Synthesis of a Tetrasaccharide Related to the Repeating Unit of the Antigen from Shigella dysenteriae Type 9 in the Form of Its 2-(Trimethylsilyl)ethyl Glycoside

Article abstract of DOI:10.1081/SCC-120028645

Starting from D-mannose, D-galactose and D-glucosamine hydrochloride, two disaccharide blocks were synthesized. Schmidt's inverse addition technique for trichloroacetimidate was utilized for the construction of a disaccharide with a β-mannosidic linkage i

Full text of DOI:10.1081/SCC-120028645

SYNTHETIC COMMUNICATIONSw
Vol. 34, No. 6, pp. 1125–1139, 2004
Synthesis of a Tetrasaccharide Related to
the Repeating Unit of the Antigen from
Shigella dysenteriae Type 9 in the Form
of Its 2-(Trimethylsilyl)ethyl Glycoside
*
Samarpita Roy, Sujit Kumar Sarkar, and Nirmolendu Roy
Department of Biological Chemistry, Indian Association for the
Cultivation of Science, Jadavpur, Kolkata, India
ABSTRACT
Starting from D-mannose, D-galactose and D-glucosamine hydro-
chloride, two disaccharide blocks were synthesized. Schmidt’s inverse
addition technique for trichloroacetimidate was utilized for the construc-
tion of a disaccharide with a b-mannosidic linkage in good yield. The two
disaccharides in the appropriate form were then allowed to react in the
presence of N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid
(TfOH) to give the tetrasaccharide derivative from which removal of
protecting groups gave the desired tetrasaccharide in the form of its
2-(trimethylsilyl)ethyl glycoside.
*
Correspondence: Nirmolendu Roy, Department of Biological Chemistry, Indian
Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India; E-mail:
bcnr@mahendra.iacs.res.in; nirmolendu@yahoo.com.
1125
DOI: 10.1081/SCC-120028645
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

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Roy, Sarkar, and Roy
Key Words: Tetrasaccharide; Shigella dysenteriae; N-iodosuccinimide;
Trifluoromethanesulfonic acid.
Shigella are facultative, non-motile, Gram-negative bacilli. They are
pathogenic primarily due to their ability to invade intestinal epithelial cells.
They are responsible for the intestinal diseases including dysentery and has the
potential for causing catastrophic public health problem in developing
countries.[1] They are much resistant to the anti-microbial drugs. It, therefore,
necessitates the exploration of other medical approaches for control of
diseases caused by these pathogens.[1] It has been suggested[2] that circulating
antibodies to the O-specific polysaccharide of Shigella may protect the host
against shigellosis and the conjugate vaccines consisting of the O-specific
polysaccharide of Shigella, covalently attached to an immunogenic protein
could, indeed, confer protective immunity to human against shigellosis.
Recently, a glycoconjugate vaccine against Shigella dysenteriae type 1 was
synthesized,[3] which was shown to have better antigenicity than the native
antigen. It is, therefore, probable that the O-specific polysaccharide from
Shigella dysenteriae type 9 (O-SP) may also play a protective role against
bacillary dysentery and shigellosis in human. The O-SP have been reported to
contain two b-D-galactoses, one N-acetyl-b-D-glucosamine and one b-D-
mannose in its repeating unit (I). It also contains a pyruvic acid acetal at the
4,6-position of one D-galactose moiety and an acetyl group at the 2- or
3-position of the other galactose moiety. Immunochemical studies,[4] on
Shigella dysenteriae type 9 showed that the presence of pyruvic acid acetal
and the O-acetyl group in the related oligosaccharides make negligible
contribution to the specificities in the immune precipitation.
We have already reported,[5] the synthesis of a blocked tetrasaccharide
derivative related to the repeating unit I in the form of its methyl (R)-pyruvate
ester and 2-(trimethylsilyl)ethyl glycoside. It is therefore deemed interesting
to synthesize the tetrasaccharide analog related to the repeating unit of the
Shigella dysenteriae type 9 as its 2-(trimethylsilyl)ethyl glycoside but without
the pyruvate acetal. The blocked oligosaccharides can be easily transformed
into the corresponding hemiacetal for the preparation of trichloroacetimidate
donor and its transformation into a neoglycoconjugate.

