D25 +2.87 (c 15.7, CHCl3).
Anal. Calcd for C16H19O6NS: C, 54.38; H, 5.42. Found: 54.25, H, 5.21.
Ethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio- -D-glucopyranoside (4).
A solution of compound 3 (3 g, 9.4 mmol) in acetonitrile (40 mL) was stirred at room
˚
temperature with a,a-dimethoxytoluene (2 mL, 13.1 mmol) and MS 3A (4 g) for
30 min. p-Toluenesulfonic acid (p-TsOH, 300 mg) was then added and stirring was
continued overnight. TLC showed about 80% conversion. The reaction was quenched
with Et3N and the solution was concentrated to a syrup. Column chromatography (5:I
25
toluene-EtOAc) gave compound 4 (3.2 g, 77.5%) as white foam. [a]D À 4.98° (c 1.2,
1
CHCl3). H NMR: d 7.82, 7.76 (2m, 4 protons of phthalimido), 7.45—7.31 (m, other
aromatic protons), 5.51 (s, CHPh), 5.36 (d, J = 10.5 Hz, H-1), 4.62 (t, J = 10.0 Hz,
H-3), 4.35 (m, H-4), 4.28 (t, J = 10.4 Hz, H-2), 3.76, 3.66, 3.56 (m, 3H, H-5, H-6),
2.63 (m, SCH2CH3), 1.13 (t, J = 7.4 Hz, SCH2CH3).
Anal. Calcd for C23H23O6NS: C, 62.57; H, 5.25. Found: 62.74, H, 5.31.
Ethyl 4,6-O-benzylidene-3-O-chloroacetyl-2-deoxy-2-phthalimido-1-thio- -D-
glucopyranoside (5). To a solution of compound 4 (317 mg, 0.72 mmol) in dichlo-
romethane (3 mL) at 0°C, triethylamine (0.5 mL, 5 eq) and chloroacetic anhydride
(0.36 g, 2.16 mmol) were added and the mixture was stirred for 3 h when TLC showed
completion of the reaction. The reaction mixture was then diluted with CH2Cl2, washed
with water, saturated NaHCO3 solution and water in succession and dried (Na2SO4).
The organic layer was concentrated and the syrupy product on column chromatography
with 7:1 toluene-EtOAc gave 7 (310 mg, 83%) which crystallized from ethyl ether: mp
25
1
153–155°C; [a]D À 8.87° (c 1.6, CHCl3). H NMR: d 7.84, 7.74 (2m, 4 H, phthali-
mido protons), 7.73-7.15 ( other aromatic protons), 6.01(t, J2,3 = J3,4 = 9 Hz, H-3),
5.57 (d, J1,2 = 10.7 Hz, H-1), 5.35 (s, 1H, CHPh) 4.45 (m, 1H, H-2), 4.23 (m, 1H,
H-2), 3.71 (s, 2 H, COCH2Cl), 2.49 (m, SCH2CH3), 1.03 (t, J = 7.4 Hz, SCH2CH3).
13C NMR (CDCl3): d 167.1 (COCH2Cl), 137.1 À 124.1 (aromatic carbons), 102.1
(C6H5CH), 82.2 (C-1), 79.5, 72.6, 70.9, 69.0, 54.3 (C-2) 40.7 (ClCH2CO), 24.8
(SCH2CH3), and 15.3 (SCH2CH3).
Anal. Calcd for C25H24O7NS: C, 57.97; H, 4.67. Found: C, 57.79, H, 4.81.
4-Methoxyphenyl 2,3,4-tetra-O-acetyl- -L-rhamnopyranoside (6). The com-
pound 6 was prepared from L-rhamnose tetraacetate in 72% yield according to the
1
known[9] method; [a]D À 65.0° (c 1.4, CHCl3). H NMR: d 6.95, 6.75 (2d, 4H, aromatic
protons), 5.43 (dd, 1H, J2,3 = 3.5 Hz, J3,4 = 10.0 Hz, H-3), 5.35 (dd, 1H, J2,3 = 3.5
Hz, J1,2 = 1.8 Hz, H-2), 5.27 (d, 1H, J1,2 = 1.6 Hz, H-1), 5.07 (t, 1H,
J3,4 = J4,5 = 9.9 Hz, H-4), 3.95 (m, 1H, H-5), 3.7 (s, 3H, OCH3), 2.11, 1.99, 1.95
(3s, 9H, 3COCH3), 1.14 (d, 1H, J = 6.2 Hz, H-6).
