Convergent Synthesis of the Hexasaccharide Repeating Unit of the O-Antigenic OPS of Escherichia coli O133

Article abstract of DOI:10.1002/ejoc.201900815

Synthesis of the hexasaccharide repeating unit of the O-antigen from E. coli O133 has been accomplished with rational protecting group manipulations on commercially available monosaccharides and stereoselective glycosylations through a convergent protocol. A late stage TEMPO mediated oxidation is used to install the required uronic acid moiety. Chloroacetate group is used extensively as a temporary protecting group.

Full text of DOI:10.1002/ejoc.201900815

A Journal of
Accepted Article
Title: Convergent Synthesis of the Hexasaccharide Repeating Unit of
the O-antigenic OPS of Escherichia coli O133
Authors: Ankita Mitra and Balaram Mukhopadhyay
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201900815
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10.1002/ejoc.201900815
European Journal of Organic Chemistry
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Convergent Synthesis of the Hexasaccharide Repeating Unit of
the O-antigenic OPS of Escherichia coli O133
Ankita Mitra,[a] and Balaram Mukhopadhyay*[a]
Abstract: Synthesis of the hexasaccharide repeating unit of the O-
antigen from E. coli O133 has been accomplished with rational
protecting group manipulations on commercially available
the O-antigenic OPS of Escherichia coli O133 in the form of its
aminoethyl glycoside (1, Figure 1).
monosaccharides and stereoselective glycosylations through
a
convergent protocol. A late stage TEMPO mediated oxidation is
used to install the required uronic acid moiety. Chloroacetate group
is used extensively as a temporary protecting group.
Introduction
Escherichia coli (E. coli) is a group of gram-negative bacteria
which has both commensal and pathogenic forms. The
pathogenic class of E. colibacteria also happens to be the
biggest cause of diarrhoea with life threatening complications,
for example, haemorrhagic colitis (HC) and haemolytic–uraemic
syndrome (HUS).1 The other general clinical syndromes caused
by E. coli are urinary tract infection and meningitis.2 Although till
date antibiotics are the main therapeutic agent to treat the
infections caused by E. coli, increasing drug resistance to the
bacterial infection are making the vaccines a more attractive
choice of therapeutics. The O-specific polysaccharide (OPS) or
O-antigens in the outer membrane of the bacteria is highly
immunogenic and nontoxic in general3 and thus can be utilized
to develop vaccine in the form of glycoconjugates. Though
currently more than 180 O-antigens from E. coli has been
recognized, isolation of them rendered the vaccine too costly
and development of the vaccine ends in the early stages of
clinical trials.4 As it turns out, scalable chemical synthesis of
these O-antigens becomes the inevitable route to achieve the
glycoconjugate vaccine. In the recent years, number of
articles5has appeared in the literature describing both the
structure elucidation and the synthesis of different O-antigenic
oligosaccharides from different E. coli strains. Recently Knirel
and his co-workers reported6 the structure of the O-specific
polysaccharide (OPS) of E. coli O133. In the endeavour to
develop glyco-conjugate vaccine against E. coli, herein we
report the total synthesis of the hexasaccharide repeating unit of
Figure 1. Structure of the target hexasaccharide (1)
Results and Discussion
The retrosynthetic analysis (Figure 2) for the hexasaccharide
indicated a [2 + 2 + 1 + 1] glycosylation strategy will be the best
suitable approach to achieve the target structure. 2-Aminoethyl
glycoside7 serve as an ideal choice for the aglycone at the
reducing end of the hexasaccharide 1 as it can facilitate
glycoconjugate formation using the free amine functionality
without hampering the anomeric stereochemistry.
The synthesis would start with the preparation of two
disaccharide building blocks, the reducing end disaccharide 4
and the disaccharide donor 10. All the stereoselective
glycosylations were achieved by the activation of thioglycoside
using NIS and H2SO4-silica. The metal free and environmental
friendly H2SO4-silica serves as an efficient alternative to
traditional toxic and hygroscopic promoters like TfOH and
TMSOTf. H2SO4-silica is particularly beneficial as (a) it is solid
material and (b) it also acts as a desiccant during the
glycosylation reactions thus improving the yields. Glycosylation
between the reducing end disaccharide
4 and the thio-
[a]
[b]
Prof. Balaram Mukhopadhyay, Dr. Ankita Mitra
Sweet Lab, Department of Chemical Sciences
Indian Institute of Science Education and Research Kolkata
Mohanpur, Nadia 741246, INDIA
E-mail: sugarnet73@hotmail.com (BM)
Dr. Ankita Mitra
Centre for Analysis and Synthesis, Lund University
Box-117, SE 221 00, Lund, Sweden
E-mail: ankmitra1989@gmail.com (AM)
disaccharide 10 would furnish the protected tetrasaccharide12.
Rational protecting group manipulations and stereoselective
glycosylation of the tetrasaccharide acceptor 13 with D-
galactosyl donor 17 would then furnish the fully protected
pentassacharide 18. Further cleavage of the temporary 4-
methoxybenzyl group followed by glycosylation with suitably
protected D-glucosyl donor 21 will give the hexasaccharide 22.
Hydrolysis of the 6-O-chloroacetate group followed by TEMPO-
mediated oxidation was planned for the installation of the
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10.1002/ejoc.201900815
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required uronic acid moiety. Finally, global de-protection would
furnish the target hexasaccharide 1 (Figure 2).
It was envisaged that the tetrasaccharide 12 can be most
conveniently synthesized by performing a [2+2] glycosylation
between the disaccharide acceptor 5 and thio-disaccharide
donor 11. In order to prepare disaccharide 4, the known linker-
equipped rhamnose acceptor 28 was coupled with known
rhamnose donor 314 through activation of the thioglycoside by
NIS in the presence of H2SO4-silica15 to furnish the disaccharide
4 in 87% yield. This disaccharide was essentially prepared in the
same way as reported in the literature.8 However, since the
required disaccharide acceptor is different from that of the
reported one, we had to change the donor rhamnose derivative
accordingly. The formation of the disaccharide was confirmed by
1
NMR spectroscopy showing H NMR signals at 5.05 ppm (J1,2
1.5 Hz, H-1) for the newly formed 1,2-trans glycoside and at
4.89 ppm (J1,2 1.0 Hz, H-1) for the other anomeric
proton.The13C NMR signals for the two anomeric carbons at
99.5 ppm (C-1) and 99.0 ppm (C-1) further affirmed the
formation of the desired disaccharide. The chloroacetate group
in donor 3 was strategically used to provide anchimeric
assistance as well as a temporary protecting group. It was
selectively cleaved using thiourea and 2,4,6-collidine16 to afford
the required disaccharide acceptor 5 in 81% yield (Scheme 1).
Scheme 1. Synthesis of the disaccharide acceptor 5
A
chemoselective
glycosylation
between
known
trichloroacetimidate donor 617 and thioglycoside acceptor 718 at -
45 oC in the presence of H2SO4-silica furnished the disaccharide
8 in 84% yield. The presence of a 6-OAc group on the D-
glucosamine donor
6 was expected to provide a distant
neighbouring group participation leading to the required 1,2-cis
linked glycoside 8 which is clearly evident from 1H NMR peaks at
5.01 ppm (H-1) and 5.44 ppm (J1,2 3.0 Hz, H-1). The 13C NMR
peaks for the two anomeric carbons at 92.8 ppm (C-1)and 85.2
ppm (C-1) also confirmed the formation of the desired
disaccharide. The acetyl groups were then removed by trans-
esterification using NaOMe in MeOH to afford the disaccharide
derivative 9 in 96% yield. Further, reaction with benzaldehyde
dimethyl acetal in the presence of CSA19 afforded the
disaccharide derivative 10 in 82% yield. Chloroacetylation of the
Figure 2. Retrosynthetic analysis for the synthesis of the target
hexasaccharide 1
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10.1002/ejoc.201900815
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free 3-OH group in 10 using chloroacetic anhydride and
pyridine20 afforded the fully protected disaccharide donor 11 in
89% yield (Scheme 2).
In a separate experiment, known galactose derivative 1421 was
benzylated using BnBr and NaH22 to give compound 15 in 94%
yield.Further,regio-selective ring opening of the benzylidene
acetalusing cyanuric chloride (TCT)23 and NaBH4 gave the
galactosyl derivative 16 in 80% yield. Subsequently, the 6-OH
group was chloroacetylated to give the desired galactose
donor17 in 91% yield. Glycosylation of donor 17 with
tetrasaccharide acceptor 13 using NIS and H2SO4-silica at -45
oC afforded the desired pentasaccharide 18 in 84% yield. Low
reaction temperature and presence of non-participating 2-OBn
adjacent to the anomeric position resulted the reaction towards
1
α-selectivity. The H and 13C NMR analysis showing 1H NMR
peak at 5.72 ppm (J1,2 2.0 Hz, H-1) and 13C NMR signal at
96.9 ppm (C-1), clearly suggests the exclusive formation of
1,2-cis glycosidic linkage in the newly formed pentasaccharide
18. Selective oxidative cleavage of the 4-methoxybenzyl ether in
compound 18 using DDQ24 furnished the pentasaccharide
acceptor 19 in 79% yield (Scheme 4).
Scheme 2. Synthesis of the disaccharide donor 11
With the two disaccharide building blocks in hand, NIS/H2SO4-
silica mediated glycosylation was performed between the
disaccharide acceptor
5 and disaccharide donor 11 that
ultimately furnished the fully protected tetrasaccharide 12 in 83%
1
yield. H NMR signal at 5.06 ppm (J1′″,2″′ 3.0 Hz, H-1) and 13C
NMR signal at 93.6 ppm (C-1) were assigned to the newly
formed glycosidic linkage. The exclusive formation of the 1,2-
trans glycoside was also evident from the JCH coupling value of
169.7 ppm. Further, the chloroacetyl ester group in the
tetrasaccharide 12 was selectively cleaved using thiourea and
2,4,6-collidine16 to furnish the required tetrasaccharide acceptor
13 in 82% yield (Scheme 3).