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Synthesis of Tetrasaccharide
1127
The basic strategy utilized in the synthesis of the tetrasaccharide related to
the repeating unit of Shigella dysenteriae type 9 was first to synthesize the two
disaccharide derivatives, convert them to a suitable disaccharide donor and a
disaccharide acceptor and then allow them to react in the presence of an
appropriate promoter to form the desired tetrasaccharide.
The known ethyl 3,4-O-isopropylidene-1-thio-b-D-galactopyranoside[6]
(1) was treated with tert-butyldiphenylsilyl chloride (TBDPSCl)[7] followed
by acetylation of the product to give 2 which on treatment with 80% acetic
gave the acceptor 3 in 91% yield (Sch. 1). The structure of 3 was confirmed
from its 1H NMR spectrum.
The acceptor 3 with two hydroxyl groups of different reactivity was
allowed to react with the known 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
D-glucopyranosyl trichloroacetimidate[8] (4) in the presence of triethylsilyl-
trifluoromethane sulfonate (TESOTf)[9,10] in dichloromethane to afford ethyl
3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1 ! 3)-2-O-
acetyl-6-O-(tert-butyldiphenylsilyl)-1-thio-b-D-galactopyranoside (5) in 69%
yield. The formation of 1 ! 3 linked disaccharide resulting from the better
reactivity of the 3-OH compared to the 4-OH group of galactose. This was
confirmed by the downfield shift of H-3C proton which showed a double
doublet at d 4.39. As expected, acetylation of 5 gave a product, which showed
1
a downfield shift of the signal for H-4C from d 4.15 to d 5.39 in its H NMR
spectrum. The b-anomeric configuration of the disaccharide was confirmed
from the high coupling constant (J1,2 ¼ 8.4 Hz) of the signal for H-1D.
Compound 5 has characteristic signals for phthalimido, 4 O-acetyl, TBDPS
and C-2D together with anomeric protons and carbons in the NMR spectra.
Removal of TBDPS from 5 was effected with TBAF[11] in THF to afford 6
which was then stirred with a,a-dimethoxytoluene[12] and catalytic amount of
10-camphorsulphonic acid (CSA) to give the corresponding benzylidene
derivative 7. A sharp singlet at d 5.44 in the 1H NMR spectrum confirmed the
formation of benzylidene in the disaccharide. Dephthaloylation of the
disaccharide 7 was performed with ethylenediamine in 1-butanol[13] to
generate the free amine which was converted into the acetamido derivative 8
with acetic anhydride and pyridine (Sch. 2).
Scheme 1. a. TBDPSCl, Pyr, 8 hr, 258C, 78%; b. Ac2O, Pyr, RT, 3 hr, 94%; c 80%
acetic acid, 808C, 2 hr, 91%.

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Roy, Sarkar, and Roy
Scheme 2. a. TES-OTf, CH2Cl2, 2308C, 30 min, 69%; b. TBAF, THF, 08C, 6 hr,
76%; c. PhCH(OCH3)2, CSA, CH3CN, RT, 16 hr, 89%; d. (i) 1-BuOH, N2NCH2CH2-
NH2, 908C, 20 hr; (ii) Ac2O, Pyr, RT, 24 hr, 85.3%.
In a separate experiment, allylation[15] of known 2-(trimethylsilyl)ethyl
4,6-O-benzylidene-3-O-benzyl-b-D-galactopyranoside[14](9) afforded the
2-O-allyl compound 10, the benzylidene ring of which was regioselectively
opened[16] with NaCNBH3 and HCl-OEt2 giving 2-(trimethylsilyl)ethyl 2-O-
allyl-3,6-di-O-benzyl-b-D-galactopyranoside (11) (Sch. 3).
Mannopyranosyl donor, 4,6-O-benzylidene-2,3-di-O-benzyl-a-D-man-
nopyranosyl trichloroacetimidate[5] (12) was allowed to react with the
acceptor 11 in dichloromethane in the presence of TESOTf under inverse
condition,[17] i.e the trichloroacetimidate donor being added to the mixture of
acceptor and TESOTf, to afford the disaccharide 2-(trimethylsilyl)ethyl 4,6-O-
benzylidene-2,3-di-O-benzyl-b-D-mannopyranosyl-(1 ! 4)-2-O-allyl-3,6-di-
O-benzyl-b-D-galactopyranoside (13) in 89% yield with 2 : 7 ratio of a and b
˚
Scheme 3. a. AllBr, NaH, DMF, RT, 6 hr; b. NaCNBH3, HCl.OEt2, MS 3 A, 08C,
45 min.

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Synthesis of Tetrasaccharide
1129
Scheme 4. a. TES-OTf, CH2Cl2, 2458C, 45 min, 69%; b. NaCNBH3; HCl.OEt2, 08C,
30 min, 68%.
anomers. The major amount of the b-product was probably due to the presence
of 4,6-benzylidene ring in the mannose moiety. Regioselective opening[16] of
the benzylidene ring of 13 using NaCNBH3 and HCl-ether in THF afforded the
disaccharide acceptor 14 (Sch. 4) in 70% yield together with a small amount
(ꢀ5%) of the 6-hydroxy compound. The compound 14 was characterized
1
from its signals in H and 13C NMR spectra.
The disaccharide donor 8 was allowed to react with the disaccharide
acceptor 14 in the presence of N-iodosuccinimide (NIS) and trifluoro-
methanesulfonic acid (TfOH)[18] to afford the tetrasaccharide derivative
2-(trimethylsilyl)ethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-gluco-
pyranosyl-(1 ! 3)-2-O-acetyl-4,6-O-benzylidene-b-D-galactopyranosyl
-
(
1 ! 4)-2,3,6-tri-O-benzyl-b-D-mannopyranosyl-(1 ! 4)-2-O-allyl-3,6-di
-
O-benzyl-b-D-galactopyranoside (15) in 74% yield (Sch. 5). The formation of
b-glycosidic linkage was confirmed from high coupling value of H-1 proton in
1H NMR. The signals in 13C also support the formation of b-glycosidic
linkage. Compound 15 was deallylated,[19] debenzylated,[19] and deacetylated
in succession to afford the desired tetrasaccharide 16. Compound 16 has
characteristic signals for NHCOCH3, C-2D,OCH2CH2SiMe3 together with 4
anomeric protons and carbons in its NMR spectra.
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,