Anal. Calcd for C17H21O7: C, 63.54; H, 6.59. Found: C, 63.83, H, 6.70.

Escherichia coli Type O26:H
101
4-Methoxyphenyl 2,3-O-isopropylidene- -L-rhamnopyranoside (8). Compound
6 (4 g, 10.1 mmol) was de-O-acetylated as described for compound 5 to give
4-methoxyphenyl a-L-rhamnopyranoside (7, 2.58 g, 94.5%). To a solution of 7 (5 g,
18.5 mmol) dissolved in DMF (20 mL), 2,2-dimethoxypropane (32 mL) and p-TsOH
(250 mg) were added and the mixture was stirred at room temperature for 5 h. The
reaction was then quenched with NEt3 and the solvents were removed under reduced
pressure. Column chromatography with 4:1 toluene-EtOAc gave 8 (4.2g, 73%); mp 62°C
1
(Et2O-pet ether); [a]D À 49.7° (c 1.1, CHCl3). H NMR signals at d 7.0–6.8 (m, 4H,
aromatic protons), 5.62 (bs, 1H, H-1), 4.36 (d, J2,3 = 5.7 Hz, H-2), 4.24 (t, J2,3 = 5.7 Hz,
H-3), 3.78 (m, 1H, H-4), 3.76 (s, O-CH3), 3.49 (dd, 1H, J = 1.8 Hz, J = 7.5 Hz, H-5),
1.57 (2s, 6H, CMe2),1.26 (d, J = 6.3 Hz,C-CH3).
Anal. Calcd for C16H22O6: C, 61.92; H, 7.14. Found: C, 61.79; H, 7.31.
4-Methoxyphenyl 4-O-benzoyl-2,3-O-isopropylidene- -L-rhamnopyranoside (9).
Compound 8 (4.7 g, 15.2 mmol) was dissolved in pyridine (25 mL) and benzoyl
chloride (4.4 mL) was added with a syringe while cooling the reaction mixture at 0°C.
The reaction mixture was then stirred at room temperature for 4 h when TLC (3:1
toluene-Et2O) showed completion of the reaction. Water (1.2 mL) was added to
decompose the excess benzoyl chloride and the mixture was concentrated and co-
evaporated thrice with toluene. The product was then dissolved in CH2Cl2 (50 mL) and
the solution was washed with water, dried (Na2SO4) and concentrated to a syrup.
Column chromatography with 3:1 toluene-Et2O gave pure 9 (5.8 g, 92%) which
25
1
crystallized from hot ethanol (mp 88–90°C). [a]D À 1.03° (c 1.6, CHCl3); H NMR: d
8.21–6.83 (m, 9H, aromatic protons), 5.68 (s, 1H, H-2) 5.19 (dd, 1H, J3,4 = 7.5 Hz,
J4,5 = 9.9 Hz, H-4), 4.51 (dd, 1H, J3,4 = 7.8 Hz, J2,3 = 5.4 Hz, H-3), 4.42 (d, 1-H,
J2,3 = 5.4 Hz, H-2), 4.04 (m,1H, H-5), 3.78 (s, 3H, OCH3) 1.66, 1.40 (2s, 6H, CMe2),
1.18 (d, J = 6.3 Hz, H-6).
Anal. Calcd for C23H26O7: C, 66.65; H, 6.32. Found: C, 66.47, H, 6.21.
4-Methoxyphenyl 4-O-benzoyl- -L-rhamnopyranoside (10). Compound 9 (5 g,
12.1 mmol) was stirred with 80% AcOH (35 mL) at 80°C for 2 h, when TLC showed
one major spot. The reaction mixture was concentrated to a syrup which on column
chromatography with 3:1 toluene-EtoAc gave 10 (3.77 g, 83.5%) as amorphous solid.
25
[a]D À 101.8° (c l.6, CHCl3).