Scheme 4. Synthesis of the pentasaccharide acceptor 19
The known compound 2025 was treated with benzoyl chloride
and pyridine11 in order to protect the 6-OH with benzoyl ester
and thus furnishing the final monosaccharide donor 21 in 86%
yield. Subsequently, the pentasaccharide acceptor 19 was
coupled with the donor 21 in presence of NIS and H2SO4-silica
o
at -55 C to afford the desired hexasaccharide 22 in 78% yield
(Scheme 5). Here, in addition to the presence of non-
participating 2-OBn group in D-glucosyl moiety and distant
participation26 of the 6-OBz group likely have facilitated the
exclusive formation of 1,2-cis glycosidic linkage. The newly
Scheme 3. Synthesis of the tetrasaccharide acceptor 13
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10.1002/ejoc.201900815
European Journal of Organic Chemistry
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formed α-glycosidic bond in 22 was confirmed by NMR analysis
which shows H NMR peak at 5.15 ppm (J1‴ʺ,2‴ʺ 2.5 Hz, H-1‴ʺ)
and 13C NMR peak at 96.8 ppm (C-1‴ʺ).
challenging 1,2-cis glycosylations required in the total synthetic
route were successfully achieved by rational protecting group
manipulations and utilizing the remote participation of the 6-O-
1
acyl groups.
A
late stage TEMPO-mediated oxidation
successfully installed the uronic acid moiety in the target
hexasaccharide structure.
Scheme 5. Synthesis of the protected hexasaccharide 22
Once the hexasaccharide was obtained the chloroacetate group
was cleaved using thiourea and 2,4,6-collidine furnishing 6-OH
free derivative 23 in 81% yield which was immediately oxidized
to the corresponding uronic acid derivative using 2,2,6,6-
tetramethylpiperidin-1-oxyl
(TEMPO)
together
with
(diacetoxyiodo)benzene (BAIB)27 as co-oxidant. The 6-O-
chloroacetyl moiety was strategically installed so that it can be
selectively cleaved to furnish a free primary hydroxyl group that
can undergo TEMPO mediated oxidation to give the desired
uronic acid derivative. In the subsequent steps global
deprotection was performed to obtain the target hexasaccharide
1. At first,the benzylidine group was hydrolyzed using aqueous
o
AcOH12 at 80 C to afford the di-ol derivative which was directly
treated with thioacetic acid28 for 72 hours at room temperature to
permit the conversion of azido to the corresponding acetamido
derivative 24 in 71%yield.In the next step, Zemplen de-O-
acetylation9 unmasked all the ester protections on the
hexasaccharide derivative. Finally, Hydrogenolysis using 10%
Pd-C cartridge on a ThalesNano continuous flow hydrogenation
assembly removed the benzyl and NHCBz protection and
furnished the target hexasaccharide 1 in 63% overall yield
(Scheme 6).
Conclusions
Scheme 6. Synthesis of the target hexasaccharide 1
Convergent total synthesis of the hexasaccharide repeating unit
of the O-antigen from E. coli O133 is achieved in the form of its
2-aminoethyl glycoside. The 2-aminoethyl glycoside is useful for
further glycoconjugate formation utilizing the free terminal amine
without disturbing the anomeric stereochemistry. The
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2-(benzyloxycarbonyl)-aminoethyl
rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-
4-O-benzoyl-3-O-benzyl-α-L-
Experimental Section
rhamnopyranoside (5):
General Methods
A mixture of pure disaccharide 4 (1.6 g, 1.7 mmol), thiourea (1.2 g, 17.0
mmol) and 2,4,6-collidine (2.3 mL, 17.0 mmol) was stirred in 25 mL
CH2Cl2-MeOH (2:3) and refluxed for 10 hours. The completion of the
reaction was evident by a complete conversion of the starting material to
a slower moving spot as evident from the TLC using (n-hexane-EtOAc;
3:2). The solvents were evaporated in vacuo and the solid residue
obtained was dissolved in CH2Cl2 and washed with brine (2 × 30 mL).
The organic layer was separated, dried (Na2SO4), filtered and evaporated
in vacuo to obtain a syrupy residue. This was further subjected to flash
All solvents and reagents were dried prior to use according to
standard methods.29 The commercially purchased reagents were used
without any further purification unless mentioned otherwise.
Dichloromethane was dried and distilled over P2O5 to make it anhydrous
and moisture-free. All reactions were monitored by Thin Layer
Chromatography (TLC) on Silica-Gel 60-F254 with detection by
fluorescence followed by charring after immersion in 10% ethanolic
solution of H2SO4. Flash chromatography was performed with Silica Gel
230-400 mesh. Optical rotations were measured on sodium-line at
ambient temperature. 1H and 13C NMR were recorded at 500 MHz and
125 MHz respectively on a Bruker Avance 500 MHz spectrometer. The
assignments of 1H and 13C peaks were done with the help of 1H-1H
chromatography using n-hexane-EtOAc (1:1) as eluent to furnish the
25
pure disaccharide acceptor 5 (1.2 g, 81%) as colorless foam. [α]D
=
+98 (c 1.1, CHCl3). 1H NMR (CDCl3, 500 MHz) : 8.16-7.27 (m, 25H,
ArH), 5.48 (m, 2H, H-4, H-4), 5.23 (s, 3H, COOCH2Ph, H-1), 4.94 (d, 1H,
J1,2  1.0 Hz, H-1), 4.78, 4.61 (ABq, 2H, JAB 12.0 Hz, CH2Ph), 4.70, 4.66
(ABq, 2H, JAB 12.0 Hz, CH2Ph), 4.43 (d, 1H, J1,2 1.0 Hz, J2,3 3.0 Hz, H-
2), 4.17 (d, 1H, J1,2 1.0 Hz, J2,3 2.5 Hz, H-2), 4.09 (m, 1H, H-5), 4.06 (dd,
1H, J2,33.0 Hz, J3,4 9.5 Hz, H-3), 4.03 (dd, 1H, J2,3 2.5 Hz, J3,4 10.0 Hz,
H-3), 3.95 (m, 1H, H-5), 3.88, 3.63 (m, 2H, OCH2), 3.57, 3.50 (m, 2H,
CH2NH), 2.90 (bs, 1H, OH), 1.35 (d, 3H, J5,6 6.5 Hz, C-CH3), 1.32 (d, 3H,
1
COSY and H-13C HSQC spectra. In the target hexasaccharide structure
1, the 1H NMR values were denoted as H for the reducing end unit A, H
for the unit B, H for the unit C, H for the unit D, H for the unit E and
H″″ʹ for the unit F as marked in the Figure 1.
Preparation of H2SO4-Silica
J
5,66.5 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) : 165.8, 165.7
To slurry of silica gel (10 g, 230-400 mesh) in dry diethyl ether (50 mL)
was added commercially available concentrated H2SO4 (1 mL) and the
slurry was shaken for 5 min. The solvent was evaporated under reduced
pressure, resulting in free flowing H2SO4-Silica, which was dried at 110
oC for 3 hours and then used for reactions.
(2COPh), 156.3 (COOCH2Ph), 137.5, 137.3, 136.2, 133.1 (2), 129.8 (2),
129.7 (2), 128.5 (2), 128.4 (2), 128.3 (8), 128.2, 128.1, 128.0 (3), 127.7
(4) (ArC), 101.2 (C-1), 99.2 (C-1), 76.1, 76.0, 75.0, 73.1, 72.7, 72.0, 71.9,
67.9, 67.1, 66.9, 66.8 (2), 17.6 (C-CH3), 17.4 (C-CH3). HRMS calcd for
C50H53NO13Na (M+Na)+: 898.3415, found: 898.3410.