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Roy, Sarkar, and Roy
Scheme 5. a. NIS, TfOH, CH2Cl2, 2208C, 1 hr, 74%; b. PdCl2, MeOH; c. H2, 10%
Pd/C, EtOH; d. NaOMe, MeOH.
India) using 15–20 times (by weight) of the crude product. The organic
extracts were dried over anhydrous Na2SO4. All solvents were distilled and/
or dried before use and all evaporations were conducted at or below 408C
under reduced pressure unless stated otherwise. Optical rotations were
measured at 248C with a Perkin-Elmer 241 MC polarimeter. The 1H and 13
C
NMR spectra were recorded with a Bruker DPX 300 spectrometer using
CDCl3 as solvent and tetramethylsilane as internal standard unless otherwise
stated. Melting points were determined on a paraffin oil bath and are
uncorrected.
Ethyl 2-O-Acetyl-6-O-tert-butyldiphenylsilyl-3,4-O-
isopropylidene-1-thio-b-D-galactopyranoside (2)
To a solution of ethyl 3,4-O-isopropylidene-1-thio-b-D-galactopyrano-
side (1) (3 g, 11.34 mmol) in pyridine (15 mL) was added tert-butyldi-
phenylsilyl chloride (2.1 mL, 17.0 mmol). The mixture was stirred for 8 hr at
258C and then concentrated under reduced pressure. Column chromatography
of the residue in 1 : 1 toluene-EtOAc gave the pure TBDPS derivative (4.45 g,
78%) which was acetylated conventionally to afford pure 2 (4.3 g, 94%);

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Synthesis of Tetrasaccharide
1131
[a]2D5 þ 21.48 (c 0.7, CHCl3); 1H NMR: d 7.64–7.29 (m, 10H, aromatic
protons), 4.93 (dd, 1H, J2,3 ¼ 7.5 Hz, J1,2 ¼ 10.3 Hz, H-2), 4.27 (dd, 1H,
J3,4 ¼ 5.1 Hz, J4,5 ¼ 2.0 Hz, H-4), 4.25 (d, 1H, J ¼ 10.1 Hz, H-1), 4.09 (dd,
1H, J2,3 ¼ 7.5 Hz, J3,4 ¼ 5.3 Hz, H-3), 3.88–3.77 (m, 3H, H-6, H-5), 2.58
(m, 2H, SCH2CH3), 2.02 (s, 3H, OCOCH3), 1.46, 1.27 [2 s, 6H, C(CH3)2],
1.14 (t, 3H, J ¼ 7.3 Hz, SCH2CH3), 0.98 [s, 9H, Ph2SiC(CH3)3]; 13C NMR:
170.2 (OCOCH3), 136.1–125.7 (aromatic carbons), 110.7 [C(CH3)2], 83.0
(C-1), 77.9, 77.6, 73.9, 72.1, 63.1 (C-6), 28.2, 26.8 [C(CH3)2], 27.0
[Ph2SiC(CH3)3], 24.2 (SCH2CH3), 21.5 (OCOCH3), 19.6 [Ph2SiC(CH3)3],
15.2 (SCH2CH3). Anal. Calcd for C29H40O6SiS: C, 63.93; H, 7.40. Found: C,
63.77; H, 7.78.
Ethyl 2-O-Acetyl-6-O-tert-butyldiphenylsilyl-1-thio-b-D-
galactopyranoside (3)
A solution of 2 (3.8 g, 6.98 mmol) in 80% acetic acid (20 mL) was stirred
at 808C for 2 hr when TLC showed complete conversion to the product.
Solvents were then evaporated off. Column chromatography with 3 : 1
toluene-EtOAc gave pure 3 (3.20 g, 91%) as a glassy mass. [a]2D5-25.48 (c 0.5,
1
CHCl3); H NMR: d 7.72–7.38 (m, 10H, aromatic protons), 5.09 (t, 1H,
J ¼ 9.6 Hz, H-2), 4.35 (d, 1H, J ¼ 9.9 Hz, H-1), 4.16 (d, 1H, J ¼ 3.0 Hz, H-4),
3.95–3.91 (m, 2H, H-6), 3.62 (dd, 1H, J2,3 ¼ 9.3 Hz, J3,4 ¼ 3.2 Hz, H-3), 3.51
(t, 1H, J ¼ 5.0 Hz, H-5), 2.68 (m, 2H, SCH2CH3), 2.15 (s, 3H, OCOCH3), 1.24
(t, 3H, J ¼ 7.5 Hz, SCH2CH3), 1.06 [s, 9H, Ph2SiC(CH3)3]; 13C NMR:
d 136.1–128.3 (aromatic carbons), 83.5 (C-1), 78.0, 74.2, 71.6, 70.3 64.1
(C-6), 27.2 [Ph2SiC(CH3)3], 23.9 (SCH2CH3), 21.5 (OCOCH3), 19.5
[Ph2SiC(CH3)3], 15.3 (SCH2CH3). Anal. Calcd for C26H36O6SiS: C, 61.87;
H, 7.19. Found: C, 61.65; H, 7.07.
Ethyl 3,4,6-tri-O-Acetyl-2-deoxy-2-phthalimido-b-D-
glucopyranosyl-(1 ! 3)-2-O-acetyl-6-O-tert-
butyldiphenylsilyl-1-thio-b-D-galactopyranoside (5)
To a solution of the acceptor 3 (1.01 g, 2.0 mmol) in CH2Cl2 (10 mL)
˚
was added 4A molecular sieve (2.5 g) and stirred for 2 hr under N2 at 2308C.
To this cold solution, were added TESOTf (81.7 mL, 0.36 mmol) and 3,4,6-
tri-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl
trichloroacetimi-
date (4) donor (1.4 g, 2.41 mmol) and the solution was allowed to stir for
30 min at that temperature. Then the solution was diluted with CH2Cl2
(25 mL) and washed successively with saturated NaHCO3 solution and

