One-pot iterative glycosylations toward a tetrasaccharide related to the O-specific polysaccharide from Escherichia coli O132

Article abstract of DOI:10.1080/07328303.2021.1928153

An efficient synthetic strategy has been developed for the synthesis of a tetrasaccharide related to the O-specific polysaccharide from Escherichia coli O132 as their 2-aminoethyl glycosides in a very good yield by adopting sequential glycosylation followed by in-situ removal of the p-methoxybenzyl (PMB) group in the same reaction pot. Furthermore, the synthetic route was adapted by carrying out three stereoselective iterative glycosylations followed by in situ removal of the PMB group in one pot. The stereochemical outcomes of all the glycosylation steps were excellent with satisfactory yields.

Full text of DOI:10.1080/07328303.2021.1928153

Journal of Carbohydrate Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lcar20
One-pot iterative glycosylations toward a
tetrasaccharide related to the O-specific
polysaccharide from Escherichia coli O132
Vijay Nath Mishra & Pintu Kumar Mandal
To cite this article: Vijay Nath Mishra & Pintu Kumar Mandal (2021): One-pot iterative
glycosylations toward a tetrasaccharide related to the O-specific polysaccharide from Escherichia
coli O132, Journal of Carbohydrate Chemistry, DOI: 10.1080/07328303.2021.1928153
To link to this article: https://doi.org/10.1080/07328303.2021.1928153
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JOURNAL OF CARBOHYDRATE CHEMISTRY
https://doi.org/10.1080/07328303.2021.1928153
One-pot iterative glycosylations toward a
tetrasaccharide related to the O-specific polysaccharide
from Escherichia coli O132
Vijay Nath Mishraa and Pintu Kumar Mandala,b
aMedicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow, India;
bChemical Sciences Division, Academy of Scientific and Innovative Research (AcSIR),
Ghaziabad, India
ABSTRACT
ARTICLE HISTORY
Received 5 October 2020
Accepted 5 May 2021
An efficient synthetic strategy has been developed for the
synthesis of a tetrasaccharide related to the O-specific polysac-
charide from Escherichia coli O132 as their 2-aminoethyl glyco-
KEYWORDS
Escherichia coli;
oligosaccharide; one-pot;
glycosylation; H2SO4-silica
sides in
a
very good yield by adopting sequential
glycosylation followed by in-situ removal of the p-methoxy-
benzyl (PMB) group in the same reaction pot. Furthermore,
the synthetic route was adapted by carrying out three stereo-
selective iterative glycosylations followed by in situ removal of
the PMB group in one pot. The stereochemical outcomes of
all the glycosylation steps were excellent with satisfactory
yields.
GRAPHICAL ABSTRACT
Introduction
Escherichia coli is a facultative Gram-negative bacterium that presents pre-
dominantly in the guts of human and other warmblooded animals.[1,2]
Although most of the E. coli strains are usually harmless and beneficial to
the host’s body, there are some E. coli variants having virulence factors and
contributing to a variety of infections in humans and animals.[3,4] These
pathotypes cause mainly three type of diseases, i.e., enteric/diarrheal dis-
ease, sepsis/meningitis and urinary tract infections (UTIs), and are also
CONTACT Pintu Kumar Mandal
pintuchem06@gmail.com, pk.mandal@cdri.res.in
Medicinal and Process
Chemistry Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram extension, Sitapur Road,
P.O. Box 173, Lucknow, 226 031, India; Chemical Sciences Division, Academy of Scientific and Innovative
Research (AcSIR), Ghaziabad- 201002, India.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2021 Taylor & Francis Group, LLC

2
V. N. MISHRA AND P. K. MANDAL
→2)-α-D-Galf-(1→3)-α-L-Rhap2Ac-(1→4)-α-D-Glcp-(1→2)-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1→
Figure 1. Structure of the pentasaccharide repeating unit of the O-antigen of E. coli O132.
responsible for losing of life for more than two million humans per year
due to both intraintestinal and extraintestinal diseases.[5,6]
Being a major component of the cell wall of Gram-negative bacteria, the
O-antigens often play important roles during host infections, subsequent
immune responses in the host and in controlling its virulence property.
The virulent E. coli strains causing diarrhea are classified into six subgroups
based on their mechanisms of action, which include enteropathogenic E.
coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC),
diffusely adherent E. coli (DAEC), enteroaggregative E. coli (EAEC), and
enterohaemorrhagic E. coli (EHEC).[7]
Recently, Knirel et al.[8] reported the structure of the O-polysaccharide
repeating unit (O-unit) of E. coli O132. It is a Shiga toxin-producing E.
coli, and its pathogenic strain is reported to be the causative agent for
septicemia in chicken,[9] diarrhea in children under three years of age,[10]
diarrhea in calves and rabbits.[11,12] The structure of E. coli O132 comprises
a linear repeating unit of pentasaccharide (Fig. 1), which consists of a D-
glucosamine (GlcNAcp), two L-rhamnose (Rhap), a D-glucose (Glcp), and
a D-galactofuranose (Galf) sugar residues.
The recent thrust in the drug discovery program is to develop newer
approaches to control bacterial infections due to the emergence of multi-
drug resistant bacterial strains. It is well recognized that bacterial antigenic
character regulates the immunochemical activity of glycoconjugate vaccines,
making them more attractive targets for the design of potential vaccine
candidates.[13] For the study of the involvement of these O-antigens in the
pathogenicity and the immunological properties, desirable amount of the
O-polysaccharide is required. Whereas the O-antigen from naturally occur-
ring biological sources is difficult to isolate and very expensive to produce
appropriate quantities of material with sufficient purity. Subsequently,
chemical synthetic strategies for the preparation of oligosaccharides as well
as their glycoconjugate derivatives are beneficial to get significant quantity
of pure compounds.
The stepwise oligosaccharide syntheses demand extensive protecting
group manipulation and purification after each step making them expen-
sive, time consuming, and tedious. The oligosaccharide synthesis related to
the O-antigen of E. coli O132 repeating unit was reported only by
Mukhopadhyay et al.[14] using a [3 þ 2] convergent strategy. However, the
introduction of an expeditious, step-economic, one-pot total synthesis is
necessary to be relatively environmentally friendly and cost-effective. We
report herein a concise one-pot synthesis of the tetrasaccharide fragment 1