Anal. Calcd for C20H22O7: C, 64.16; H, 5.92. Found: C, 64.39, H, 6.11.
4-Methoxyphenyl 2,4-di-O-benzoyl- -L-rhamnopyranoside (11). Compound 10
(2.65 g, 7.09 mmol) in dry CH2Cl2 (27 mL) was treated with trimethyl orthobenzoate
(6 mL) and ( ) camphor-10-sulfonic acid (catalytic amount) with stirring. After 15 min
TLC (3:1 toluene-EtOAc) showed a single faster moving spot and the reaction was
quenched by adding a few drops of NEt3. The solution was concentrated to a syrup and
treated with 80% aqueous acetic acid (52 mL) at room temperature for 30 min. The
solution was then concentrated and the resulting syrupy product was purified by
column chromatography with 3:1 toluene-EtOAc to afford compound 11 (2.68 g) which
crystallized from hot ethanol: mp 112°C; [a]D À 18.1° (c 2.8, CHCl3). 1H NMR: d
8.10–6.83 (m, aromatic protons, 14 H), 5.58 (bs, 1H, H-1), 5.56 (m, 1H, H-2),
5.34 (t, J = 9.8 Hz, H-4), 4.52 (dd, J2,3 = 3.26 Hz, J3,4 = 9.8 Hz, H-3), 4.23 (m,

102
Sarkar, Mukherjee, and Roy
1H, H-5), 3.78 (s , 3H, OCH3), 1.32 (d, J = 6.2 Hz, CCH3), 13C NMR d: 167.5,
166.4 (2 COCH3), 155.7–115.1 (aromatic carbons), 97.0 (C-1), 75.9, 73.5, 69.3.
67.3, 56.1 (OCH3), 18.1 (CCH3).
Anal. Calcd for C27H26O8: C, 67.78; H, 5.48. Found: C, 67.94, H, 5.31.
2-Azido-3,4-di-O-acetyl-2-deoxy- -L-fucopyranosyl nitrate (13). To a solution
of 12 (800 mg, 3.75 mmol) in acetonitrile (20 mL) at À 15°C was added sodium azide
(0.36 gm, 5.53 mmol) and ceric ammonium nitrate (7.4 g, 13.5 mmol). The suspension
was vigorously stirred for 10 h when TLC showed completion of the reaction. The
mixture was diluted with cold ethyl ether and washed with ice-water. The organic
layer was concentrated and the resulting syrupy product was column chromatographed
(5:1 toluene-EtOAc) to afford compound 13 (620 mg, 52%) which crystallized from
ether (mp 114–116°C); [a]D À 126.7° (c 0.8, CHCl3). 1H NMR: d 6.32 (d,1H,
25
J1,2 = 4.2 Hz, H-1), 5.35 (m, I H, H-4), 5.28 (dd,1H, J2,3 = 11.2 Hz, J3,4 = 3.1 Hz,
H-3), 4.31 (m, 1H, H-5), 4.09 (dd, 1H, J1,2 = 4.16 Hz, J2,3 = 11.2 Hz, H-2), 2.19,
2.07 (2s, 6H, 2COCH3), 1.22 (d, J = 6.4 Hz, CCH3); I.R: 2131 cm À 1 (N3).
Anal. Calcd for C10H14O8N4: C, 41.96; H, 4.93. Found: C, 42.02, H, 4.77.
3,4-Di-O-acetyl-2-azido-2-deoxy- -L-fucopyranose (14). To a solution of com-
pound 13 (90 mg, 283 mmol) in 20:1 dioxane-water (2.1 mL), sodium nitrite (42.6 mg,
0.62 mmol) was added. The mixture was stirred at 80°C for 20 h when TLC showed
only a trace of the starting compound. Water (25 mL) was added and the mixture was
extracted with CH2Cl2 (15 mL ꢁ 3). The extract was dried (Na2SO4), filtered and the
filtrate was concentrated to dryness. Column chromatography (3:1 toluene-EtOAc) gave
14 (60 mg, 88%) as a syrup.
Anal. Calcd for C10H15O6N3: C, 43.95; H, 5.53. Found: C, 44.12, H, 5.72.
2-Azido-3,4-di-O-acetyl-2-deoxy- -L-fucopyranosyl trichloroacetimidate (15).