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1132
Roy, Sarkar, and Roy
water. The organic phase was collected, dried (Na2SO4) and concentrated.
Column chromatography of the residue with 10 : 1 toluene-EtOAc gave pure
5 (1.28 g, 69%); [a]2D5 þ 7.28 (c 0.7, CHCl3); 1H NMR: d 7.87–7.73 [m, 4H,
N(CO)2C6H4], 7.69–7.27 (m, 10H, aromatic protons), 5.77 (dd, 1H,
J2,3 ¼ 9.2 Hz, J3,4 ¼ 10.8 Hz, H-3D), 5.55 (d, 1H, J ¼ 8.5 Hz, H-1D), 5.18
(t, 1H, J ¼ 9.6 Hz, H-4D), 5.09 (t, 1H, J ¼ 9.7 Hz, H-2C), 4.38 (dd, 1H,
J1,2 ¼ 8.4 Hz, J2,3 ¼ 10.7 Hz, H-2D), 4.27 (dd, 1H, J5,6a ¼ 4.3 Hz,
J6e,6a ¼ 12.5 Hz, H-6Da ), 4.21 (d, 1H, J ¼ 9.8 Hz, H-1C), 4.13 (dd, 1H,
J5,6e ¼ 2.2 Hz, J6a,6e ¼ 12.4 Hz, H-6De ), 3.71 (dd, 1H, J2,3 ¼ 9.4 Hz,
J3,4 ¼ 3.1 Hz, H-3C), 3.55 (t, 1H, J ¼ 6.0 Hz, H-5C), 2.52 (m, 2H,
SCH2CH3), 2.03, 2.03, 1.86, 1.57 (4s, 12H, 4OCOCH3), 1.12 (t, 3H,
J ¼ 7.5 Hz, SCH2CH3), 1.04 [s, 9H, Ph2SiC(CH3)3]; 13C NMR: d 171.0,
170.6, 169.8, 169.6 (4 COCH3), 136.0–128.1 (aromatic carbons), 98.8
(C-1D), 83.4 (C-1C), 82.0, 78.8, 72.5, 70.8, 69.1, 68.8, 68.5, 62.9, 62.0 (C-6C,
C-6D), 54.8 (C-2D), 27.2 [Ph2SiC(CH3)3], 23.4 (SCH2CH3), 21.1, 21.0, 20.9,
20.8 (4 COCH3), 19.6 [Ph2SiC(CH3)3], 15.0 (SCH2CH3). Anal. Calcd for
C46H55O15SiNS: C, 59.91; H, 6.01; N, 1.51. Found: C, 59.85; H, 5.98;
N, 1.40.
Ethyl 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-D-
glucopyranosyl-(1 ! 3)-2-O-acetyl-1-thio-b-D-
galactopyranoside (6)
Tetrabutylammonium fluoride (366 mg, 1.16 mmol) was added to a
solution of 5 (800 mg, 0.86 mmol) in THF (3 mL) at 08C, then the temperature
was slowly raised to 258C. After 6 hr the solution was concentrated. Column
chromatography of the residue with 1 : 1 toluene-EtOAc gave pure 6 (450 mg,
76%); [a]2D5 2 16.68 (c 0.6, CHCl3); 1H NMR: d 7.86–7.73 [m, 4H,
N(CO)2C6H4], 5.75 (dd, 1H, J2,3 ¼ 9.3 Hz, J3,4 ¼ 10.5 Hz, H-3D), 5.53 (d, 1H,
J ¼ 8.4 Hz, H-1D), 5.13 (t, 1H, J ¼ 9.5 Hz, H-4D), 5.06 (t, 1H, J ¼ 9.5 Hz,
H-2C), 4.34 (dd, 1H, J1,2 ¼ 8.4, J2,3 ¼ 10.7 Hz, H-2D), 4.30–4.18 (m, 2H,
H-6D), 4.23 (d, 1H, J ¼ 10.0 Hz, H-1C), 4.15 (bs, 1H, H-4C), 3.89 (m, 2H,
H-6C), 3.55 (bs, 1H, H-5D), 2.54 (m, 2H, SCH2CH3) 2.10, 2.03, 1.83, 1.50 (4 s,
12 H, OCOCH3), 1.14 (t, 3H, J ¼ 7.4 Hz, SCH2CH3); 13C NMR: d 171.2,
170.5, 169.8, 169.6 (4 COCH3), 134.9–124.1 (aromatic carbons), 98.8 (C-1D),
83.6 (C-1C), 81.9, 72.5, 70.7, 69.3, 69.1, 68.6, 62.5, 62.2 (C-6C, C-6D), 54.8
(C-2D), 23.7 (SCH2CH3), 21.2, 21.1, 20.7, 20.6, (4 COCH3), 15.1 (SCH2CH3).
Anal. Calcd for C30H37O15SN: C, 52.70; H, 5.45; N, 2.04. Found: C, 52.43;
H, 5.26; N, 1.97.