JOURNAL OF CARBOHYDRATE CHEMISTRY
3
Figure 2: Retrosynthetic strategy for target tetrasaccharide 1 as its 2-aminoethyl glycoside and
related building blocks.
(Fig. 2), which is part of the pentasaccharide repeating unit of the O-anti-
gen of E. coli O132. It was achieved by using a linear and one-pot iterative
glycosylations approach. The 2-aminoethyl moiety was applied as a protect-
ing group at the reducing end, which will facilitate further glycoconjugate
formation without hampering the stereochemistry of the anomeric center.
Its terminally free amine can provide glycoconjugates to elucidate further
biological implications of the O-antigen and vaccine potential.
Results and discussion
Tetrasaccharide 1 as its 2-aminoethylglycoside could be obtained from pro-
tected tetrasaccharide 2 through global deprotection. The retrosynthetic dis-
connection of tetrasaccharide 2 revealed that it could be prepared either via
a sequential glycosylation-deprotection technique or by one-pot sequential
glycosylation. The sequential disconnection approach indicates that pro-
tected tetrasaccharide 2 can be achieved by stereoselective glycosylation of
trisaccharide acceptor 8 with rhamnosyl donor 6, whereas trisaccharide 8
can be prepared with thioglucosyl donor 5 and disaccharide acceptor 7,
which is achievable with monosaccharide acceptor 3 and rhamnosyl donor
4 (Fig. 2).
We have designed to have all the thioglycoside donors with a suitable in
situ removable protecting group like p-methoxybenzyl (PMB) group. This
serves two purposes. Firstly, most glycosylation reactions rely on the same
activation methods, and secondly, in-situ removable of the PMB group by
increasing the temperature of the same reaction mixture, which can reduce
the number of reaction steps by providing glycosyl acceptor. This was

4
V. N. MISHRA AND P. K. MANDAL
Scheme 1. Synthesis of glycosyl acceptor 3. Reagents and conditions: (a) HO(CH2)2NHCbz
(Cbz ¼ benzyloxycarbonyl), BF3ꢀOEt2, (CH2Cl)2, 55 ꢁC, 12 h, 89%; (b) CH3ONa, CH3OH, rt, 1 h; (c)
PhCH(OCH3)2, p-TsOH, DMF, rt, 12 h, 82% over two steps.
achieved by using a combination of N-iodosuccinimide (NIS) and silica-
supported sulfuric acid (H2SO4-SiO2) as the promoter.[15,16] Here, H2SO4-
silica has been used successfully as a noncorrosive solid acid promoter for
all glycosylation reaction.
One-pot sequential glycosylation was achieved from the same suitable
protected monosaccharide building blocks, 3[17], 4[18], 5[19] and 6,[20]
derived from commercially available D-glucosamine hydrochloride, L-
rhamnose, and D-glucose (Fig. 2). For each pathway, a retro-synthetic ana-
lysis of the fully protected tetrasaccharide derivative 2 led to common
building blocks.
Firstly, 2-(benzyloxycarbonylamino)ethyl 4,6-O-benzylidene-2-deoxy-2-N-
phthalimido-b-D-glucopyranoside 3 was prepared from 1,3,4,6-tetra-O-ace-
tyl-2-deoxy-2-N-phthalimido-b-D-glucopyranoside 9[21] in a 76% yield
using a three-step reaction sequence involving glycosylation with 2-N-car-
boxybenzylamino ethanol with the promotion of boron trifluoride-diethyl
ether (BF3ꢀOEt2) and then deacetylation using sodium methoxide and ben-
zylidene acetal formation using benzaldehyde dimethylacetal[22] in the pres-
ence of p-toluenesulfonic (Scheme 1).
Synthesis of 2 via a sequential glycosylation-deprotection strategy
The stereoselective glycosylation of acceptor 3 with ʟ-rhamnosyl thioglyco-
side donor 4 in the presence of NIS and H2SO4-silica[15,16] followed by
removal of the PMB group[23] from the product in the same pot by tuning
the reaction conditions led to the formation of disaccharide 7 in an 82%
yield (Scheme 2). The quantity of H2SO4-silica present in the reaction mix-
ture was very low, which allowed the selective deprotection of the highly
acid labile PMB group without affecting the benzylidene acetal in the mol-
ecule. The stereochemical outcome of the newly formed glycosidic linkage
was confirmed by NMR spectroscopic analysis [appearance of signals at d
5.52 (s, 1 H, PhCH), 5.26 (d, J ¼ 8.5 Hz, 1 H, H-1A), 4.60 (d, J ¼ 1.6 Hz, 1 H,
1
H-1B) in H NMR spectrum, and d 102.0 (PhCH), 99.5 (C-1B), 98.9 (C-1A)
in the 13C NMR spectrum]. Although the C-2 position of the ʟ-rhamnosyl
donor 4 was protected with a nonparticipating PMB group, the