To a solution of 14 (68 mg, 0.25 mmol) in dichloroethane (1 mL), potassium
carbonate (91 mg, .68 mmol) was added followed by the addition of trichloroace-
tonitrile (0.13 mL, 1.2 mmol) with vigorous stirring under N2. The reaction was
complete in 3.5 h (TLC) after which the mixture was filtered through Celite and
washed with CH2Cl2 (5 mL). The filtrate was concentrated to afford 15 (83.5 mg,
1
80%) as a syrup. H NMR: d 8.76 (s, 1H, C = NH), 5.69 (d, 1H, J = 8.4 Hz, H-1),
5.25 (d, 1H, J3,4 = 3.27 Hz H-4), 4.89 (dd, 1H, J2,3 = 10.7 Hz, J3,4 = 3.3 Hz, H-3),
3.99 (m, 1H, H-2), 3.89 (m, 1H, H-5), 2.18, 2.07 (2s, 6H, 2COCH3), 1.25 (d, 3H,
J = 6.33 Hz, CCH3).
4-Methoxyphenyl 4,6-O-benzylidene-3-O-chloroacetyl-2-deoxy-2-phthalimido-
-D-glucopyranosyl-(1 3)-2,4-di-O-benzoyl- -L-rhamnopyranoside (16). To a
solution of the donor 5 (390 mg, 0.75 mmol) and the acceptor 11 (300 mg, 0.63
!
˚
mmol) in CH2Cl2 (12 mL), 4A MS (3 g) were added and the mixture was stirred
overnight at room temperature under N2. The mixture was then cooled to À 25°C and
NIS (203 mg, 0.90 mmol) was added. After 15 min TfOH (9.5 mL, 0.11 mmol) was
introduced and stirring was continued. After 45 min the acceptor was completely
consumed, and the reaction mixture was filtered through a Celite bed and washed with

Escherichia coli Type O26:H
103
CH2Cl2. The combined filtrate and washings were washed successively with 5%
Na2S2O3 solution, aqueous saturated NaHCO3 and water, dried (Na2SO4), concentrated
and purified by column chromatography with 12:1 toluene-Et2O to afford the
25
1
disaccharide 16 as a foam (300 mg, 54.9%); [a]D À 5.5° (c 0.9, CHCl3). H NMR:
d 8.08–6.75 (23 H, aromatic protons), 5.74 (m, 1H, H-3II), 5.60 (d, J1,2 = 8.1 Hz,
H-1II), 5.57 (m, 1H, H-4I), 5.34 (bs, 1H, CHPh), 4.44 (dd, 1H, J2,3 = 3.6 Hz, J3,4 = 9.4
Hz, H-3I), 4.33 (m, 1H, H-4II), 4.18 (dd, 1H, J1,2 = 8.3 Hz, J2,3 = 10.2 Hz, H-2II),
3.97 (m, 1H, H-5I), 3.70 (s, 2H, ClCH2CO), 3.68 (s, 3H, OCH3), 3.60 (m, 1H, H-6II),
3.56 (m, 2H, H-5II), 1.02 (d, J5,6 = 6.6 Hz, H-6I). 13C NMR: d 165.6,165.1 (2OCOPh),
163.9 (ClCH2CO), 154.2–113.7 (aromatic carbons), 100.6 (CHPh), 97.9 (C-1II), 95.3
(C-1I), 77.6, 74.9, 71.4, 71.0, 70.3, 67.4, 65.9, 64.9, 54.3 (OCH3), 53.9 (C-2II), 39.2
(ClCH2CO), 16.5 (C-6I).
Anal. Calcd for C50H44O15NCl: C, 64.27; H, 4.75. Found: C, 64.42, H, 4.87.