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Synthesis of Tetrasaccharide
1133
Ethyl 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-D-
glucopyranosyl-(1 ! 3)-2-O-acetyl-4,6-O-benzylidene-
1-thio-b-D-galactopyranoside (7)
To a solution of 6 (350 mg, 0.51 mmol) in DMF (5 mL), a,a-dimethoxy-
toluene (115 mL, 0.76 mmol) and CSA (80 mg) were added. The mixture was
stirred at r.t. for 16 hr. The reaction was quenched with Et3N, concentrated and
column chromatographed with 2 : 1 toluene-EtOAc to afford 7 (351 mg, 89%),
1
[a]2D5 2 258 (c 0.5, CHCl3); H NMR: d 7.76–7.63 [m, 4H, N(CO)2C6H4],
7.42–7.27 (m, 5H, aromatic protons), 5.68 (dd, 1H, J2,3 ¼ 9.3 Hz,
J3,4 ¼ 10.8 Hz, H-3D), 5.49 (d, 1H, J ¼ 8.3 Hz, H-1D), 5.44 (bs, 1H, CHPh),
5.12 (t, 1H, J ¼ 9.6 Hz, H-4D), 5.11 (t, 1H, J ¼ 9.6 Hz, H-2C), 4.32 (dd, 1H,
J4,5 ¼ 2.2 Hz, J6e,6a ¼ 8.7 Hz, H-3C), 4.19 (d, 1H, J ¼ 9.4 Hz, H-1C), 4.17
(d, 1H, J ¼ 2.3 Hz, H-4C), 3.93 (d, 1H, J ¼ 12.3 Hz, H-6aC), 3.77 (m, 2H,
H-6D), 3.37 (bs, 1H, H-5C), 2.70–2.42 (m, 2H, SCH2CH3) 2.02, 1.96, 1.76,
1.62 (4 s, 12 H, OCOCH3), 1.09 (t, 3H, J ¼ 7.4 Hz, SCH2CH3); 13C NMR:
d 171.0, 170.6, 169.8, 169.7 (4 COCH3), 138.2–124.0 (aromatic carbons),
101.3 (CHPh), 99.2 (C-1D), 83.2 (C-1C), 79.3, 76.3, 72.4, 71.0,70.5, 69.5,
69.3, 68.2 (C-6C), 62.1 (C-6D), 54.8 (C-2D), 22.9 (SCH2CH3), 21.9, 21.3, 21.1,
20.8, 20.4 (4 COCH3), 15.1 (SCH2CH3). Anal. Calcd for C37H41O15SN:
C, 57.57; H, 5.35; N, 1.81. Found: C, 57.50; H, 5.26; N, 1.97.
Ethyl 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-
glucopyranosyl-(1 ! 3)-2-O-acetyl-4,6-O-benzylidene-
1-thio-b-D-galactopyranoside (8)
To compound 7 (300 mg, 0.43 mmol) dissolved in n-butanol (5 mL)
ethylenediamine (1 mL) was added. The solution was stirred at 908C for 20 hr.
Solvents were then removed under reduced pressure and finally by co-
evaporation twice with toluene to give yellow syrup. Acetic anhydride (3 mL)
and pyridine (3 mL) were then added and stirring was continued. After 14 hr,
the solution was concentrated to syrup which was purified by column
chromatography using 1 : 1 toluene-EtOAc to give 8 (151.2 mg, 85.3%) as a
thick glass, [a]2D5 þ 188 (c 0.6, CHCl3). 1H NMR: d 7.38–7.16 (m, 5H,
aromatic protons), 5.90 (d, 1H, J ¼ 7.2 Hz, NHCOCH3), 5.60 (dd, 1H,
J2,3 ¼ 9.7 Hz, J3,4 ¼ 9.9 Hz, H-3D), 5.36 (bs, 1H, CHPh), 5.20 (t, 1H,
J ¼ 9.3 Hz, H-4D), 5.19 (d, 1H, J ¼ 7.2 Hz, H-1D), 4.88 (t, 1H, J ¼ 9.5 Hz,
H-2C), 4.11 (d, 1H, J3,4 ¼ 3.0 Hz, H-4C), 3.72 (dd, 1H, J2,3 ¼ 3.2 Hz,
J3,4 ¼ 9.8 Hz, H-3C), 3.30 (bs, 1H, H-5C), 3.06 (m, 1H, H-5D), 2.76–2.53
(m, 2H, SCH2CH3), 1.98, 1.95, 1.88, 1.86, 1.70 (5 s, 15H, OCOCH3), 1.16
(t, 3H, J ¼ 7.4 Hz, SCH2CH3); 13C NMR: d 171.3, 171.0, 170.6, 170.5, 170.1