JOURNAL OF CARBOHYDRATE CHEMISTRY
5
Scheme 2. Synthesis of tetrasaccharide 2 by a sequential glycosylation-deprotection strategy.
Reagents and conditions: (a) NIS, H2SO4-silica, CH2Cl2/Et2O (2:1), MS 4 Å, –30 ꢁC, 30 min, then
0 ꢁC, 30 min, 82% for compound 7, 71% for compound 8, and 77% for compound 2.
glycosylation reactions furnished exclusively 1,2-trans-glycosidic linkages
under the control of anomeric effect.[24] Subsequently, the disaccharide
acceptor 7 was reacted with 4-O-PMB protected thioglycoside donor 5 in
the presence of NIS and H2SO4-silica[15,16] followed by removal of the
PMB group from the product in the same pot by raising the temperature
of the reaction mixture to furnish trisaccharide 8 in a 71% yield (Scheme
2) together with a minor quantity (ꢂ1%) of its b-isomer, which was sepa-
rated by column chromatography. Gratifyingly, compound 8 was obtained
in satisfactory yield with good stereochemical outcome. The reaction tem-
perature and promoter combination might have an influence on the stereo-
chemical outcome of the glycosylation. Compound 8 formation was
confirmed by spectroscopic study [signals at d 5.29 (d, J ¼ 8.5 Hz, 1 H, H-
1
1A), 4.56 (d, J ¼ 1.6 Hz, 1 H, H-1B), 4.01 (d, J ¼ 2.4 Hz, 1 H, H-1C) in H
NMR spectrum and d 98.8 (C-1A), 97.8 C-1B), 96.2 (C-1C) in 13C NMR
spectrum, respectively]. The stereoselective glycosylation of trisaccharide
acceptor 8 with compound 6 followed by the removal of the PMB group
from the intermediate products was repeated by the same protocols to fur-
nish tetrasaccharide 2 in a 77% yield (Scheme 2). The structure of com-
pound 2 was confirmed by spectroscopic data [signals at d 5.27 (d,
J ¼ 8.52 Hz, 1 H, H-1A), 4.79 (brs, 1 H, H-1D), 4.60 (brs, 1 H, H-1B), 3.94

6
V. N. MISHRA AND P. K. MANDAL
1
(d, J ¼ 3.8 Hz, 1 H, H-1C) in the H NMR spectrum and d 98.0 (C-1A), 97.0
(C-1D), 96.9 (C-1B), 93.6 (C-1C) in the 13C NMR spectrum, respectively].
Synthesis of 2 via one-pot iterative glycosylations
Having obtained the protected tetrasaccharide 2 in a minimum number of
steps and a very good yield by the sequential synthetic glycosylation strat-
egy, we decided to explore the possibility of achieving compound 2 by car-
rying out three iterative glycosylation steps and in situ removal of the
temporary protecting PMB group from the glycosylation products in the
same flask. For this purpose, temporary protecting groups that are sensitive
to acidic conditions, e.g., TBS,[25] PMB,[26] and TBDPS,[27] are typically
employed for glycosylation ꢃ deprotection ꢃ glycosylation in one-pot.[28]
For example, Hung and co-workers[29] employed a TBS[26] protecting
group as a temporary masking group for one-pot glycosylation. Similarly,
Nilsson and co-workers[26] employed PMB ether as an in situ removable
protecting group in the one-pot glycosylation. In that case, glycosylations
were usually conducted at subzero temperatures under acidic conditions.
The temporary protecting group is removed postglycosylation either by
warming up the reaction mixture or by adding more acid such as TfOH,
TMSOTf, or BF3ꢀEt2O.
Here, we explored the synthesis of tetrasaccharide 2 via a one-pot glyco-
sylation technique using the same building-blocks as above. For this pur-
pose, compound 3 was first glycosylated with 2-PMB protected L-
rhamnosyl thioglycoside donor 4 via the previously standardized procedure
using NIS and H2SO4-silica as the promoter in dichloromethane-diethyl
ether at ꢃ30 ꢁC for 30 min to consume all of the donor 4 as indicated by
TLC. Once the donor 4 was completely consumed, the temperature of the
reaction mixture was raised to 0 ꢁC for in-situ removal of p-methoxybenzyl
ether in 30 min according to TLC results, and produced the desired disac-
charide acceptor. The reaction mixture was again cooled to ꢃ30 ꢁC, and to
this cold reaction mixture, 4-PMB protected glucothioglycoside donor 5
and fresh NIS was added with the reaction kept at that temperature for
30 min similar as previously standardized procedure. Then, the temperature
of reaction mixture was raised again to 0 ꢁC for 30 min for in-situ removal
of p-methoxybenzyl ether group, resulting in the desired trisaccharide
acceptor. Thereafter, the same reaction vessel was again cooled to ꢃ30 ꢁC,
and 3-PMB protected L-rhamnosyl thioglycoside donor 6 in dry dichloro-
methane-diethyl ether solution was injected into the cold reaction vessel
together with fresh NIS. The reaction was allowed at this temperature for
30 min, which followed by rising the temperature to 0 ꢁC for 30 min for in-
situ removal of p-methoxybenzyl ether group to produce the desired