4-Methoxyphenyl 4,6-O-benzylidene-2-deoxy-2-phthalimido- -D-glucopyrano-
syl-(1 3)-2,4-di-O-benzoyl- -L-rhamnopyranoside (17). Thiourea was added to a
!
solution of 16 (40 mg, 0.046 mmol) in 2:1 CH3OH-CH2Cl2 (0.9 mL) and excess
NaHCO3 (80 mg) was added. The mixture was stirred at 25°C for 1 h (TLC) and the
solution was concentrated. The residue was treated with CH2Cl2 and washed with
water, dried (Na2SO4) and concentrated. Column chromatography (5:1 toluene-EtOAc)
25
of the residue afforded compound 17 (32 mg, 91%) as a foam; [a]D À 25.9° (c 1.0,
CHCl3). 1H NMR: d 8.15–6.82 (23H, aromatic protons), 5.63 (dd, J1,2 = 1.8 Hz,
J2,3 = 3.4 Hz, H-2I), 5.54 (d, 1H, J1,2 = 1.2 Hz, H-1I), 5.48 (d, 1H, J1,2 = 8.3 Hz,
H-1II), 5.43 (s, 1H, CHPh), 5.42 (t, 1H, J = 9.7 Hz, H-4I), 4.51 (m, 1H, H-3I), 4.47
(m, 1H, H-3II), 4.33 (m, 1H, H-4II), 4.14 (dd, 1H, J1,2 = 8.4 Hz, J2,3 = 10.5 Hz, H-
2II), 4.04 (m, 1H, H-5II), 3.77 (s, 2H, ClCH2CO), 3.76 (s, 3H, OCH3), 3.58 (m, 2H,
H-6II), 3.41 (m, 1H, H-5II), 1.1 (d, 3H, J5,6 = 6.2 Hz, H-6I). 13C NMR: d 166.6,
165.3 (2 CO of OBz), 155.6–115.1 (aromatic carbons), 102.2 (CHPh), 99.7 (C-1II),
96.8 ((C-1I), 82.2, 76.1, 72.9, 72.6, 68.85, 68.7, 67.3, 66.4, 56.8 (C-2II), 56.1 (OCH3),
17.9 (C-6I).
Anal. Calcd for C48H43O14N: C, 67.20; H, 5.05. Found: C, 67.02, H, 5.17.
4-Methoxyphenyl 2-azido-3,4-di-O-acetyl-2-deoxy- -L-fucopyranosyl-(1 3)-
!
4,6-O-benzylidene-2-deoxy-2-phthalimido- -D-glucopyranosyl-(1 3)-2,4-di-O-ben-
!
zoyl- -L-rhamnopyranoside (18). A solution of 17 (210 mg, 0.27 mmol) and 15
˚
(260 mg, 0.63 mmol) in CH2Cl2 (3 mL) was stirred in the presence of MS 4A (1 g)
under Ar for 1 h. The mixture was then cooled to À 30°C and a solution of TESOTf
(6 mL, 0.027 mmol) in dry CH2Cl2 (2 mL) was added dropwise. The reaction was
allowed to proceed at À 30°C for 30 min when TLC showed the disappearance of the
donor 22. The reaction was quenched with the addition of Et3N and the mixture was
filtered through a Celite bed. The filtrate was concentrated and the syrupy product was
purified by column chromatography with10:1 toluene-Et2O to afford 18 (175 mg,
25
62.2%) which crystallized as fine needles: mp 148°C (ethanol); [a]D À 29.4° (c 1,
CHCl3). 1H NMR: d 8.15–6.83 (aromatic protons), 5.60 (dd, 1H, J1,2 = 1.9 Hz,
J2,3 = 3.5 Hz, H-2I), 5.54 (d, 1H, J1,2 = 1.6 Hz, H-1III), 5.42 (s, 2H, H-1I, CHPh), 5.40
(d, 1H, J = 8.6 Hz, H-1II), 4.99 (bs, 1H, H-4III), 4.96 (m, 1H, H-4I), 4.37 (dd, 1H,

104
Sarkar, Mukherjee, and Roy
J = 4.0 Hz, J = 9.8 Hz, H-2II), 4.22 (dd, 1H, J = 10.4 Hz, J = 8.4 Hz, H-4II), 3.78 (s,
3H, OCH3), 3.33 (m, 1H, H-5II), 1.95, 1.86 (2s, 6H, 2COCH3), 1.08 (d, 3H, J = 6.3
Hz, H-6I), 0.33 (d, 3H, J = 6.6 Hz, H-6III). 13C NMR: d 169.2, 168.5 (CO of 2OAc),
165.2, 163.9 (CO of 2 OBz), 101.3 (CHPh), 98.3 (C-1II), 97.6, 95.3 (C-1I, C-1III), 79.4,
74.1, 71.6, 71.1, 69.5, 68.3, 65.9, 65.4, 64.0 (C-2III), 56.6 (C-2II), 54.6 (OCH3), 19.5,
19.4 (2COCH3), 16.5 (C-6I), 13.7 (C-6III).