2-(benzyloxycarbonyl)-aminoethyl
chloroacetyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-
4-O-benzoyl-3-O-benzyl-2-O-
p-Tolyl 2-azido-2-deoxy-3,4,6-tri-O-acetyl-α-D-glucopyranosyl-(1→3)-
2,4-di-O-benzyl-α-L-rhamnopyranoside (8):
L-rhamnopyranoside (4):
A mixture of known acceptor 718(1.1 g, 2.3 mmol), trichloroacetamidate
donor 6(1.4 g, 3.0 mmol) and activated MS 4Å (2.0 g) in anhydrous
CH2Cl2 (20 mL) was stirred under N2 for 15 min. The reaction mixture
was cooled to -45 oC and H2SO4-Silica (55 mg) was added followed by
stirring the reaction mixture at same temperature. Complete consumption
of the acceptor within few minutes was evident from the TLC (n-hexane-
EtOAc; 3:1). The reaction mixture was immediately filtered through a pad
of Celiteand the filtrate was successively washed with Na2S2O3 (2×30
mL), NaHCO3 (2×30 mL) and brine (30 mL). Organic layer was collected,
dried over anhydrous Na2SO4, filtered and evaporated in vacuo. The
crude product thus obtained was purified by flash chromatography using
n-hexane-EtOAc (3:1) to afford pure compound 8(1.6 g, 84%) as
A mixture of compound 2 (1.1 g, 2.1 mmol), known compound 314 (1.4 g,
2.7 mmol) and activated MS 4Å (2.0 g) in dry CH2Cl2 (20 mL) was stirred
in ice-water bath for 1 hour. NIS (790 mg, 3.5 mmol) was added and the
reaction mixture was stirred under nitrogen for 20 min. H2SO4-silica (45
mg) was then added to the reaction vessel and allowed to stir until
complete consumption of the acceptor as evident by TLC using (n-
hexane-EtOAc; 3:1). Within 10 minutes, the entire acceptor was
consumed and the mixture was immediately filtered through a pad of
Celite. The filtrate was successively washed with Na2S2O3 (230 mL),
NaHCO3 (230 mL) and brine (30 mL). The organic layer was collected,
dried over NaSO4 and evaporated to syrup. The crude product thus
25
amorphous solid. [α]D = +112 (c0.9, CHCl3). 1H NMR (CDCl3, 500
obtained was purified by flash chromatography using n-hexane-EtOAc
25
(2:1) to afford the disaccharide 4 (1.7 g, 87%) as white foam. [α]D
=
MHz) : 7.43-7.11 (m, 14H, ArH), 5.56 (dd, 1H, J2,310.0 Hz, J3,4 6.0 Hz,
H-3), 5.44 (dd, 1H, J1,2 3.0 Hz, H-1), 5.07-4.97 (m, 2H, H-1, H-4), 4.91,
4.61 (ABq, 2H, JAB 11.0 Hz, CH2Ph), 4.72(d, 2H, CH2Ph), 4.21-4.17 (m,
2H, H-5, H-5), 4.08-4.02 (m, 3H, H-2, H-3, H-6a), 3.92 (dd, 1H, J5,6a 2.0
Hz,J6a,6b 12.5 Hz, H-6b), 3.73 (t, 1H, J3,4,J4,5 9.5 Hz, H-4), 3.34 (dd, 1H,
+116 (c 1.1, CHCl3).1H NMR (CDCl3, 500 MHz) : 8.15-7.18 (m, 25H,
ArH), 5.73 (t, 1H, J1,2, J2,3 2.5 Hz, H-2), 5.46 (t, 1H, J3,4, J4,5 9.5 Hz, H-
4), 5.29 (t, 1H, J3,4,J4,5 9.5 Hz, H-4), 5.20 (d, 2H, COOCH2Ph), 5.16 (bs,
1H, NH), 5.05 (d, 1H, J1,2 1.5 Hz, H-1), 4.89 (d, 1H, J1,2 1.0 Hz, H-1),
4.71, 4.57 (ABq, 2H, JAB 12.5 Hz, CH2Ph), 4.65 (s, 2H, CH2Ph), 4.22,
4.14 (ABq, 2H, JAB 15.5 Hz, COCH2Cl), 4.11-4.05 (m, 3H, H-2, H-3, H-5),
4.01 (dd, 1H, J2,3 3.0 Hz, J3,4 9.5 Hz, H-3), 3.94 (m, 1H, H-5), 3.84 (m,
1H, OCH2), 3.61 (m, 1H, OCH2), 3.55 (m, 1H, CH2NH), 3.46 (m, 1H,
CH2NH), 1.33 (d, 1H, J5,6 6.5 Hz, C-CH3), 1.29 (d, 1H, J5,6 6.5 Hz, C-
CH3). 13C NMR (CDCl3, 125 MHz) : 166.3, 165.7, 165.5 (3 COPh),
156.3 (COOCH2Ph), 137.6, 137.2, 136.2, 133.7, 133.1, 129.9 (2), 129.8,
129.7 (2), 129.6, 128.5 (2), 128.4 (4), 128.3 (4), 128.2 (4), 128.1, 127.7
(3), 127.6 (ArC), 99.5 (C-1), 99.0 (C-1), 76.2, 76.1, 73.4, 73.1, 72.4 (2),
71.1, 70.0, 67.3, 67.1, 66.9 (2), 40.8 (COCH2Cl), 40.7 (CH2NH), 17.6 (C-
CH3), 17.5 (C-CH3). HRMS calcd for C52H54ClNO14Na (M+Na)+:974.3131,
found 974.3129.
J
1,2 3.5 Hz,J2,3 10.0 Hz, H-2), 2.34 (s, 3H, SC6H4CH3), 2.09, 2.07, 1.91
(3s, 9H, 3 COCH3), 1.35 (d, 3H, J5,6 6.5 Hz, C-CH3). 13C NMR (CDCl3,
125 MHz) : 170.6, 169.8, 169.6 (3COCH3), 137.9, 137.8, 132.8, 132.1
(2), 129.4, 130.0 (2), 128.4 (2), 128.3 (4), 128.0, 127.6 (3) (ArC), 92.8 (C-
1), 85.2 (C-1), 79.2, 75.5, 74.4, 74.0, 71.7, 70.4, 69.2, 68.1, 67.4, 61.6,
60.6, 21.0 (SC6H4CH3), 20.7, 20.6, 20.4 (3COCH3), 17.7 (C-CH3).
HRMS calcd for C39H45N3NaO11S (M+Na)+:786.2672, found 786.2669.
p-Tolyl 2-azido-2-deoxy-α-D-glucopyranosyl-(1→3)-2,4-di-O-benzyl-
α-L-rhamnopyranoside (9):
To a solution of pure disaccharide 8 (1.5 g, 1.9 mmol) in MeOH (25 mL),
NaOMe in MeOH (2 mL, 0.5 M) was added and the reaction was allowed
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10.1002/ejoc.201900815
European Journal of Organic Chemistry
FULL PAPER
to stir at room temperature for 5 hours. Complete consumption of the
starting material to a slower moving spot as evident from TLC (n-hexane-
EtOAc; 1:2) ensures the formation of the product. The reaction mixture
was neutralized with Dowex 50W X8 H+ resin, filtered and evaporated in
vacuo. The crude product was further purified by flash chromatography to
137.1, 136.8, 132.2 (3), 130.4, 130.0 (2), 129.1, 128.5 (5), 128.3 (2),
128.1 (2), 128.0, 127.8, 126.4 (2) (ArC), 101.7 (CHPh), 94.0 (C-1), 85.1
(C-1), 79.5, 79.0, 76.3, 74.6, 74.1, 71.7, 70.9, 69.2, 68.5, 62.9, 61.2, 40.5
(COCH2Cl), 21.1 (SC6H4CH3), 17.7(C-CH3). HRMS calcd for
C39H45ClN3O11Na (M+Na)+: 824.2384, found: 824.2381.
25
afford the desired compound 9(1.2 g, 96%) as amorphous powder. [α]D
= +98 (c 1.0, CHCl3).1H NMR (CDCl3, 500 MHz) : 7.40-7.09 (m, 14H,
ArH), 5.45 (d, 1H, J1,2 1.0 Hz, H-1), 4.95 (d, 1H, J1,2 1.0 Hz, H-1), 4.81,
4.62 (ABq, 2H, JAB 11.0 Hz, CH2Ph), 4.70, 4.64 (ABq, 2H, JAB 12.0 Hz,
CH2Ph), 4.16 (m, 1H, H-5), 4.07 (m, 1H, H-2), 4.02-3.99 (m, 2H, H-3, H-
3), 3.81 (m, 1H, 6a), 3.71-3.67 (m, 4H, H-4, H-4, H-5, H-6b), 3.61 (dd,
1H, J1,2 1.0 Hz, J2,3 7.0 Hz, H-2), 2.33 (s, 3H, SC6H6CH3), 1.31 (d, 3H,
J5,6 6.5 Hz, C-CH3). 13C NMR (CDCl3, 125 MHz) : 138.0, 137.8,
137.4,132.1 (3), 130.4, 129.9 (2), 128.5 (2), 128.4 (3), 128.2 (2), 128.1,
127.9 (ArC), 93.7 (C-1), 85.6 (C-1), 79.5, 75.4, 74.8, 74.7, 72.0, 71.7,
71.0, 70.8, 69.2, 62.5, 61.5, 21.1 (SC6H4CH3), 17.8 (C-CH3). HRMS calcd
for C33H39N3O8Na (M+Na)+:660.2356, found 660.2351.
2-(benzyloxycarbonyl)-aminoethyl
2-azido-2-deoxy-4,6-O-
benzylidene-3-O-chloroacetyl-α-D-glucopyranosyl-(1→3)-2,4-di-O-
benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-
rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-
rhamnopyranoside (12):
A suspension of the disaccharide acceptor 5 (810 mg, 0.9 mmol), the
disaccharide donor 11 (935 mg, 1.2 mmol) and MS 4Å (2.0 g) in dry
CH2Cl2 (20 mL) was stirred under N2 for 30 minutes. NIS (263 mg, 1.2
mmol) was added and the reaction mixture was cooled to -5 ºC followed
by addition of H2SO4-silica (45 mg). The reaction mixture was stirred at
the same temperature for 20 minutes when TLC (n-hexane-EtOAc; 5:2)
revealed complete consumption of the disaccharide acceptor 5. The
reaction mixture was immediately filtered through a pad of Celite and
washed with CH2Cl2. The combined filtrate obtained was washed
successively with Na2S2O3 (230 mL), NaHCO3 (230 mL) and brine (30
mL). The organic layer was separated, dried over anhydrous NaSO4,
filtered and evaporated in vacuo. The residue was then purified by flash
chromatography using n-hexane-EtOAc (4:1) as eluent to afford the
desired tetrasaccharide 12 (1.2 g, 83%) as white powder. [α]D25 = +108
(c0.9, CHCl3). 1H NMR (CDCl3, 500 MHz) : 8.08-7.10 (m, 40H, ArH),
5.75 (t, 1H, J2,3, J3,4 10.0 Hz, H-3), 5.48 (s, 1H, CHPh), 5.37 (t, 1H,
p-Tolyl
2-azido-2-deoxy-4,6-O-benzylidene-α-D-glucopyranosyl-
(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranoside (10):
To a solution of pure compound 9 (1.2 g, 1.8 mmol) in dry CH3CN (20
mL), benzaldehyde dimethyl acetal (0.4 mL, 2.3mmol) and CSA (50 mg)
was added and stirred at room temperature. The reaction mixture was
continued to stir for 3 hours till the TLC showed complete consumption of
the starting material (n-hexane-EtOAc; 3:1). Et3N was added to neutralise
the reaction mixture and the solvent was evaporated in vacuo. The crude
residue obtained was subjected to flash chromatography using n-hexane-
EtOAc (2:1) as eluent to furnish the pure disaccharide derivative 10 (1.1
g, 82%) as colorless foam. [α]D25 = +110 (c 1.0, CHCl3).1H NMR (CDCl3,
500 MHz) : 7.43-7.12 (m, 19H, ArH), 5.53 (s, 1H, CHPh), 5.50 (d, 1H,
J1,2 1.0 Hz, H-1), 4.97 (d, 1H, J1,2 4.0 Hz, H-1), 4.91, 4.61 (ABq, 2H,
JAB 11.0 Hz, CH2Ph), 4.73, 4.64 (ABq, 2H, JAB11.5 Hz, CH2Ph), 4.27 (m,
2H, H-3, H-6a), 4.21 (m, 1H, H-5), 4.10 (m, 3H, H-2, H-3, H-5), 3.75-
3.68 (m, 2H, H-4, H-6b), 3.54 (t, 1H, J3,4, J4,5 9.5 Hz, H-4), 3.32 (dd,
1H, J1,2 4.0 Hz,J2,3 10.0 Hz, H-2), 2.35 (s, 3H, SC6H4CH3), 1.35 (d, 3H,
J5,6 6.5 Hz, C-CH3). 13C NMR (CDCl3, 125 MHz) : 137.8, 137.7, 137.3,
136.9, 132.2 (2), 130.5, 129.9 (2), 129.3, 128.5 (2), 128.4 (2), 128.3 (2),
128.2 (2), 128.1 (2), 127.9, 127.8, 126.4 (2) (ArC), 102.0 (CHPh), 94.1
(C-1), 85.4 (C-1), 81.8, 79.7, 75.9, 74.8, 74.6, 71.9, 69.2, 68.8, 68.7,
62.8, 62.5, 21.1 (SC6H4CH3), 17.7 (C-CH3). HRMS calcd for
C40H43N3O8Na (M+Na)+:748.2669, found 748.2662.