JOURNAL OF CARBOHYDRATE CHEMISTRY
7
Scheme 3. Synthesis of tetrasaccharide 2 via a one-pot glycosylation-deprotection-glycosyla-
tion technique.
tetrasaccharide 2. Eventually, we got the target tetrasaccharide 2 in a 55%
overall yield via the one-pot glycosylation strategy (Scheme 3). Compound
2 formation was confirmed by NMR spectroscopy. Carrying out the glyco-
sylation-deprotection reactions in a one pot setup significantly reduced the
number of purification steps. The PMB ether acted as an in-situ removable
temporary protecting group for the hydroxyl functionality for next glycosy-
lation reaction. Important advantages of our one-pot glycosylation strategy
are that only one class of glycosyl donors (simple and stable thioglycosides)
and one promotor system (NIS/H2SO4-silica) are required.
Finally, tetrasaccharide 2 was subjected to a series of deprotection reac-
tions, (a) elimination of the phthalimido group through hydrazinolysis,[30]
followed by N-acetylation and O-deacetylation with sodium methoxide[31]

8
V. N. MISHRA AND P. K. MANDAL
.
Scheme 4. Synthesis of the target compound 1. Reagents and conditions: (a) (i) NH2NH2 H2O,
C2H5OH, 80 ꢁC, 6 h; (ii) acetic anhydride, pyridine, rt, 2 h; (iii) NaOMe/MeOH; (b) H2, 20%
Pd(OH)2- C, CH3OH, rt, 24 h, 56% overall yield.
and (b) removal of all benzyl groups through hydrogenolysis using
Pearlman’s catalyst,[32] to furnish the desired free tetrasaccharide 1 in an
overall yield of 56% (Scheme 4). Compound 1 has been confirmed by its
spectral data and its anomeric protons have been identified at d 5.10 (d, J
= 7.7 Hz, 1 H, H-1A), 4.92 (brs, 1 H, H-1B) and 4.56 (brs, 2 H, H-1D, H-
1C)) ppm, and their corresponding carbon signals are at 102.8 (C-1A),
101.9 (C-1D), 100.4 (C-1B), and 98.4 (C-1C) ppm, respectively (See
Supporting information).
Conclusions
In summary, we have developed an expeditious synthetic strategy for a tet-
rasaccharide related to the O-antigen of E. coli O132 as its 2-aminoethyl
glycoside (1) through one-pot glycosylation-deprotection and sequential
glycosylation-deprotection techniques. The synthetic strategy relies on in-
situ removable PMB ether that serves as a temporary protecting group for
hydroxyl functionality undergoing glycosylation reaction in one pot, which
greatly reduced the number of product purification steps. A general iodo-
nium ion-mediated glycosylation condition was applied to all glycosyla-
tion steps.

JOURNAL OF CARBOHYDRATE CHEMISTRY
9
Experimental section
General methods
All reactions were monitored by using a thin layer chromatography (TLC)
over silica gel-coated TLC plates. The spots on TLC were visualized with
UV when there is an active UV group or by warming ceric sulfate [2%
Ce(SO4)2 in 5% H2SO4 in EtOH]-sprayed plates on a hot plate if there is
no UV active moiety. The spots on TLC were also confirmed by the latter
method even when there is an active UV group. Silica gel (230–400 mesh)
1
was used for purification by column chromatography. All NMR spectra, H
and 13C NMR, DEPT 135, 2 D COSY and HSQC, were recorded on
Brucker DPX 400 MHz spectrometers using CDCl3 and D2O as solvents
and TMS used as an internal reference unless stated otherwise. Chemical
shift values are represented in d ppm. ESI-MS data were recorded on a
JEOL spectrometer. Optical rotations were determined on an Autopol III
polarimeter. IR spectra were recorded on Shimadzu Spectrophotometers.
Commercially available grades of organic solvents of adequate purity were
used in all reactions.
Preparation of H2SO4-silica
To a 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 for uniformly acidic strength. Then the solvent
was evaporated under reduced pressure resulting in free-flowing H2SO4-sil-
ica, which was dried at 110 ꢁC for 3 h and used for the reactions.
2-(Benzyloxycarbonylamino)ethyl 4,6-O-benzylidene-2-deoxy-2-N-
phthalimido-b-D-glucopyranoside (3)
To a solution of compound 9 (5 g, 10.4 mmol) in DCE (100 mL) were
.
added 2-N-carboxybenzylamino ethanol (2.5 g, 12.6 mmol) and BF3 Et2O
(1.8 mL, 12.6 mmol) at 0 ꢁC and and the reaction mixture was allowed to
stir at 55 ꢁC for 12 h. The reaction mixture was diluted with water and
extracted with CH2Cl2 (100 mL). The organic layer was washed with satd.
NaHCO3 and water, dried (Na2SO4) and concentrated. The crude product
was purified over SiO2 using hexane/EtOAc (2:1) as eluant to give pure
compound. To a solution of the pure compound in 0.1 M CH3ONa
(50 mL) was allowed to stir at room temperature for 1 h. The re-action
mixture was neutralized with Amberlite-IR 120 (Hþ) resin,filtered, and
concentrated. To a solution of the deacetylated product (3.9 g, 8.16 mmol)
in anhydrous DMF (20 mL) were added benzaldehyde dimethyl acetal
(1.40 mL, 9.24 mmol) and p-TsOH (300 mg, 1.6 mmol), and the reaction