Anal. Calcd for C58H56O19N4: C, 63.86; H, 5.27. Found: C, 64.02, H, 5.40.
4-Methoxyphenyl 2-acetamido-3,4-di-O-acetyl-2-deoxy- -L-fucopyranosyl-
(1 3)-2-deoxy-2-phthalimido- -D-glucopyranosyl-(1 3)-2,4-di-O-benzoyl- -L-
!
!
rhamnopyranoside (19). A solution of 18 (50 mg, 0.05 mmole) in aldehyde free
ethanol (4 mL) containing acetic anhydride (0.2 mL) was stirred with 10% Pd on
charcoal under hydrogen for three days when all the starting material was transformed
into a slower moving compound as observed in the TLC (EtOAc). The mixture was
filtered through a Celite bed, the filtrate was concentrated to a syrupy product which
on column chromatography with 3:1 EtOAc-toluene gave pure 19 (27 mg, 58%);
25
1
[a]D À 46.1 (c 0.9, CHCl3). H NMR: d 8.17–6.82 (18H, aromatic protons), 5.92 (d,
IH, J = 3.6 Hz, N-H), 5.49 (d, 1H, J1,2 = 7.8 Hz, H-1II), 5.43 (d, 1H, J1,2 = 1.7 Hz,
H-1I), 5.39 (t, 1H, J = 9.8 Hz, H-4I), 5.12 (d, 1H, J = 2.3 Hz, H-2I), 4.94 (m, 1H, H-
4II), 4.67 (d, 1H, J = 3.7 Hz, H-1III), 4.44 (dd, 1H, J = 3.7 Hz, H-2II), 2.08, 1.87 (2s,
6H, 2OCOCH3), 1.1 (d, 1H, J = 2.1 Hz, H-6I), 1.08 (d, 1H,J = 2.4 Hz, H-6III). 13C
NMR: d 170.8, 170.5 (2 COPh), 169.8, 167.0 (2 COCH3) 164.8 (NHCOCH3), 155.2,
149.9, 134.1, 133.7, 132.9, 130.6, 130.2, 129.5, 129.0, 128.7, 128.3, 128.2, 117.7, 114.7
(aromatic carbons), 98.9 (C-1II), 98.7 (C-1III), 96.4 (C-1I), 81.2, 76.4, 76.0, 72.5, 71.9,
70.1, 70.0, 88.3, 66.9, 66.4, 62.0, 55.7 (OCH3), 55.0 (C-2II), 47.7 (C-2III), 22.10, 20.64,
20.62 (3COCH3), 17.56 (C-61), 15.96 (C-6III).
Anal. Calcd for C53H56O20N2: C, 61.15; H, 5.42. Found: C, 61.32, H, 5.49.
4-Methoxyphenyl 2-acetamido-3,4-di-O-acetyl-2-deoxy- -L-fucopyranosyl-
(1 3)-4,6-di-O-acetyl-2-acetamido-2-deoxy- -D-glucopyranosyl-(1 3)-2,4-di-O-
!
!