J
J
3,4, J4,5 9.5 Hz, H-4), 5.33 (t, 1H, J3,4, J4,5 9.5Hz, H-4), 5.25 (d, 1H,
1,2 1.0 Hz, H-1), 5.13 (d, 2H, COOCH2Ph), 5.10 (bs, 1H, NH), 5.06 (d,
1H, J1‴,2‴ 3.0 Hz, H-1), 5.04 (d, 1H, J1,2 1.0 Hz, H-1), 4.92, 4.54 (ABq,
2H, JAB 11.5 Hz , CH2Ph), 4.82 (d, 1H, J1,2 1.0 Hz, H-1), 4.64, 4.61 (d,
2H, JAB 11.5 Hz, CH2Ph), 4.59, 4.48 (d, 2H, JAB 12.0Hz, CH2Ph), 4.42,
4.29 (d, 2H, JAB 11.5 Hz, CH2Ph), 4.40 (m, 1H, H-2), 4.30 (m, 1H, H-
6a), 4.18 (dd, 1H, J2,32.5 Hz, J3,410.0 Hz, H-3), 4.16 (s, 2H,
COCH2Cl), 4.11 (m, 1H, H-5), 4.06 (m, 1H, H-2), 4.02-3.94 (m, 3H, H-2,
H-3, H-5), 3.90 (dd, 1H, J2,3 3.0 Hz, J3,4 10.0 Hz, H-3), 3.80 (m, 2H, H-5,
H-6b), 3.75 (m, 1H, OCH2), 3.72-3.64 (m, 3H, H-4, H-4, OCH2), 3.47,
3.41 (m, 2H, CH2NH), 3.21 (dd, 1H, J1,23.0 Hz, J2,3 10.5 Hz, H-2),
3.08 (m, 1H, H-5), 1.38 (d, 3H, J5,6 6.5 Hz, C-CH3), 1.27 (d, 3H, J5,6
6.5 Hz, C-CH3), 1.18 (d, 3H, J5,6 6.0 Hz, C-CH3).13C NMR (CDCl3, 125
MHz) : 166.3 (COCH2Cl), 165.7, 165.6 (2COCH3), 156.3 (COOCH2Ph),
137.6, 137.5, 137.4, 136.9, 136.2, 133.2, 133.1, 129.9, 129.8 (3), 129.0,
128.5 (2), 128.4 (8), 128.3 (6), 128.2 (4), 128.1 (4), 128.0 (3), 127.8 (4),
127.6 (2), 126.3 (3) (ArC), 101.6 (CHPh), 101.2 (C-1), 99.2 (C-1), 97.9
(C-1), 93.6 (C-1), 79.3, 79.0, 76.4, 76.1, 75.7, 75.4, 73.8, 73.6, 73.2,
73.0, 72.3, 72.2, 71.8, 71.6, 71.0, 68.5, 68.4, 67.5, 67.1, 66.9 (2), 62.6,
61.1, 45.7 (CH2NH), 40.6 (COCH2Cl), 17.8 (C-CH3), 17.6 (C-CH3).HRMS
calcd for C85H89ClN4O22Na (M+Na)+ : 1575.5555, found: 1575.5553.
p-Tolyl
2-azido-2-deoxy-4,6-O-benzylidene-3-O-chloroacetyl-α-D-
glucopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranoside (11):
To a solution of pure disaccharide derivative 10 (1.0 g, 1.4 mmol) in dry
CH2Cl2 (20 mL), Pyridine (0.5 mL, 5.6 mmol) was added and the reaction
mixture was stirred at -5 °C. After stirring for 10 minutes, chloroacetic
anhydride (479 mg, 2.8 mmol) was added to the reaction vessel and the
reaction was stirred for 1 hour at same temperature. After complete
conversion of the starting material (TLC in n-hexane-EtOAc; 3:1), the
solvent was evaporated in vacuo. The crude residue thus obtained, was
purified by flash chromatography using n-hexane-EtOAc (4:1) as eluent
to afford the desired disaccharide derivative 11 (983 mg, 89%) as white
powder. [α]D25 = +126 (c 1.0, CHCl3).1H NMR (CDCl3, 500 MHz) : 7.42-
2-(benzyloxycarbonyl)-aminoethyl
2-azido-2-deoxy-4,6-O-
benzylidene-α-D-glucopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-
rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-
rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-
rhamnopyranoside (13):
7.12 (m, 19H, ArH), 5.74 (t, 1H, J2,3,J3,4 9.5 Hz, H-3), 5.53 (d, 1H, J1,2

A mixture of tetrasaccharide 12 (1.0 g, 0.6 mmol) in 20 mL of CH2Cl2-
CH3OH (2:3), thiourea (456 mg, 6.0 mmol) and 2,4,6-collidine (0.8 mL,
6.0 mmol) was refluxed at 50 oC for 8 hours till the TLC (n-hexane-
EtOAc; 2:1) showed complete conversion of the starting material to a
slower moving spot. The solvents were evaporated in vacuo, the solid
residue thus obtained was dissolved in CH2Cl2 (50 mL) and washed with
brine (2  20 mL). The organic layer was collected, dried (NaSO4),
filtered and evaporated in vacuo. The crude product was further purified
1.0 Hz, H-1), 5.48 (s, 1H, CHPh), 5.01 (d, 1H, J1,2 3.5 Hz, H-1), 4.94,
4.64 (ABq, 2H, JAB 10.5 Hz, CH2Ph), 4.73, 4.57 (ABq, 2H, JAB 12.0 Hz,
CH2Ph), 4.26 (dd, 1H, J5,6a 5.0 Hz,J6a,6b 10.5 Hz, H-6a), 4.20 (m, 1H, H-
5), 4.15 (s, 2H, COCH2Cl), 4.13-4.08 (m, 3H, H-2, H-3, H-5), 3.75 (t, 1H,
J
3,4,J4,59.5 Hz, H-4), 3.69 (m, 2H, H-4, H-6b), 3.24 (dd, 1H, J1,2 3.5 Hz,
J2,3 10.5 Hz, H-2), 2.34 (s, 3H, SC6H4CH3), 1.38 (d, 3H, J5,6 6.5 Hz, C-
CH3).13C NMR (CDCl3, 125 MHz) : 166.4 (COCH2Cl), 137.8, 137.4,
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201900815
European Journal of Organic Chemistry
FULL PAPER
by flash chromatography using n-hexane-EtOAc (2:1) as eluent to afford
CH2Ph), 4.69 (s, 2H, CH2Ph), 4.60 (d, 1H, J1,2 9.5 Hz, H-1), 3.91 (t, 1H,
J1,2J2,3 9.5 Hz, H-2), 3.86 (m, 1H, H-6a), 3.82 (d, 1H, J3,4 2.5 Hz, H-4),
3.81 (s, 3H, CH2C6H4OCH3), 3.59 (dd, 1H, J2,3 9.5 Hz, J3,4 2.5 Hz, H-3),
3.53 (m, 1H, H-6b), 3.43 (m, 1H, H-5), 2.31 (s, 3H, SC6H4CH3). 13C NMR
(CDCl3, 125 MHz) : 159.3, 138.3, 137.3, 132.1 (2), 129.9, 129.6 (2),
129.3 (2), 128.4, 128.3 (6), 128.2 (2), 128.1 (2), 127.7 (2), 127.5, 113.8
(2) (ArC), 88.0 (C-1), 83.9, 78.7, 77.4, 75.6, 74.1, 73.3, 72.6, 62.2, 55.2
(CH2C6H4OCH3), 21.0 (SC6H4CH3). HRMS calcd for C35H38O6SNa
(M+Na)+: 609.2287, found: 609.2283.