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1134
Roy, Sarkar, and Roy
(5 COCH3), 138.3–126.8 (aromatic carbons), 101.7 (CHPh), 100.0 (C-1D),
83.3 (C-1C), 78.3, 76.5, 72.0, 71.1, 70.5, 69.5, 69.4, 68.4 (C-6C), 62.4 (C-6D),
57.1 (C-2D), 23.6 (SCH2CH3), 23.2 (NHCOCH3), 21.7, 21.2, 21.1, 21.0
(4 COCH3), 15.2 (SCH2CH3). Anal. Calcd for C31H41O14SN: C, 54.45; H,
6.04; N, 2.04. Found: C, 54.50; H, 6.16; N, 2.10.
2-(Trimethylsilyl)ethyl 2-O-Allyl-3-O-benzyl-4,6-O-
benzylidene-b-D-galacto-pyranoside (10)
To a solution of 9[14] (2.5 g, 5.45 mmol) in DMF (15 mL) was added NaH
(654 mg, 13.6 mmol, 60% oil coated) at 08C with stirring. After 30 min, allyl
bromide (0.7 mL, 8.18 mmol) was added and stirring was continued at room
temperature for 7 hr. Excess NaH was decomposed by the addition of MeOH
(5 mL). The reaction mixture was then diluted with CH2Cl2 and filtered. The
organic layer was washed with water, dried (Na2SO4) and concentrated.
Column chromatography of the syrupy product with 5 : 1 toluene-EtOAc gave
pure 10 (2.4 g, 92%); [a]2D5 þ 7.78 (c 0.8, CHCl3); H NMR: d 7.38–7.08
1
(m, 10H, aromatic protons), 5.80 (m, 1H, CH2CH55CH2), 5.31 (s, 1H, CHPh),
5.16–4.97 (m, 2H, CH2CH55CH2), 4.52, 4.46 (2d, 2H, J ¼ 12.5 Hz, CH2Ph),
4.25 (dd, 1H, J5,6 ¼ 5.7 Hz, J6a,6e ¼ 12.3 Hz, H-6e), 4.16 (d, 1H, J ¼ 7.8 Hz,
H-1), 4.11 (dd, 1H, J5,6a ¼ 5.9 Hz, J6e,6a ¼ 12.4 Hz, H-6a), 3.91 (d, 1H,
J ¼ 3.4 Hz, H-4), 3.53 (dd, 1H, J1,2 ¼ 7.8 Hz, J2,3 ¼ 9.6 Hz, H-2), 3.30 (dd,
1H, J2,3 ¼ 9.6 Hz, J3,4 ¼ 3.6 Hz, H-3), 3.12 (bs, 1H, H-5), 3.87–3.80 [m, 2H,
OCH2CH2Si(Me)3], 0.85 [m, 2H, OCH2CH2Si(CH3)3], 20.15 [s, 9H,
OCH2CH2Si(CH3)3]; 13C NMR: d 138.4–126.5 (aromatic carbons), 135.3
(CH2CH55CH2), 116.4 (CH2CH55CH2), 103.1 (C-1), 101.3 (CHPh), 78.9,
78.0, 74.1, 73.9, 72.0, 69.1, 67.2 [OCH2CH2Si(CH3)3], 66.2 (C-6), 18.2
[OCH2CH2Si(CH3)3], 21.5 [OCH2CH2Si(CH3)3]. Anal. Calcd for
C28H38O6Si: C, 67.44; H, 7.68. Found: C, 67.33; H, 7.56.
2-(Trimethylsilyl)ethyl 2-O-Allyl-3,6-di-O-benzyl-b-D-
galactopyranoside (11)
To a solution of 10 (2 g, 4.01 mmol) in dry THF (20 mL) was added
NaCNBH3 (2.3 g, 36.1 mmol) at 08C. A saturated solution of HCl in ethylether
was added drop-wise with vigorous stirring till pH came down to about 2–3.
The suspension was stirred for 45 min at 08C, filtered through a Celite bed and
washed with CH2Cl2. The combined filtrate and washings was washed with aq
NaHCO3 and water, dried (Na2SO4) and concentrated. Column chromato-
graphy of the syrupy product with 5 : 1 toluene-EtOAc gave 11 (1.4 g, 70%);

ORDER
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Synthesis of Tetrasaccharide
1135
[a]2D5 2 4.78 (c 0.5, CHCl3); 1H NMR: d 7.22–7.09 (m, 10H, aromatic
protons), 5.86–5.74 (m, 1H, CH2CH55CH2), 5.16–4.97 (m, 2H, CH2-
CH55CH2), 4.56 (s, 2H, CH2Ph), 4.42 (s, 2H, CH2Ph), 4.23 (dd, 1H,
J5,6e ¼ 5.7 Hz, J6a,6e ¼ 12.3 Hz, H-6e), 4.13 (d, 1H, J ¼ 7.5 Hz, H-1), 4.13
(dd, 1H, J5,6a ¼ 5.7 Hz, J6e,6a ¼ 12.3 Hz, H-6a), 3.86 (dd, 1H, J1,2 ¼ 7.5 Hz,
J2,3 ¼ 9.6 Hz, H-2), 3.83 (bs, 1H, H-4), 3.65–3.52 [m, 2H, OCH2CH2-
Si(Me)3], 3.38 (m, 1H, H-5), 3.27 (dd, 1H, J2,3 ¼ 9.3 Hz, J3,4 ¼ 3.3 Hz, H-3),
0.85 [m, 2H, OCH2CH2Si(CH3)3], 20.15 [s, 9H, OCH2CH2Si(CH3)3]; 13C
NMR: d 138.6–128.1 (aromatic carbons), 135.8 (CH2CH55CH2), 117.1
(CH2CH55CH2), 103.6 (C-1), 81.0, 79.1, 74.1, 74.4, 73.7, 72.9, 69.7 (C-6),
67.7, 67.4 [OCH2CH2Si(CH3)3], 18.8 [OCH2CH2Si(CH3)3], 20.9 [OCH2-
CH2Si(CH3)3]. Anal. Calcd for C28H40O6Si: C, 67.16; H, 8.05. Found: C,
67.33; H, 8.26.
2-(Trimethylsilyl)ethyl 2,3-Di-O-benzyl-4,6-O-benzylidene-b-
D-mannopyranosyl-(1 ! 4)-2-O-allyl-3,6-di-O-benzyl-b-D-
galactopyranoside (13)
To a solution of the acceptor 11 (560 mg, 1.12 mmol) and TESOTf
˚
(45 mL, 0.20 mmol) in CH2Cl2 (5.0 mL) was added 4 A molecular sieves
(300 mg) and the mixture was stirred for 2 hr at 2458C. To this mixture 2,3-
di-O-benzyl-4,6-O-benzylidene-a,b-D-mannopyranosyl trichloroacetimidate
(12) (800 mg, 1.34 mmol) in CH2Cl2 (1.0 mL) was added drop-wise and the
mixture was allowed to stir for 45 min at that temperature when TLC showed
optimum amount of the product. The reaction was quenched with the
addition of Et3N, diluted with CH2Cl2 (50 mL), filtered and washed
successively with water, sat. NaHCO3 and water. The organic phase was
collected, dried (Na2SO4) and concentrated. Column chromatography of the
residue with 10 : 1 toluene-EtOAc gave pure disaccharide 13 (690 mg, 69%);
[a]2D5 2 188 (c 0.7, CHCl3); 1H NMR: d 7.36–7.04 (m, 25H, aromatic
protons), 5.84–5.73 (m, 1H, CH2CH55CH2), 5.42 (s, 1H, CHPh), 5.09–4.96
(m, 2H, CH2CH55CH2), 4.90, 4.74 (2d, 2H, CH2Ph), 4.47 (bs, 1H, H-1B),
4.37 (d, 1H, J ¼ 2.2 Hz, H-4A), 4.16 (d, 1H, J ¼ 4.7 Hz, H-1A), 3.71–3.64
[m, 2H, OCH2CH2Si(Me)3], 3.01 (m, 1H, H-5B), 0.86 [m, 2H, OCH2CH2-
Si(CH3)3], 20.15 [s, 9H, OCH2CH2Si(CH3)3]; 13C NMR: d 138.6–125.9
(aromatic carbons), 135.2 (OCH2CH55CH2), 116.4 (OCH2CH55CH2) 103.2
(C-1A), 102.3 (C-1B), 101.2 (CHPh), 81.5, 79.2, 78.3, 78.2, 74.3, 73.7, 73.3,
73.3, 71.9, 69.3, 67.5, 67.1 (C-6A,C-6B), 18.3 [OCH2CH2Si(CH3)3], 21.5
[OCH2CH2Si(CH3)3]. Anal. Calcd for: C55H66O11Si: C, 70.93; H, 7.14;
Found: C, 71.02; H, 6.97.