10
V. N. MISHRA AND P. K. MANDAL
mixture was allowed to stir at room temperature for 12 h. The reaction
mixture was neutralized with Et3N (1.5 mL) and evaporated to dryness
under reduced pressure. The reaction mixture was diluted with water and
extracted with CH2Cl2 (100 mL). The organic layer was washed with satd.
NaHCO3 and water, dried (Na2SO4) and concentrated. The crude product
was purified over SiO2 using hexane/EtOAc (1:1) as eluant to give pure
25
compound 3 (3.7 g, 82%) as semi solid, [a]D ꢃ30 (c 1.0, CHCl3); IR
(KBr): 3400, 3020, 1716, 1389, 1215, 1084, 761, 669 cmꢃ1; 1H NMR
(400 MHz, CDCl3): d 7.82–7.79 (m, 2 H, Ar-H), 7.66–7.65 (m, 2 H, Ar-H),
7.50–7.48 (m, 2 H, Ar-H), 7.39–7.31 (m, 6 H, Ar-H), 7.29–7.26 (m, 2 H, Ar-
H), 5.55 (s, 1 H, PhCH), 5.27 (d, J ¼ 8.5 Hz, 1 H, H-1), 5.01–4.90 (m, 3 H,
NH, COOCH2Ph), 4.65–4.60 (m, 1 H, H-6A), 4.36 (dd, J ¼ 4.2, 10.2 Hz, 1H,
H-6B), 4.23 (dd, J ¼ 8.5, 10.5 Hz, 1H, H-2), 3.82–3.78 (m, 2 H, H-4,
–OCH2), 3.66–3.57 (m, 3 H, H-3, H-5, –OCH2), 3.28-3.27 (m, 2 H, NCH2),
2.57 (brs, 1 H, OH); 13C NMR (100 MHz, CDCl3): d 168.3, 168.1 (COPhth),
156.3–123.6 (Ar-C), 101.9 (PhCH), 99.1 (C-1), 82.0 (C-3), 69.1 (-OCH2),
68.6 (2 C, C-6, C-4), 66.6 (NHCOOCH2Ph), 66.2 (þC-5), 56.6 (C-2), 40.8
(NCH2-); HRMS (ESI-TOF) calcd for C31H31N2O9 [M þ H]þ 575.2024,
found 575.2020.
2-(Benzyloxycarbonylamino)ethyl (3,4-di-O-benzyl-a-L-rhamnopyranosyl)-
(1fi3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-b-D-glucopyranoside (7)
To a solution of compound 3 (2.5 g, 4.35 mmol) and thioglycoside donor 4
(2.93 g, 5.22 mmol) in anhydrous CH2Cl2/Et2O (2:1 v/v; 10 mL) was added
flame-activated MS-4 Å (1.5 g) and the reaction mixture was cooled to
ꢃ30 ꢁC under N2. To the cold reaction mixture was added NIS (1.4 g,
6.25 mmol) followed by H2SO4-SiO2 (30 mg), and the reaction mixture was
stirred at the same temperature for 30 min. After complete consumption of
compound 4 [TLC; hexane/EtOAc 2:1], the reaction mixture was allowed
to stir at 0 ꢁC for 30 min, filtered and washed with CH2Cl2 (30 mL). The
organic layer was successively washed with 5% Na2S2O3 (25 mL), satd.
NaHCO3 (25 mL), and H2O (25 mL), dried (Na2SO4), and concentrated to
give the crude product, which was purified on SiO2 using hexane/EtOAc
25
4:1 as eluent to give pure compound 7 (3.5 g, 82%) as a colorless oil. [a]D
þ18 (c 1.0, CHCl3); IR (neat): 3401, 2926, 1713, 1386, 1219, 1090,
769 cmꢃ1. 1H NMR (400 MHz, CDCl3): d 7.83–7.81 (m, 2 H, Ar-H),
7.67–7.66 (m, 2 H, Ar-H), 7.50–7.48 (m, 2 H, Ar-H), 7.38–7.25 (m, 16 H,
Ar-H), 7.22–7.20 (m, 2 H, Ar-H), 5.52 (s, 1 H, PhCH), 5.26 (d, J ¼ 8.5 Hz,
1 H, H-1A), 5.01–4.91 (m, 3 H, NH, COOCH2Ph), 4.73 (d, J ¼ 11 Hz, 1 H,
PhCH2), 4.66–4.61 (m, 1 H, H-4A), 4.60 (d, J ¼ 1.6 Hz, 1 H, H-1B),
4.56–4.46 (m, 3 H, PhCH2), 4.37 (dd, J ¼ 4.16, 10.24 Hz, 1 H, H-6A), 4.24