25
the pure tetrasaccharide acceptor 13 (780 mg, 82%) as foam. [α]D
=
+114 (c0.8, CHCl3). 1H NMR (CDCl3, 500 MHz) : 8.10-7.15 (m, 40H,
ArH), 5.54 (s, 1H, CHPh), 5.38 (m, 2H, H-4, H-4), 5.25 (d, 1H, J1,2 1.0
Hz, H-1), 5.15 (d, 2H, COOCH2Ph), 5.08 (m, 2H, H-1, H-1), 4.93, 4.51
(ABq, 2H, JAB 11.0 Hz, CH2Ph), 4.85 (d, 1H, J1,2 1.0 Hz, H-1), 4.62 (m,
2H, CH2Ph), 4.56 (m, 2H, CH2Ph), 4.44 (s, 2H, CH2Ph), 4.40 (m, 1H, H-
2), 4.30 (m, 2H, H-5, H-6a), 4.21 (m, 1H, H-3), 4.10-3.98 (m, 5H, H-2,
H-2, H-3, H-3, H-5), 3.92 (m, 1H, H-3), 3.84 (m, 2H, H-5, H-5), 3.74
(m, 2H, H-6b, OCH2), 3.65 (t, 1H, J3,4J4,5 9.5 Hz, H-4), 3.55 (m, 2H,
H-4, OCH2), 3.47, 3.42 (m, 2H, CH2NH), 3.30 (dd, 1H, J1,2 3.5 Hz,
6-O-chloroacetyl-2,4-di-O-benzyl-3-O-(4-methoxybenzyl)-α-D-
J
2,3 10.0 Hz, H-2), 2.94 (bs, 1H, OH), 1.28 (m, 6H, 2C-CH3), 1.22 (d,
galactopyranoside (17):
3H, J5,6 6.0 Hz, C-CH3). 13C NMR (CDCl3, 125 MHz) : 165.8, 165.7
(2COPh), 156.4 (COOCH2Ph), 138.0,137.9, 137.6(2), 137.3, 136.4,
133.2 (2), 130.1, 130.0 (2), 129.9, 129.2, 128.6 (2), 128.5 (6), 128.4 (5),
128.3 (6), 128.2 (10), 127.9 (2), 127.6 (2), 126.5 (2) (ArC), 102.0 (CHPh),
101.2 (C-1ʹ), 99.3 (C-1), 98.4 (C-1ʺ), 94.0 (C-1ʺʹ), 81.9,79.7, 76.5, 75.9,
75.8, 75.6, 74.3, 73.7, 73.3, 73.2, 73.1, 72.3, 72.0 (2), 69.0, 68.8, 68.6,
67.7, 67.3, 67.0 (2), 63.1, 62.5, 40.8 (CH2NH), 18.0 (2C-CH3), 17.7 (C-
CH3). HRMS calcd for C83H88N4O21Na (M+Na)+:1499.5839, found:
1449.5834.
Compound 16 (480 mg, 0.8 mmol) was dissolved in dry CH2Cl2 (20 mL)
and pyridine (0.3 mL, 3.2 mmol) was added to the reaction mixture and
continued to stir at -5ºC for 15 minutes. Chloroacetic anhydride (272 mg,
1.6 mmol) was then added and the reaction was stirred at the same
temperature for 2 hours till the TLC in n-hexane-EtOAc (5:2) confirmed
complete consumption of the starting material. The solvents were
evaporated and co-evaporated with toluene for complete removal of
pyridine. The crude residue thus obtained was purified by flash
chromatography using n-hexane-EtOAc (2:1) as eluent to afford
25
p-Tolyl
(15):
4-O-benzyl-3-O-(4-methoxybenzyl)-α-D-galactopyranoside
compound 17 (493 mg, 91%) as white powder. [α]D = +132 (c0.8,
CHCl3).1H NMR (CDCl3, 500 MHz) : 7.52-6.91 (m, 18H, ArH), 5.05, 4.66
(ABq, 2H, JAB 11.5 Hz, CH2Ph), 4.90, 4.82 (ABq, 2H, JAB 10.0 Hz,
CH2Ph), 4.74 (s, 2H, CH2Ph), 4.61 (d, 1H, J1,2 9.5 Hz, H-1), 4.41 (dd, 1H,
J5,6a, 7.5 Hz, J6a, 6b 11.0 Hz, H-6a), 4.17 (dd, 1H, J5,6b 5.5 Hz, J6a,6b 11.0
Hz, H-6b), 3.96 (m, 3H, H-2, COCH2Cl), 3.84 (m, 4H, H-4,
CH2C6H4OCH3), 3.64 (m, 2H, H-3, H-5), 2.35 (s, 3H, SC6H4CH3). 13C
NMR (CDCl3, 125 MHz) : 166.7 (COCH2Cl), 159.2, 138.2, 138.1, 137.3,
132.3 (2), 130.0, 129.9, 129.4 (2), 129.2 (2), 128.4, 128.2 (4), 128.1 (4),
127.6 (2), 127.4, 88.0 (C-1), 83.6, 77.2, 75.5, 75.4, 74.0, 73.0, 72.7, 64.7,
55.1 (CH2C6H4OCH3), 40.5 (COCH2Cl), 21.0. HRMS calcd for
C37H39ClO7SNa (M+Na)+: 685.2003, found: 685.2000.
To a solution of known compound 14 (750 mg, 1.5 mmol) in DMF at 5ºC,
NaH (108 mg, 4.5 mmol) was added and stirred for 10 minutes.
Thereafter, BnBr (0.3 mL, 2.3 mmol) was added to the reaction mixture
and it was stirred at room temperature for 3 hours until the TLC (n-
hexane: EtOAc; 5:2) showed complete consumption of the starting
material to a faster moving spot. Immediately, the excess NaH was
quenched with MeOH (5 mL) and DMF was evaporated invacuo. The
mixture was diluted with CH2Cl2 (20 mL) and successively washed with
H2O (2×30 mL) and brine (2×30 mL). The organic layer was separated,
dried (Na2SO4), filtered and evaporated invacuo. The residue was
purified by flash chromatography using usingn-hexane-EtOAc (3:2) to
2-(benzyloxycarbonyl)-aminoethyl 6-O-chloroacetyl-2,4-di-O-benzyl-
3-O-(4-methoxybenzyl)-α-D-galactopyranosyl-(1→3)-2-azido-2-deoxy-
4,6-O-benzylidene-α-D-glucopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-
rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-
rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-
rhamnopyranoside (18):
25
afford the pure compound 15 (833 mg, 94%) as colourless gel.[α]D
=
+89 (c0.8, CHCl3).1H NMR (CDCl3, 500 MHz) : 7.68-6.86 (m, 18H, ArH),
5.52 (s, 1H, CHPh), 4.77, 4.72 (m, 4H, H-6a, H-6b, CH2Ph), 4.61 (d, 1H,
J1,2 9.5 Hz, H-1), 4.38, 3.99 (ABq, 2H, JAB 12.0 Hz, CH2Ph), 4.15 (d, J3,4
2.5 Hz, H-4), 3.89 (t, 1H, J1,2,J2,3 9.5 Hz, H-2), 3.81 (s, 3H,
CH2C6H4OCH3), 3.65 (dd, 1H, J2,3 9.5 Hz, J3,4 2.5 Hz, H-3), 3.39 (m, 1H,
H-5), 2.35 (s, 3H, SC6H4CH3). 13C NMR (CDCl3, 125 MHz) : 159.1,
138.5, 137.8, 137.4, 133.2 (2), 130.0, 129.7, 129.5 (2), 129.3 (2), 128.8,
128.1 (2), 128.0 (2), 127.9 (2), 127.6, 127.5, 126.5 (2), 113.6, 101.1
(CHPh), 86.4 (C-1), 80.9, 75.2, 73.4, 71.2, 69.6, 69.2, 55.1
(CH2C6H4OCH3), 21.0 (SC6H4CH3). HRMS calcd for C28H30O6SNa
(M+Na)+: 517.1661, found: 517.1659.
A
mixture of tetrasaccharide acceptor 13 (750 mg, 0.5 mmol),
monosaccharide donor 17 (430 mg, 0.7 mmol) and MS 4Å (2.0g) in dry
CH2Cl2 (30 mL) was stirred under N2 for 10 min at 0ºC. NIS (190 mg, 0.8
mmol) was added followed by addition of H2SO4-silica (50 mg) at 0°C.
The reaction mixture was stirred for 10 minutes at same temperature
when TLC n-hexane-EtOAc (5:2) indicated complete consumption of the
acceptor 13. The mixture was immediately filtered through a pad of Celite.
The filtrate was diluted with CH2Cl2 (30 mL) and washed successively
with Na2S2O3 (2 × 30 mL), NaHCO3 (2 × 30 mL) and brine (30 mL). The
organic layer was collected, dried over anhydrous Na2SO4 and
evaporated in vacuo. The crude product thus obtained was purified by
flash chromatography using n-hexane-EtOAc (2:1) to afford pure
pentasaccharide 18 (866 mg, 84%) as colourless foam. [α]D25 = +126 (c
1.0, CHCl3). 1H NMR (CDCl3, 500 MHz) : 8.12-6.91 (m, 54H, ArH), 5.72
(d, 1H, J1,22.0 Hz, H-1), 5.42 (s, 1H, CHPh), 5.38 (m, 2H, H-4, H-4),
5.29 (d, 1H, J1,21.0 Hz, H-1), 5.14 (d, 2H, COOCH2Ph), 5.07 (d, 1H,
p-Tolyl
2,4-di-O-benzyl-3-O-(4-methoxybenzyl)-α-D-
galactopyranoside (16):
To a solution of known compound 15 (650 mg, 1.1 mmol) in anhydrous
CH3CN (15 mL), NaBH4 (1.6 g, 8.8 mmol) was slowly added at 0ºC. After
10 min, TCT (416 mg, 11.0 mmol) was added and the reaction mixture
was stirred at room temperature for 8 hours till the TLC showed (n-
hexane-EtOAc; 3:2) complete consumption of the starting material to a
slower moving spot. The reaction mixture was immediately filtered
through a pad of Celite followed by evaporation of the filtrate in vacuo.