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1136
Roy, Sarkar, and Roy
2-(Trimethylsilyl)ethyl 2,3,6-Tri-O-benzyl-b-D-
mannopyranosyl-(1 ! 4)-2-O-allyl-3,6-di-O-benzyl-
b-D-galactopyranoside (14)
The benzylidene ring of 13 (500 mg, 0.53 mmol) was regioselectively
opened up (as described for compound 11) to afford pure 14 (340.7 mg, 68%)
as a thick syrup; [a]2D5 2 27.48 (c 1.3, CHCl3); 1H NMR: d 7.30–7.01 (m, 25H,
aromatic protons), 5.86–5.76 (m, 1H, CH2CH55CH2), 5.16–4.97 (m, 2H,
CH2CH55CH2), 4.63 (bs, 1H, H-1B), 4.59 (bs, 2H, CH2Ph), 4.43, 4.22 (2 d,
2H, CH2Ph), 4.15 (d, 1H, J1,2 ¼ 7.5 Hz, H-1A), 3.96 (d, 1H, J2,3 ¼ 2.3 Hz,
H-2B), 3.78 (d, 1H, J3,4 ¼ 2.9 Hz, H-4A), 3.80 [m, 2H, OCH2CH2Si(Me)3],
3.29 (dd, 1H, J5,6e ¼ 2.9 Hz, J6e,6a ¼ 9.6 Hz, H-6Be ), 3.14 (m, 1H, H-5B), 2.94
(dd, 1H, J5,6a ¼ 2.9 Hz, J6e,6a ¼ 9.4 Hz, H-6Ba ), 2.43 (bs, 1H, 4-OHB), 0.88 [m,
2H, OCH2CH2Si(CH3)3], 20.15 [s, 9H, OCH2CH2Si(CH3)3]; 13C NMR:
d 139.0–127.4 (aromatic carbons), 135.2 (OCH2CH55CH2), 116.6 (OCH2-
CH55CH2), 103.4 (C-1A), 102.0 (C-1B), 82.0, 81.8, 79.6, 75.5, 74.0, 73.9,
73.8, 73.6, 73.3, 72.9, 71.2, 70.9, 70.3 (C-6A), 68.0, 67.4 (C-6B), 18.6
[OCH2CH2Si(CH3)3], 21.2 [OCH2CH2Si(CH3)3]. Anal. Calcd for:
C55H68O11Si: C, 70.78; H, 7.34. Found: C, 70.62; H, 7.49.
2-(Trimethylsilyl)ethyl 2-Acetamido-3,4,6-tri-O-acetyl-2-
deoxy-b-D-glucopyranosyl-(1 ! 3)-2-O-acetyl-4,6-O-
benzylidene-b-D-galactopyranosyl-(1 ! 4)-2,3,6-tri-O-
benzyl-b-D-mannopyranosyl-(1 ! 4)-2-O-allyl-3,6-di-O-
benzyl-b-D-galactopyranoside (15)
To a solution of the donor 8 (80 mg, 0.11 mmol) and the acceptor 14
˚
(91 mg, 0.09 mmol) in dry CH2Cl2 (3 mL) was added MS 4 A (100 mg) stirred
under N2 for 2 hr. The mixture was then cooled to 2208C, NIS (34.2 mg,
0.15 mmol) and TfOH (1.5 mL, 0.016 mmol) were added and the mixture was
allowed to stir for 1 hr at this temperature. The reaction mixture was then
diluted with CH2Cl2 (25 mL) and filtered through a Celite bed. The filtrate was
washed successively with 10% Na2S2O3, saturated aqueous NaHCO3 and
water, dried (Na2SO4) and concentrated. Column chromatography of the
resulting syrupy product with 3 : 1 toluene-EtOAc gave 15 (112.2 mg, 74%) as
1
a foam, 0.63, [a]2D5 2 8.38 (c 0.6, CHCl3). H NMR: d 7.27–7.04 (m, 30H,
aromatic protons), 5.81 (m, 1H, CH2CH55CH2), 5.80 (d, 1H, J ¼ 6.6 Hz,
NHCOCH3), 5.47 (s, 1H, CHPh), 5.35 (t, 1H, J ¼ 9.7 Hz, H-3D), 5.15–4.97
(m, 2H, CH2CH55CH2), 4.84 (t, 1H, J ¼ 9.5 Hz, H-2C), 4.40 (d, 1H,
J ¼ 8.9 Hz, H-1C), 4.15 (d, 1H, J ¼ 7.5 Hz, H-1A), 3.97 (bs, 1H, H-4C), 3.79
(d, 1H, J ¼ 2.4 Hz, H-4A), 3.29 (dd, 1H, J3,4 ¼ 2.6 Hz, J2,3 ¼ 9.4 Hz, H-3A),