JOURNAL OF CARBOHYDRATE CHEMISTRY
11
(dd, J ¼ 8.56, 10.2 Hz, 1 H, H-2A), 3.95–3.88 (m, 1 H, H-5B), 3.82–3.76 (m,
2 H, H-6B, OCH2), 3.74–3.57 (m, 5 H, OCH2, H-3B, H-2B, H-5A, H-3A),
3.27–3.20 (m, 3 H, NCH2, H-4B), 0.78 (d, J ¼ 6.1 Hz, 3 H, CH3);13C NMR
(100 MHz, CDCl3): d 168.3 (COPhth), 156.2–123.8 (Ar-C), 102.0 (PhCH),
99.5 (C-1B), 98.9 (C-1A), 80.7 (H-3A), 79.9 (C-4B), 79.7 (C-3B), 75.0
(PhCH2), 74.1 (C-4A), 71.8 (2 C, PhCH2), 69.2 (-OCH2), 68.6 (2 C, C-6A, C-
2B), 67.7 (C-5B), 66.6 (NHCOOCH2Ph), 66.5 (C-5A), 56.7 (C-2A), 40.þ8
(NCH2-), 17.3 (CH3); HRMS (ESI-TOF) calcd for C51H56N3O13
[M þ NH4]þ 918.3808, found 918.3808.
2-(Benzyloxycarbonylamino)ethyl (2,3,6-tri-O-benzyl-a-D-glucopyranosyl)-
(1fi2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1fi3)-4,6-O-benzylidene-2-
deoxy-2-N-phthalimido-b-D-glucopyranoside (8)
To a solution of compound 7 (3 g, 3.32 mmol) and compound 5 (2.45 g,
3.99 mmol) in anhydrous CH2Cl2/Et2O (2:1 v/v; 40 mL) was added flame-
activated MS 4 Å (1.5 g) and the mixture was allowed to stir under N2 at
room temperature for 1 h. The reaction mixture was cooled to ꢃ30 ꢁC and
NIS (1.07 g, 4.78 mmol) and H2SO4-SiO2 (40 mg) were added to it and
stirred at same temperature for 30 min. After complete consumption of
compound 5 [TLC; hexane/EtOAc 2:1], the reaction mixture was allowed
to stir at 0 ꢁC for 30 min, filtered and washed with CH2Cl2 (30 mL). Then
the organic layer was successively washed with Na2S2O3 (5%), NaHCO3
(satd. aq.), and water, then it was dried (Na2SO4) and concentrated. The
crude product was purified over SiO2 using hexane/EtOAc (4:1) as eluent
25
to give pure compound 8 (3.4 g, 71%) as a colorless oil. [a]D þ14 (c 1.0,
CHCl3); IR (neat): 3740, 3443, 3020, 2926, 1716, 1516, 1385, 1216, 1091,
1
766, 669 cmꢃ1; H NMR (400 MHz, CDCl3): d 7.50–7.46 (m, 4 H, Ar-H),
7.36–7.19 (m, 35 H, Ar-H), 5.53 (s, 1 H, PhCH), 5.26 (d, J ¼ 8.52 Hz, 1 H,
H-1A), 5.00–4.89 (m, 3H, NH, COOCH2Ph), 4.81–4.75 (m, 2 H, PhCH2),
4.67–4.62 (m, 3 H, H-4A, H-1B, PhCH2), 4.55–4.48 (m, 4 H, PhCH2),
4.38–4.34 (m, 3 H, PhCH2), 4.28–4.23 (m,1 H, H-2A), 4.11–4.07 (m, 3 H,
PhCH2, H-1C), 3.91–3.86 (m, 2 H, H-5B), 3.79–3.76 (m, 3 H, OCH2, H-4C,
H-5C), 3.66–3.62 (m, 4 H, H-5A, H-6A, H-3A, H-4B), 3.52–3.46 (m, 1H, H-
2B), 3.38 (d, J ¼ 9.7 Hz, 1 H, H-2C), 3.27–3.24(m, 3 H, NCH2, H-6abC),
3.18–3.14 (m, 1 H, H-6abC), 3.10–3.07 (m, 1 H, H-3B), 0.90 (d, J ¼ 6.3 Hz,
3 H, CH3); 13C NMR (100 MHz, CDCl3): d 168.4 (COPhth), 138.8–126.4
(Ar-C), 101.9 (PhCH), 98.9 (C-1A), 97.4 (C-1B), 95.1 (C-1C), 80.6 (C-3A)
80.5 (C-4B), 80.0 (C-2C), 79.1 (C-3B), 78.4 (C-5C), 74.9 (PhCH2), 73.7 (C-
4A), 73.5 (PhCH2), 73.3 (C-4C), 71.8 (PhCH2), 71.4 (PhCH2), 70.8 (C-2B),
69.8 (C-6aA), 69.3 (C-6abC), 69.2 (–OCH2), 68.6 (C-5B), 66.6 (C-5A), 67.8
(C-2B), 66.7 (2 C, C-5A, NHCOOCH2Ph), 56.6 (C-2A), 40.8 (NCH2-), 17.4