The crude compound thus obtained was purified by flash
chromatography using n-hexane-EtOAc (1:2) to afford the pure
J
1,2 1.0 Hz, H-1), 4.99 (d, 1H, J1,2 3.5 Hz, H-1), 4.94, 4.91 (ABq, 2H,
JAB 11.0 Hz, CH2Ph), 4.85 (d, 1H, J1,2 ˂ 1.0 Hz, H-1), 4.84, 4.71 (ABq, 2H,
JAB 11.0 Hz, CH2Ph), 4.67-4.59 (m, 6H, 3×CH2Ph), 4.57, 4.52 (ABq, 2H,
JAB 12.5 Hz, CH2Ph), 4.48, 4.45 (ABq, 2H, JAB 12.5 Hz, CH2Ph), 4.42 (m,
2H, H-2′, H-6aʺʺ), 4.29 (m, 2H, H-6a‴, H-6b‴ʹ), 4.22-4.14 (m, 4H, H-3ʺ, H-
3‴, H-5‴, H-5ʺʺ), 4.07, 3.75 (ABq, 2H, JAB 14.5 Hz, COCH2Cl), 4.05-3.98
25
compound 16 (522 mg, 80%) as white foam. [α]D = +112 (c0.8,
CHCl3).1H NMR (CDCl3, 500 MHz) : 7.48-6.87 (m, 18H, ArH), 4.97, 4.62
(ABq, 2H, JAB 11.5 Hz, CH2Ph), 4.85, 4.76 (ABq, 2H, JAB 10.5 Hz,
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201900815
European Journal of Organic Chemistry
FULL PAPER
(m, 5H, H-2, H-2ʺ, H-2ʺʺ, H-3ʹ, H-5), 3.93 (m, 3H, H-3, H-5ʹ, H-5ʺ), 3.87-
3.70 (m, 5H, H-3ʺʺ, H-4‴, H-4ʺʺ, H-6bʺ′, OCH2), 3.84 (s, 3H,
CH2C6H4OCH3), 3.63 (t, 1H, J3ʺ4ʺJ3ʺ4ʺ 9.5 Hz, H-4ʺ), 3.55 (m, 1H, OCH2),
3.50.3.42 (m, 3H, H-2‴, CH2NH), 3.43 (m, 1H, OCH2), 1.32 (d, 3H,
J5ʺ,6ʺ6.5 Hz, C-CH3), 1.28 (d, 3H, J5ʹ,6ʹ6.5 Hz, C-CH3), 1.20 (d, 3H, J5, 6 6.0
Hz, C-CH3). 13C NMR (CDCl3, 125 MHz) : 167.4 (COCH3), 165.6 (2)
(COPh), 156.3 (COOCH2Ph), 138.1, 138.0, 137.8, 137.5, 137.4 (2),
134.0, 136.3, 133.1, 133.0, 130.7, 130.0, 129.8, 129.7 (2), 129.1 (2),
128.9, 128.5 (10), 128.3 (12), 128.2 (6), 128.1 (2), 128.0 (2), 127.9,
127.7 (5), 127.6, 127.5, 127.4, 127.3, 127.2 (2), 126.4 (2), 113.7 (2)
(ArC), 101.9 (CHPh), 101.2 (C-1ʹ), 99.2 (C-1), 97.7 (C-1ʺ), 96.9 (C-1ʺʺ),
92.9 (C-1‴), 82.9, 79.4, 78.1, 76.5, 75.8 (2), 75.5, 75.3, 74.6, 74.4, 73.5,
73.3 (2), 73.1, 72.3, 72.1, 71.9 (2), 71.8, 71.6, 71.5, 68.8 (2), 68.5, 67.5,
67.1, 66.9, 66.8, 65.4, 62.5, 62.1, 55.2 (CH2C6H4OCH3), 40.9 (COCH2Cl),
40.7 (CH2NH), 17.9 (C-CH3), 17.6 (2×C-CH3). HRMS calcd for
C113H119ClN4O28Na (M+Na)+: 2037.7597, found: 2037.7593.
compound was then subjected to flash silica gel chromatography using n-
hexane-EtOAc (3:1) as eluent to furnish the desired compound 21(357
mg, 86%) as colorless foam. [α]D25 = +95 (c0.8, CHCl3).1H NMR (CDCl3,
500 MHz) : 8.20-6.94 (m, 24H, ArH), 4.99, 4.77 (ABq, 2H, JAB 10.5 Hz,
CH2Ph), 4.92, 4.66 (ABq, 2H, JAB 11.0 Hz, CH2Ph), 4.95, 4.90 (ABq, 2H,
JAB 10.5 Hz, CH2Ph), 4.69 (m, 1H, H-6a), 4.64 (d, 1H, J1,2 9.5 Hz, H-1),
4.46 (dd, 1H, J5,6b 4.6 Hz, J6a, 6b 11.5 Hz, H-6b), 3.79 (t, 1H, J2,3J3,4 8.5
Hz, H-3), 3.67 (m, 2H, H-4, H-5), 3.52 (t, 1H, J1,2J2,3 9.5 Hz, H-2), 2.29
(SC6H4CH3).13C NMR (CDCl3, 125 MHz) : 166.1 (COPh), 162.3, 138.1,
138.0, 137.8, 137.5, 134.5 (2), 133.0 (2), 130.5 (2), 129.8 (2), 129.6 (2),
128.8 (2), 128.5 (2), 128.4 (2), 128.3, 128.1 (2), 128.0 (2), 127.9 (2),
127.8 (ArC), 87.4 (C-1), 86.7, 80.7, 77.6 (2), 75.9, 75.4, 75.1, 63.5, 21.1
(SC6H4CH3) HRMS calcd for C41H40O6SNa (M+Na)+ : 683.2443, found:
683.2446.
2-(benzyloxycarbonyl)-aminoethyl 6-O-benzoyl-2,3,4-tri-O-benzyl-α-
D-glucopyranosyl-(1→3)-6-O-chloroacetyl-2,4-di-O-benzyl-α-D-
galactopyranosyl-(1→3)-2-azido-2-deoxy-4,6-O-benzylidene-α-D-
glucopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→2)-
4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-
O-benzyl-α-L-rhamnopyranoside (22):
2-(benzyloxycarbonyl)-aminoethyl 6-O-chloroacetyl-2,4-di-O-benzyl-
α-D-galactopyranosyl-(1→3)-2-azido-2-deoxy-4,6-O-benzylidene-α-D-
glucopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→2)-
4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-
O-benzyl-α-L-rhamnopyranoside (19):
A
solution of known glucose donor 21 (257 mg, 0.4 mmol),
To a 20 mL solution of pure pentasaccharide 18 (842 mg, 0.4 mmol) in
CH2Cl2-H2O (4:1), DDQ (182 mg, 0.8 mmol) was added and the reaction
mixture was vigorously stirred at room temperature. After 2 hours, the
starting material was fully consumed (TLC in n-hexane-EtOAc; 2:1) and a
slower moving spot was generated. The reaction mixture was diluted with
CH2Cl2 (30 mL), washed with H2O (220 mL) and brine (220 mL)
successively. The organic layer was then collected, dried over anhydrous
Na2SO4, filtered and evaporated in vacuo. The crude syrupy residue was
subjected to flash chromatography (n-hexane-EtOAc; 1.4:1) to afford the
desired pentasaccharide acceptor 19 (666 mg, 79%) as white foam.
[α]D25 = +148 (c0.8, CHCl3).1H NMR (CDCl3, 500 MHz) : 8.23-7.33 (m,
50H, ArH), 5.87 (d, 1H, J1ʺʺ,2ʺʺ 3.5 Hz, H-1ʺʺ), 5.57 (m, 2H, H-4, H-4′), 5.52
(s, 1H, CHPh), 5.45 (d, 1H, J1ʺ,2ʺ ˂ 1.0 Hz, H-1ʺ), 5.33 (m, 2H,
COOCH2Ph), 5.25 (d, 1H, J1′,2′ ˂ 1.0 Hz, H-1′), 5.19 (d, 1H, J1‴,2‴ 3.5 Hz,
H-1‴), 5.12, 5.08 (ABq, 2H, JAB 11.5 Hz, CH2Ph), 5.03 (d, 1H, J1,2 ˂ 1.0
Hz, H-1), 4.88-4.70 (m, 10H, 5×CH2Ph), 4.60 (m, 3H, H-2′, H-3‴, H-6aʺʺ),
4.49-4.34 (m, 5H, H-3ʺʺ, H-5‴, H-5ʺʺ, H-6a‴, H-6bʺʺ), 4.24, 3.93 (ABq, 2H,
JAB 15.0 Hz, COCH2Cl), 4.19-4.15 (m, 6H, H-2, H-2ʺ, H-3, H-3ʺ, H-5, H-
5ʺ), 4.11 (m, 1H, H-3′), 4.01 (m, 2H, H-2ʺʺ, OCH2), 3.89 (m, 4H, H-4‴, H-
4ʺʺ, H-5′, H-6b‴), 3.81 (t, 1H, J3ʺ,4ʺ J4ʺ,5ʺ 9.5 Hz, H-4ʺ), 3.72 (m, 1H, OCH2),
3.67, 3.60 (m, 2H, CH2NH), 3.59 (m, 1H, H-2‴), 1.50 (d, 3H, J5ʺ, 6ʺ6.5Hz,
C-CH3), 1.46 (d, 3H, J5ʹ,6ʹ 6.5Hz, C-CH3), 1.39 (d, 3H, J5,6 6.0 Hz, C-CH3).
13C NMR (CDCl3, 125 MHz) : 167.2 (COCH2Cl), 165.6 (2×COPh), 156.2
(COOCH2Ph), 138.0, 137.9, 137.6, 137.5, 137.4, 136.9, 136.3, 133.1 (2),
130.0, 129.8 (2), 129.2, 128.5 (6), 128.4 (2), 128.3 (12), 128.2 (2), 128.1
(7), 128.0 (2), 127.7 (10), 127.4 (4), 126.5 (2) (ArC), 102.2 (CHPh), 101.3
(C-1ʹ), 99.2 (C-1), 97.8 (C-1ʺ), 96.3 (C-1ʺʺ), 93.0 (C-1‴), 82.9, 79.4, 76.5,
75.8, 75.5, 75.4, 75.3, 74.8,73.6, 73.3, 73.2, 73.1, 72.3, 72.2, 72.1, 71.9
(2), 71.5, 70.8, 69.8, 68.8, 68.7, 68.5, 67.6, 67.2, 66.9 (2), 65.2, 62.4,
62.2, 40.9 (COCH2Cl), 40.7 (CH2NH), 17.9 (C-CH3), 17.6 (2×C-CH3).