acetyl- -L-rhamnopyranoside (20). Ethylenediamine (0.5 mL) was added to a
solution of compound 19 (27 mg, 0.026 mmol) in 1-butanol (2.5 mL) under argon. The
solution was stirred for 20 h at 90°C when TLC (EtOAc) indicated completion of the
reaction. The solvents were evaporated and the residue was coevaporated twice with
toluene. The product was treated with pyridine (0.5 mL) and Ac2O (0.5 mL) for 20 h
when TLC (EtOAc) showed one major spot. The reaction mixture was concentrated
under reduced pressure followed by coevaporation with toluene to remove trace
reagents. Column chromatography (3:1 EtOAc-toluene) then gave compound 20 (12
1
mg, 50% overall); [a]D À 48.6° (c 0.5, CHCl3). H NMR: d 6.97, 6.82 (2d, 4H, J = 9
Hz, aromatic protons of 4-methoxyphenyl), 5.95 (d, IH, J = 7.3 Hz, NH), 5.82 (d, IH,
J = 9.1 Hz, NH), 5.34 (bs, 2H, H-1I, H-1III), 5.17 (m, 1H, H-2I), 5.12 (d, 1H, J = 7.9
Hz, H-1II), 4.89 (dd, 1H, J = 9.5 Hz, J = 9.3 Hz, H-4I), 2.17, 2.12, 2.09, 2.07, 2.01,
2.00 (8s, 24H, 6OCOCH3, 2 NHCOCH3), 1.16 (d, 3H, J = 6.2 Hz, H-6I), 1.10 (d, 3H,
J = 6.4 Hz, H-6III). 13C NMR: d 170.2, 170.1, 170.0, 169.7, 169.6, 169.3, 169.2, 168.9
(8 COCH3), 154.3, 148.9, 116.8, 113.7 (aromatic carbons), 98.4 (C-1II), 96.6 (C-1III),
95.4 (C-1I), 77.1, 74.0, 73.5, 71.6, 70.9, 70.7, 69.6, 69.4, 67.3, 65.8, 64.8, 61.4 (C-6II),

Escherichia coli Type O26:H
105
57.5 (C-2II), 54.6 (OCH3), 47.3 (C-2III), 28.7, 22.6, 22.3, 21.7, 20.1, 20.0, 19.8, 19.7 (8
COCH3), 16.5 (C-61), 14.4 (C-6III).
Anal Calcd for C41H56O21N2: C, 53.94%; H, 6.18%. Found C, 54.1; H, 6.35.
4-Methoxyphenyl 2-acetamido-2-deoxy- -L-fucopyranosyl-(1 3)-2-acetamido-
!
2-deoxy- -D-glucopyranosyl-(1 3)- -L-rhamnopyranoside (21). The acetate 20
!
(16.0 mg, 0.0175 mmol) was treated with 0.05 M NaOMe in methanol (5 mL) as
described in the case of compound 3. The deacetylated product was dissolved in water
and filtered through Sep-Pak C-18 cartridge and concentrated to dryness to afford pure
25
1
21 (10.6 mg, 91.4%); [a]D À 90.7° (c 0.7, water). H NMR: d 6.99, 6.87 (2d, 4H,
J = 8.8 Hz, aromatic protons of 4-methoxyphenyl), 5.31 (s, IH, H-1I), 4.92 (d, 1H,
J = 3.8 Hz, H-1III), 4.68 (bs, 1H, H-2I), 4.56 (d, 1H, J = 8.4 Hz), 4.25 (m, 1H, H-4III),
3.99 (dd, J = 3.8 Hz, J = 11.1 Hz, H-2III), 3.86 (dd. 1H, J = 2.9 Hz, J = 9.8 Hz, H-
3I), 3.69 (s, 3H, OCH3), 3.60 (t, 1H, J = 8.9 Hz, H-2II), 1.93, 1.86 (2s, 6H, 2
NHCOCH3), 1.10, 1.07 (2d, 6H, J = 6.2 Hz, H-6I, H-6III). 13C NMR: d 174.80, 174.77
(2NHCOCH3), 155.22, 149.75, 119.36, 115.60 (aromatic carbons), 103.40 (C-1II), 99.30
(C-1III), 98.17 (C-1I), 80.50, 78.89, 76.01, 71.60, 71.28, 70.17, 68.74, 68.16, 67.28,
60.93 (C-6II), 56.27 (C-2II), 56.11 (OCH3), 49.91 (C-2III), 22.69, 22.58 (2NHCOCH3),
16.97 (C-61), 15.72 (C-6III).
Anal. Calcd for C29H44O15N2: C, 52.72%; H, 6.71%. Found C, 52.51; H, 6.95.
ACKNOWLEDGMENT
Financial support by the Council of Scientific and Industrial Research, New Delhi
(Project No 01/1536/98/EMR-II) is thankfully acknowledged.
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Received August 10, 2002
Accepted January 24, 2003