12
V. N. MISHRA AND P. K. MANDAL
þ
(CH3); HRMS (ESI-TOF) calcd for C78H84N3O18 [M þ NH4]þ 1350.5744,
found 1350.5734.
2-(Benzyloxycarbonylamino)ethyl (2-O-acetyl-4-O-benzyl-a-L-
rhamnopyranosyl)-(1fi4)- (2,3,6-tri-O-benzyl-a-D-glucopyranosyl)-(1fi2)-(3,4-
di-O-benzyl-a-L-rhamnopyranosyl)-(1fi3)-4,6-O-benzylidene-2-deoxy-2-N-
phthalimido-b-D-glucopyranoside (2)
Flame-activated molecular sieves (4Å; 1.5 g) were added to a solution of com-
pound 8 (3 g, 2.25mmol) and compound 6 (1.24 g, 2.70 mmol) in CH2Cl2/
Et2O (2:1 v/v; 50mL), and the reaction mixture was stirred at room tempera-
ture under N2 for 1 h. The reaction mixture was cooled to ꢃ25 ꢁC, and NIS
(0.73 g, 3.23 mmol) and H2SO4-SiO2 (30 mg) were added. The reaction mixture
was stirred at the same temperature for 30min, then the temperature was
raised to 0 ꢁC, and it was stirred at 0 ꢁC for another 30 min. The reaction mix-
ture was diluted with CH2Cl2 (100 mL), and the organic layer was successively
washed with Na2S2O3 (5%), NaHCO3 (satd. aq.), and water, then it was dried
(Na2SO4) and concentrated. The crude product was purified over SiO2 using
hexane/EtOAc (3:1) as eluent to give pure compound 2 (2.9 g, 77%) as a color-
25
less oil; [a]D þ24 (c 1.0, CHCl3); IR (neat): 3395, 3020, 1392, 1215, 1044, 761,
669 cmꢃ1; 1H NMR (400 MHz, CDCl3): d 7.51–7.49 (m, 2 H, Ar-H), 7.36–7.21
(m, 37 H, Ar-H), 7.18–7.14 (m, 5 H, Ar-H), 5.54 (s, 1 H, PhCH), 5.27 (d,
J ¼ 8.52 Hz, 1 H, H-1A), 5.01–4.89 (m, 4 H, NH, COOCH2Ph, H-4A), 4.84-4.80
(m, 4 H, PhCH2, H-2D), 4.79 (brs, 1 H, H-1D), 4.72-4.51 (m, 6 H, PhCH2, H-
1B), 4.48–4.34 (m, 4 H, PhCH2, H-5D), 4.26 (dd, J ¼ 8.7, 10.0 Hz, 1 H, H-2A),
4.06–4.00 (m, 2 H, PhCH2, H-5B), 3.95–3.88 (m, 4 H, H-1C, H-2B, H-3D, H-3B),
3.80–3.76 (m, 5 H, OCH2, H-4C, H-5C, H-3C), 3.67–3.60 (m, 4 H, H-5A, H-6abA
,
H-2C), 3.46–3.41 (m, 1 H, H-4B), 3.27–3.18 (m, 5 H, NCH2, H-3A, H-6abC),
3.10–3.06 (m, 1 H, H-4D), 2.04 (s, 3 H, COCH3), 0.91 (d, J ¼ 6.2 Hz, 3 H, CH3),
0.89 (d, J ¼ 6.2 Hz, 3 H, CH3); 13C NMR (100 MHz, CDCl3): d 170.6 (COCH3),
167.9, 167.7 (COPhth), 156.2–126.3 (Ar-C), 101.8 (PhCH), 98.0 (C-1A), 97.0
(C-1D), 96.9 (C-1B), 93.6 (C-1C), 82.1 (C-3A), 80.5 (C-4D), 80.1 (2C, C-4B, C-
5C), 78.9 (C-2C), 78.4 (C-3D), 75.0 (2 C, C-3B, PhCH2), 74.4 (C-3C), 73.7
(PhCH2), 72.9 (PhCH2), 72.8 (2 C, PhCH2), 72.2 (2 C, C-2D, C-4C), 72.1 (2C,
C-4A, PhCH2), 71.5 (C-5D), 69.8 (C-5B), 69.2 (C-6A), 68.6 (C-2B), 68.1
(-OCH2), 67.7 (C-6aBC), 66.6 (2 C, C-5A, NHCOOCH2Ph), 56.6 þ(C-2A), 40.8
(NCH2-), 17.6 (CH3); HRMS (ESI-TOF) calcd for C93H99N2O23 [Mþ H]þ
1611.6633, found 1611.6628.