HRMS calcd for C105H111ClN4O27Na (M+Na)+: 1917.7022, found:
1917.7020
pentasaccharide acceptor 19 (610 mg, 0.3 mmol) and MS 4Å (2.0 g) in
dry CH2Cl2was stirred under N2 for 20 min. NIS (114 mg, 0.5 mmol) was
added and the reaction mixture was cooled to -55 oC followed by addition
of H2SO4-silica (35 mg). After 30 minutes, TLC in n-hexane-EtOAc (5:2)
indicated complete consumption of the acceptor 19. The reaction mixture
was immediately filtered through a pad of Celite and the filtrate was
successively washed with Na2S2O3 (2 × 30 mL), NaHCO3 (2 × 30 mL)
and brine (30 mL). The organic layer was collected, dried (Na2SO4) and
evaporated in vacuo. The residue thus obtained was purified by flash
chromatography using n-hexane-EtOAc (2:1) and afforded the pure
hexasaccharide 22 (609 mg, 78%) as colorless foam. [α]D25 = +122 (c0.8,
CHCl3).1H NMR (CDCl3, 500 MHz) : 8.13-7.04 (m, 70H, ArH), 5.71 (d,
1H, J1ʺʺ,2ʺʺ 3.5 Hz, H-1ʺʺ), 5.40 (m, 2H, H-4, H-4′), 5.30 (d, 1H, J1ʺ,2ʺ ˂ 1.0
Hz, H-1ʺ), 5.24 (s, 1H, CHPh), 5.15 (m, 3H, COOCH2Ph, H-1‴ʺ), 5.11,
4.74 (ABq, 2H, JAB 11.0 Hz, CH2Ph), 5.07 (d, 1H, J1ʹ,2ʹ ˂ 1.0 Hz, H-1ʹ),
5.00 (d, 1H, J1ʹʺ,2ʺʹ 3.5 Hz, H-1ʺʹ), 4.95, 4.90 (ABq, 2H, JAB 11.5 Hz,
CH2Ph), 4.87 (d, 1H, J1,2 ˂ 1.0 Hz, H-1), 4.85 (m, 4H, 2×CH2Ph), 4.67 (m,
2H, CH2Ph), 4.63-4.54 (m, 4H, 2×CH2Ph), 4.54, 4.50 (ABq, 2H, JAB 11.5
Hz, CH2Ph), 4.43 (m, 2H, CH2Ph), 4.38 (2H, H-2ʹ, H-3‴), 4.29-4.22 (m,
4H, H-3ʺ, H-5‴, H-6aʺʺ, H-6a‴ʺ), 4.19-4.13 (m, 5H, H-3ʺʺ, H-3‴ʺ, H-6a‴, H-
6b‴′, H-6b‴ʺ), 4.07 (m, 3H, H-2ʺ, H-4‴ʺ, H-5ʺ), 4.05, 3.81 (m, 2H,
COCH2Cl), 3.99 (m, 2H, H-2, H-2ʺʺ), 3.92 (m, 3H, H-5, H-5ʹ, H-4ʺʺ), 3.87
(m, 1H, H-6b‴), 3.78 (m, 2H, H-3, H-5ʹʹʹʹ), 3.74, 3.57 (m, 2H, OCH2), 3.71-
3.61 (m, 5H, H-2‴ʺ, H-3ʹ, H-4ʺ, H-4‴, H-5‴ʺ), 3.49, 3.43 (m, 2H, CH2NH),
3.37 ((dd, 1H, J1‴,2‴ 3.5 Hz, J2‴,3‴ 10.0 Hz, H-2‴), 1.32 (d, 3H, J5,6 6.5 Hz,
C-CH3), 1.28 (d, 3H, J5,6 6.0 Hz, C-CH3), 1.21 (d, 3H, J5,6 6.5 Hz, C-CH3).
13C NMR (CDCl3, 125 MHz) : 167.3 (COCH2Cl), 166.1, 165.6 (3×COPh),
156.2 (COOCH2Ph), 139.2, 138.5, 138.4, 138.2, 138.0, 137.9, 137.8,
137.6 (2), 137.5 (2), 136.9, 136.3 (2), 133.1, 133.0, 132.9, 130.2, 130.1,
129.9, 129.8, 129.7 (4), 129.6 (2), 129.1 (2), 128.5 (4), 128.4 (4), 128.3
(10), 128.2 (10), 128.1 (5), 127.9 (4), 127.8 (4), 127.7 (2), 127.6 (4),
127.4, 126.4 (2) (ArC), 102.1 (CHPh), 101.3 (C-1ʹ), 99.3 (C-1), 97.8 (C-
1ʺ), 96.8 (C-1‴ʺ), 96.4 (C-1ʺʺ), 93.0 (C-1‴), 83.0, 82.0, 80.5, 79.5, 77.9,
76.6, 76.4, 75.8 (2), 75.7, 75.6, 75.0, 74.9, 74.8, 74.4, 73.9, 73.6, 73.3,
73.2, 72.4, 72.3, 72.0, 71.7, 71.6, 71.0, 70.5, 69.1, 68.9, 68.8, 68.5, 67.6,
67.2, 67.0, 66.9, 65.3, 63.1, 62.4, 62.2, 40.9 (COCH2Cl), 40.8 (CH2NH),
18.0 (C-CH3), 17.6 (2×C-CH3). HRMS calcd for C139H143ClN4NaO33
(M+Na)+:2453.9221, found: 2453.9218.
p-Tolyl 6-O-Benzoyl-2,3,4-tri-O-Benzyl-β-D-glucopyranoside (21):
To a solution of compound 20(350 mg, 0.6 mmol) in pyridine (10 mL),
benzoyl chloride (0.1 mL, 0.9 mmol) was added and the reaction mixture
was stirred at room temperature for 4 hours until the TLC (n-hexane:
EtOAc; 4:1) showed complete consumption of the starting material to a
faster moving spot. The solvent was then evaporated and co-evaporated
with toluene to ensure complete removal of pyridine. The crude
2-(benzyloxycarbonyl)-aminoethyl 6-O-benzoyl-2,3,4-tri-O-benzyl-α-
D-glucopyranosyl-(1→3)-2,4-di-O-benzyl-α-D-galactopyranosyl-
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201900815
European Journal of Organic Chemistry
FULL PAPER
(1→3)-2-azido-2-deoxy-4,6-O-benzylidene-α-D-glucopyranosyl-
(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-
benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-
rhamnopyranoside (23):
removed under reduced pressure followed by purification of the crude
compound using flash chromatography (n-hexane-EtOAc; 1:9) to furnish
the pure hexasaccharide derivative 24 (266 mg, 71%) as
colorlessfoam.[α]D25 = +106 (c0.8, CHCl3).8.06-7.11 (m, 65H, ArH), 5.35
(m, 2H, H-4, H-4′), 5.24 (d, 1H, J1,2 3.0 Hz, H-1ʺʺ), 5.13 (m, 3H, H-1‴ʺ,
COOCH2Ph), 5.04 (m, 2H, H-1′, H-1‴), 5.04, 4.74 (ABq, 2H, 11.5 Hz,
CH2Ph), 4.98 (d, 1H, J1ʺ,2ʺ ˂ 1.0 Hz, H-1ʺ), 4.96, 4.92 (ABq, 2H, JAB 11.0
Hz, CH2Ph), 4.90-4.80 (m, 4H, 2×CH2Ph), 4.82 (m, 1H, H-1), 4.68-4.52
(m, 6H, 3×CH2Ph), 4.49 (m, 1H, H-2ʹ), 4.50, 4.46 (ABq, 2H, JAB 11.0 Hz,
CH2Ph), 4.38 (m, 2H, H-3ʺ, H-3‴), 4.42, 4.32 (ABq, 2H, JAB 12.0 Hz,
CH2Ph), 4.29 (m, 2H, H-5‴, H-5‴ʺ), 4.23 (m, 2H, H-2ʺʺ, H-3‴ʺ), 4.12 (m,
2H, H-3ʹ, H-4ʺʺ), 4.04 (m, 1H, H-2ʺ), 4.01-3.93 (m, 5H, H-2, H-4‴ʺ, H-5, H-
6a‴ʺ, H-6b‴ʺ), 3.89 (m, 2H, H-3, H-5ʺ), 3.82 (m, 3H, H-2‴ʺ, H-5ʹ, H-6a‴),
To a solution of pure hexasaccharide 22 (552 mg, 0.2 mmol) in 25 mL of
CH2Cl2-MeOH (2:3), thiourea (152 mg, 2.0 mmol) and collidine (0.3 mL,
2.0 mmol) was added. The reaction mixture was refluxed for 10 hours at
55ºC until the TLC (n-hexane-EtOAc; 1.8:1) showed consumption of the
hexasaccharide to a slower moving spot. The solvent mixture was
evaporated under reduced pressure and the residue was dissolved in
CH2Cl2 (25 mL). It was then successively washed with H2O (25 mL) and
brine (25 mL). The organic layer was separated, dried over anhydrous
Na2SO4, filtered and evaporated in vacuo to a syrupy residue which was
further purified by flash chromatography using n-hexane-EtOAc (3:2) as
eluent to obtain the pure hexasaccharide derivative 23 (433 mg, 81%) as
foam. [α]D25 = +94 (c0.8, CHCl3).1H NMR (CDCl3, 500 MHz) : 8.09-7.11
(m, 70H, ArH), 5.75 (d, 1H, J1‴′,2‴′ 3.5 Hz, H-1‴′), 5.35 (m, 2H, H-4, H-4′),
5.25 (m, 2H, H-1ʺ, CHPh), 5.15 (m, 3H, H-1‴ʺ, COOCH2Ph), 5.11, 4.74
(ABq, 2H, JAB 12.0 Hz, CH2Ph), 5.02 (m, 2H, H-1ʹ, H-1‴), 4.93-4.82 (m,
6H, 3×CH2Ph), 4.84 (m, 1H, H-1), 4.62 (m, 4H, 2×CH2Ph), 4.55 (m, 4H,
2×CH2Ph), 4.46 (m, 2H, CH2Ph), 4.44 (m, 2H, H-2′, H-3‴), 4.38-4.31 (m,
3H, H-3ʺ, H-5‴, H-5‴ʺ), 4.23 (m, 2H, H-3‴ʺ, H-6a‴′ʹ), 4.17-4.09 (m, 3H, H-2,
H-6a‴, H-6b‴ʺ), 4.03-3.96 (m, 6H, H-2ʺ, H-2‴′, H-3′, H-4‴′, H-4‴ʺ, H-5),
3.89 (m, 1H, H-3), 3.83 (m, 1H, H-5ʺ), 3.76 (m, 2H, H-5′, OCH2), 3.68 (m,
4H, H-2‴ʺ, H-5‴′, H-6aʺʺ, H-6b‴), 3.63 (m, 4H, H-3‴′, H-4ʺ, H-4‴, H-6bʺʺ),
3.54 (m, 1H, OCH2), 3.45, 3.40 (m, 2H, CH2NH), 3.28 (dd, 1H, J1‴,2‴ 3.5
Hz, J2‴,3‴ 10.0 Hz, H-2‴), 1.25 (m, 6H, 2×C-CH3), 1.20 (d, 3H, J5,6 6.5 Hz,
C-CH3). 13C NMR (CDCl3, 125 MHz) : 166.1, 165.7, 165.6 (3×COPh),
156.3 (COOCH2Ph), 139.2, 138.7, 138.4, 138.2, 138.0 (2), 137.8 (2),
137.6, 137.5, 136.9, 136.3, 133.1, 133.0, 132.9, 130.2, 130.1, 129.9,
129.8 (3). 129.7 (2), 129.1, 128.7, 128.5 (4), 128.4 (8), 128.3 (8), 128.2
(10), 128.1 (3), 128.0, 127.9 (4), 127.8 (8), 127.7 (2), 127.6 (4), 127.5 (2),
127.4, 127.1, 126.4 (2), 126.0 (ArC), 102.0 (CHPh), 101.4 (C-1ʹ), 99.3 (C-
1), 98.2 (C-1ʺ), 96.7 (C-1‴ʺ), 96.4 (C-1ʺʺ), 94.1 (C-1‴), 83.1, 82.0, 80.6,
79.7, 76.6, 76.5, 75.8, 75.7 (2), 75.6 (2), 75.0, 74.8, 74.4, 74.2 (2), 74.1,
73.4, 73.3, 73.2 (2), 73.1, 72.3, 72.0, 71.9 (2), 71.8, 71.3, 71.1, 70.9, 69.1,
68.8, 67.6, 67.2, 67.0, 66.9, 40.8 (CH2NH), 17.9 (C-CH3), 17.7 (2×C-CH3).