ORDER
REPRINTS
Synthesis of Tetrasaccharide
1137
3.17–3.08 (m, 1H, H-5B), 2.01, 2.00, 1.87, 1.84, 1.78 (5 s, 15H, 5COCH3),
0.86 [m, 2H, OCH2CH2Si(CH3)3], 20.15 [s, 9H, OCH2CH2Si(CH3)3]; 13C
NMR: d 170.8 (NHCOCH3), 170.5, 170.3, 169.8, 169.4 (4 OCOCH3), 138.4–
126.9 (aromatic carbons), 116.5, 103.1 (C-1A), 101.7 (C-1C), 101.2 (CHPh),
100.5 (C-1B), 99.8 (C-1D), 81.3, 80.6, 79.3, 75.8, 74.4, 73.9, 73.7, 73.6, 73.5,
73.3, 73.3, 73.2, 71.7, 71.3, 71.1, 71.0, 70.2, 68.8, 67.1, 62.0, 61.8, 55.9 (C-
2D), 23.1 (NHCOCH3), 21.0, 20.5, 20.5, 20.4 (4 COCH3), 18.3 [OCH2CH2-
Si(CH3)3], 21.5 [OCH2CH2Si(CH3)3]. Anal. Calcd for: C84H103O25NSi: C,
64.89; H, 6.67; N, 0.90 Found: C, 64.73; H, 6.52; N, 0.93.
2-(Trimethylsilyl)ethyl 2-Acetamido-2-deoxy-b-D-
glucopyranosyl-(1 ! 3)-b-D-galactopyranosyl-(1 ! 4)-
b-D-mannopyranosyl-(1 ! 4)-b-D-galactopyranoside (16)
To a solution of 15 (106 mg, 0.068 mmol) in methanol (2 mL), PdCl2
(9 mg, 0.05 mmol) was added and the mixture was stirred at rt for 3 hr. The
solvent was removed and the product was purified by column chromatography
with 1 : 1 toluene-EtOAc. To the purified product, dissolved in EtOH (7 mL),
was added 10% Pd/C (100 mg) and the mixture was stirred under H2 for 72 hr
when TLC (EtOAc) showed a single spot. The mixture was then filtered
through a Celite bed and the filtrate was concentrated to a thick glass. The
product was treated with 0.05 M NaOMe in MeOH (3 mL) for 6 hr, decation-
ized with Dowex 50W Hþ resin, filtered and concentrated to dryness to afford
16 (29 mg, 53% overall in 3 steps); [a]D 2 598 (c, 0.9, water). 1H NMR (D2O)
d 5.10 (d, 1 H, J ¼ 7 Hz, H-1D), 4.59 (bs, 1 H, H-1B), 4.48 (d, 1 H, J ¼ 8.1 Hz,
H-1C), 4.24 (d, 1 H, J ¼ 7.3 Hz, H-1A), 4.10 (H-4A), 3.88 (1-H, H-2B), 1.76
(NHCOCH3), 0.84 (m, 2 H, OCH2CH2SiMe3), 20.15 [s, 9 H, OCH2CH2-
Si(CH3)3]. 13C NMR d 170.0 (NHCOCH3), 103.5 (C-1A), 103.0 (C-1D), 102.6
(C-1C), 100.8 (C-1B), 81.5, 80.8, 79.5, 78.1, 75.2, 74.6, 73.4, 73.1, 73.0, 72.6,
71.5, 70.1, 71.0, 68.4, 67.0, 64.0, 63.7, 62.1, 61.4, 56.1 (C-2D), 21.2
(NHCOCH3), 18.4 [OCH2CH2Si(CH3)3], 21.5 [OCH2CH2Si(CH3)3]. Anal.
Calcd for C31H57O21NSi: C, 46.09; H, 7.11; N, 1.73. Found: C, 46.25; H, 7.34;
N, 1.82.
ACKNOWLEDGMENT
Financial support from the Department of Science and Technology,
New Delhi, India (Project No. SP/S1/G14/95) is thankfully acknowledged.

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1138
Roy, Sarkar, and Roy
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Received in India September 8, 2003

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