JOURNAL OF CARBOHYDRATE CHEMISTRY
13
2-(Benzyloxycarbonylamino)ethyl (2-O-acetyl-4-O-benzyl-a-L-
rhamnopyranosyl)-(1fi4)-(2,3,6-tri-O-benzyl-a-D-glucopyranosyl)-(1fi2)-(3,4-
di-O-benzyl-a-L-rhamnopyranosyl)-(1fi3)-4,6-O-benzylidene-2-deoxy-2-N-
phthalimido-b-D-glucopyranoside (2). (One-Pot procedure)
A mixture of 3 (300 mg, 0.52 mmol), 4 (232.0 mg, 0.57 mmol), and flame-
activated 4 Å molecular sieves were stirred anhydrous CH2Cl2/Et2O (2:1 v/
v; 20 mL) for 1 h at room temperature under N2 atmosphere. The reaction
mixture was cooled to ꢃ30 ꢁC, and NIS (142 mg, 0.63 mmol) and H2SO4-
SiO2 (100 mg) were added in succession. After 30 min, the total consump-
tion of both starting materials was observed (checked by TLC). Then the
reaction mixture was slowly raised to 0 ꢁC, and the mixture was stirred for
30 min. The temperature of the reaction mixture was again lowered to
ꢃ30 ꢁC, and the donor 5 (369 mg, 0.60 mmol) and NIS (148 mg, 0.66 mmol)
were added to the same vessel. The reaction mixture was stirred at the
same temperature for 30 min. After full consumption of starting materials
(checked by TLC), the reaction mixture was slowly raised to 0 ꢁC, and the
mixture was stirred for 30 min. Then the reaction mixture was slowly
brought to ꢃ30 ꢁC and thioglycoside donor 6 (164 mg, 0.36 mmol) in dry
CH2Cl2/Et2O (5 mL) and NIS (88 mg, 0.39 mmol) was added. The reaction
was completed within 30 min (indicated by TLC). After that, the reaction
mixture was slowly raised to 0 ꢁC, and the mixture was stirred for 30 min.
After full consumption of starting materials (checked by TLC), the reaction
mixture was quenched by Et3N and then filtered through a celite bed fol-
lowed by washing with CH2Cl2 (50 mL). The combined organic layer was
successively washed with 5% Na2S2O3, satd. NaHCO3, water, dried
(Na2SO4), and concentrated. The crude product was purified over SiO2
using hexane-EtOAc (3:1) as eluant to give pure compound 2 (293 mg,
55%). Colorless foam. Spectral data match that of the substrate synthe-
sized previously.
2-Aminoethyl (a-L-rhamnopyranosyl)-(1fi4)-(a-D-glucopyranosyl)-(1fi2)-(a-L-
rhamnopyranosyl)-(1fi3)-2-acetamido-2-deoxy-b-D-glucopyranoside (1)
To a solution of compound 2 (200 mg, 0.12 mmol) in EtOH (20 mL) was
added hydrazine monohydrate (200 ll) and the reaction mixture was
allowed to stir at 80 ꢁC for 6 h. The solvents were removed under reduced
pressure and a solution of the crude product in acetic anhydride-pyridine
(5 mL; 1:1 v/v) was allowed to stir at room temperature for 2 h. The sol-
vents were removed under reduced pressure and the crude product in
0.1 M sodium methoxide (10 mL) was allowed to stir at room temperature
for 2 h and neutralized with Dowex 50 W X-8 (Hþ). The reaction mixture
was filtered and passed through a short pad of SiO2. To a solution of

14
V. N. MISHRA AND P. K. MANDAL
purified compound in methanol (20 mL) was added 20% Pd(OH)2/C
(200 mg) and the reaction mixture was allowed to stir at room temperature
under a positive pressure of hydrogen for 24 h. The reaction mixture was
R
V
filtered through a Celite bed and evaporated to dryness. The solution of
the crude mass in methanol was passed through a column of Dowex 50 W-
X8 (Naþ) and evaporated to give tetrasaccharide 1 (51 mg, 56%) as a white
powder, which was further purified by passing through a column of
25
Sephadex-LH 20 using CH3OH–H2O (4:1) as eluant. [a]D ꢃ16 (c 1.0,
H2O); IR (KBr): 3419, 2910, 1302, 1142, 988, 667 cmꢃ1; 1H NMR
(400 MHz, CDCl3): d 5.10 (d, J ¼ 7.7 Hz, 1 H, H-1A), 4.92 (brs, 1 H, H-1B),
4.56 (brs, 2 H, H-1D, H-1C), 4.22–4.08 (m, 3 H, H-5D, H-5B, H-5C).
3.94–3.21 (m, 17 H, H-2D, H-2B, H-3D, H-3B, H-3C, H-6abC, OCH2, H-3A,
H-2A, H-4C, H-6abA, H-4D, H-2C, H-4A), 2.88–2.84 (m, 2 H, H-4B, H-5A),
2.25–2.22 (m, 2 H, NCH2), 2.11 (s, 3 H, COCH3), 1.28 (s, 6 H, CH3); 13C
NMR (100 MHz, CDCl3): d 175.0 (COCH3), 102.8 (C-1A), 101.9 (C-1D),
100.4 (C-1B), 98.4 (C-1C), 78.2 (C-3A), 77.2 (C-4C), 75.7 (C-2B), 75.4 (C-
3D), 75.0 (C-5A), 74.3 (C-2D), 73.4 (C-4B), 72.5 (C-4D), 71.6 (C-2C), 71.1
(C-3B), 70.9 (C-3C), 70.4 (C-5B), 69.2 (C-5D), 68.6 (C-5C), 67.1 (OCH2),
65.8 (C-4A), 61.1 (C-6A), 60.1 (C-6C), 54.0 (C-2A), 40.5 (NCH2), 22.0
(COCH3), 1þ8.1 (CH3), 17.9 (CH3).; HRMS (ESI-TOF) calcd for
C28H51N2O19 [M þ H]þ 719.3081, found 719.3087.
Funding
V. M. thanks CSIR, New Delhi for providing Senior Research Fellowship. The author grate-
fully acknowledges financial support by DST-Science and Engineering Research Board
[Scheme No. EMR/2017/001791], New Delhi, India and SAIF Division of CSIR-CDRI for
providing the spectroscopic and analytical data. CDRI communication no. 10245.
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