HRMS calcd for C137H140N4NaO33 (M+Na)+:2391.9298, found 2391.9295.
3.76-3.69 (m, 5H, OCH2, H-3ʺʺ, H-4ʺʺ, H-5ʺʺ, H-6b‴), 3.61 (t, 1H, J3ʺ,4ʺ
,
J4ʺ,5ʺ 9.5 Hz, H-4ʺ), 3.54 (m, 2H, H-4‴, OCH2), 3.47, 3.41 (m, 2H, CH2NH),
2.86 (dd, 1H, J1‴,2‴ 3.0 Hz, J2‴,3‴ 10.0 Hz, H-2‴), 1.94 (s, 3H, NHCOCH3),
1.25 (d, 3H, J5ʺ,6ʺ6.5 Hz, C-CH3), 1.21 (d, 3H, J5ʹ,6ʹ 6.0 Hz, C-CH3), 1.19 (d,
3H, J5,6 6.0 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) : 177.7 (COOH),
170.6 (NHCOCH3), 166.1, 166.0, 165.6 (3×COPh), 156.3 (COOCH2Ph),
138.4, 138.3 (2), 137.9 (4), 137.7, 137.6, 136.6, 133.4, 133.1, 133.0,
130.0, 129.9 (4), 129.8, 129.7 (2), 129.0 (2), 128.6 (8), 128.5 (6), 128.4
(8), 128.2 (8), 128.1 (2), 128.0 (8), 127.9 (6), 127.8 (6), 127.6 (2), 127.5
(ArC), 101.4 (2) (C-1ʹ, C-1ʺ), 99.3 (C-1), 98.7 (C-1ʺ‴), 95.8 (C-1ʺʺ), 93.5
(C-1‴), 82.4, 82.0, 80.4, 79.4, 78.0, 76.3 (2), 75.8, 75.7 (2), 75.4, 75.1,
75.0, 74.8, 74.6 (2), 74.4, 73.4 (2), 73.2, 72.6, 72.1 (2), 71.9, 71.7, 71.4,
71.2, 69.6, 68.7, 67.6, 67.2, 67.0, 66.9, 63.2, 62.4, 62.0, 61.4, 40.8
(CH2NH), 21.1 (NHCOCH3), 18.0, 17.7, 17.5 (3×C-CH3). HRMS calcd for
C132H140N2NaO34 (M+Na)+:2319.9185, found 2319.9182.
2-amino β-D-glucopyranosyl-(1→3)-α-D-galactopyranosyluronic acid-
(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-
rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-
rhamnopyranoside (1):
A solution of the pure hexasaccharide (223 mg, 0.1 mmol) derivative 24
in NaOMe (0.05 M solution in MeOH; 20 mL) was stirred at room
temperature for 8 hours to remove all the ester protections. The reaction
mixture was neutralized with Dowex 50W X8 H+ resin, filtered and
evaporated in vacuo. The crude product thus obtained was dissolved in
MeOH (50 mL) and passed through
a 10% Pd/C cartridge in a
2-(benzyloxycarbonyl)-aminoethyl 6-O-benzoyl-2,3,4-tri-O-benzyl-α-
D-glucopyranosyl-(1→3)-2,4-di-O-benzyl-α-D-galactopyranosyluronic
acid-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-2,4-di-O-
benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-
rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-
rhamnopyranoside (24):
ThalesNano flow hydrogenation assembly under continuous flow of H2 at
atmospheric pressure.Thehydrogenolysis of the benzyl groups was
complete after 3 such cycles as evident from mass spectrometry. This
process also removed the NHCBz protection liberating the free amino
group.Finally, the residue was dissolved in water and washed with
CH2Cl2 to remove the organic impurities. The aqueous layer was
separated and freeze dried to get the pure target compound 1 (64 mg,
25
63%) as white amorphous mass. [α]D = +76 (c0.5, MeOH). Partial 1H
Compound 23 (387 mg, 0.2 mmol) was dissolved in a biphasic-mixture of
CH2Cl2-H2O (1.5:1; 25 mL) followed by addition of TEMPO (21.0 mg, 0.1
mmol) and iodosobenzene diacetate (IBDA) (343 mg, 1.1 mmol). The
reaction mixture was vigorously stirred at 5 ºC for 8 hours till the TLC (n-
hexane-EtOAc; 1:3) showed complete consumption of the starting
material to a slower moving spot. Aqueous saturated Na2S2O3 (5 mL)
was added to quench the reaction. The mixture was diluted with CH2Cl2
(30 mL) and washed with brine (2× 20 mL). The organic layer was
separated, dried (Na2SO4), filtered and evaporated in vacuo to give the
crude oxidized residue that was directly treated with 80% AcOH (15 mL)
at 80 ºC for 4 hours to selectively cleave the benzylidene acetal
protection. After the starting material was fully consumed (TLC in n-
hexane-EtOAc; 1:6) and a slower moving spot was generated, the
solvent was evaporated and co-evaporated with toluene. The crude diol
residue thus obtained was dissolved in CH3COSH (10 mL) and allowed
to stir at room temperature for 48 hours until TLC (n-hexane-EtOAc; 1:9)
showed complete consumption of the starting material. Solvents were
NMR (MeOD, 500 MHz) : 5.12 (d, 1H, J1ʹ,2ʹ < 1.0 Hz, H-1ʹ), 5.06(d, 1H,
J1ʺʺʹ,2ʺʺʹ 3.0 Hz, H-1ʺʺʹ), 4.97(d, 1H, J1,2 <1.0 Hz, H-1), 4.95 (d, 1H, J1ʺ,2ʺ
<
1.0 Hz, H-1ʺ), 4.91 (d, 1H, J1ʺʺ,2ʺʺ 3.5 Hz, H-1ʺʺ), 4.81 (d, 1H, J1ʺʹ,2ʺʹ 3.0 Hz,
H-1ʺʹ), 1.99 (s, 3H, NHCOCH3), 1.40, 1.36, 1.12 (3d, 9H, J 6.0 Hz,
3×CH3). 13C NMR (125 MHz, MeOD) : 177.8 (COOH), 172.1
(NHCOCH3), 101.9 (C-1ʺʺʹ) 101.7 (C-1ʺʺ), 100.5 (C-1ʹ), 99.4 (C-1ʺ), 98.2
(C-1), 97.7 (C-1ʺʹ), 80.7, 78.5, 77.3, 76.2, 75.6, 75.2, 74.8, 74.2, 73.9,
73.6, 73.3, 73.0, 72.6, 72.3, 71.9, 71.5, 71.1, 70.6, 70.2, 69.8, 69.2, 68.4,
67.4, 66.9, 65.9, 65.2, 52.6 (OCH2), 41.8 (CH2NH), 21.3 (NHCOCH3),
18.2, 18.1, 18.0 (3×C-CH3). HRMS calcd for C40H68N2O29Na
(M+Na)+1063.3805, found 1063.3802.
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10.1002/ejoc.201900815
European Journal of Organic Chemistry
FULL PAPER
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Acknowledgements
The work is supported by the research grant EMR/2017/003043
from Science and Engineering Research Board (SERB), New
Delhi to BM.
Keywords:O-antigen • total synthesis • TEMPO oxidation •
glycosylation • H2SO4-silica
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10.1002/ejoc.201900815
European Journal of Organic Chemistry
FULL PAPER
Entry for the Table of Contents
Total Synthesis
Text for Table of Contents: A convergent synthetic route is established for the total synthesis of the hexasaccharide repeating unit
of the O-antigen from E. coli O133. The desired glycosidic linkages were achieved through activation of either thioglycosides or
trichloroacetimidates in stereoselective manner. The required uronic acid moiety was installed at the hexasaccharide late-stage by
using TEMPO mediated oxidation of the primary hydroxyl group.
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