Total synthesis of a unique tetrasaccharide present in the human clotting factor IX and mammalian Notch 1 receptor

Article abstract of DOI:10.1016/j.tetasy.2009.02.003

The synthesis of a unique tetrasaccharide linked to the serine 61 of human clotting factor IX through an α-l-fucose residue has been achieved for the first time in excellent yield. All glycosylation and protecting group manipulation steps are high yieldin

Full text of DOI:10.1016/j.tetasy.2009.02.003

Tetrahedron: Asymmetry 20 (2009) 473–477
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Total synthesis of a unique tetrasaccharide present in the human clotting factor
IX and mammalian Notch 1 receptor q
*
Chinmoy Mukherjee, Anup Kumar Misra
Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII-M, Kolkata 700054, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a unique tetrasaccharide linked to the serine 61 of human clotting factor IX through an
a-L-fucose residue has been achieved for the first time in excellent yield. All glycosylation and protecting
group manipulation steps are high yielding and reproducible for a scale-up preparation. A sequential gly-
cosylation strategy has been used to assemble suitably protected monosaccharide synthons for the prep-
aration of the target tetrasaccharide.
Received 7 January 2009
Accepted 3 February 2009
Available online 9 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
role in a wide variety of developmental processes including somite
formation, neurogenesis, angiogenesis, and lymphoid develop-
Most of the proteins found in Nature are glycosylated and con-
tain a variety of O- and N-linked oligosaccharides attached to
them.1 These oligosaccharide structures have significant roles in
the physiochemical and biological functions of a particular glyco-
protein.2 Human clotting factor IX (Factor IX, Christmas factor), a
vitamin K-dependent plasma glycoprotein, plays an important role
in the blood coagulation process.3 The disease called hemophilia B
is the result of the deficiency of factor IX.4 Currently, hemophilia B
is being treated with the human plasma-derived factor IX. As an
alternative, a recombinant factor IX has been produced in milk to
avoid the potential risks associated with human blood-derived
products.5 It is a serine protease zymogen essential for the normal
hemostasis.6 In addition to the serine protease domain, human
ment.11 Irregularity in the Notch signaling due to modification of
its EGF domains resulted in T-cell leukemia, cerebral autosomal
arteriopathy, and leukoencephalopathy.12 Recently, Moloney et al.
reported the structure of an a-L-fucose containing tetrasaccharide,
O-glycosidically linked to the Notch 1 protein, which is identical to
the tetrasaccharide present in the human factor IX reported earlier
(Fig. 1).13
HO
OH
HO
O
OH
OH
COOH
HO
O
O
O
HO
O
HO
O
O
OSE
AcHN
NHAc
OH
HO
OH
1
clotting factor contains
c-carboxyglutamic acid domain and two
Figure 1. Structure of the targeted tetrasaccharide as its 2-(trimethylsilyl) ethyl
glycoside 1.
epidermal growth factor (EGF) domains, which often mediate pro-
tein–protein interactions.7 Due to the direct O-glycosyl linkage of
the
post-translational modifications can be found in the EGF modules.
Several glycoproteins exist in which the -fucose moiety is directly
O-glycosidically linked to serine/threonine as part of the monosac-
charide or oligosaccharide structures.8,9 The structure of a unique
tetrasaccharide O-glycosidically linked to the serine 61 of human
L-fucose moiety with serine/threonine residue, several unusual
Preparation of carbohydrate-derived molecules for their use in
therapeutics is a thrust area in medicinal chemistry research. As
mentioned earlier, treatment of hemophilia could be possible using
recombinant human factor IX produced in milk.5 However, for the
successful clinical use of the recombinant factor IX, it is essential to
have a thorough knowledge of the biological properties of the un-
L
clotting factor through an
a-L-fucose residue has been reported
ique tetrasaccharide attached to it through an
a-L-fucose residue.
by Harris et al. and Nishimura et al. separately (Fig. 1).10
In another aspect, O-linked fucose has also been found in the
mammalian Notch 1 cell surface receptor, which plays an essential
Although oligosaccharides can be isolated from natural sources,
they cannot meet the desired quantity required for their biological
studies. Therefore, chemical synthetic strategies are the only op-
tion left to achieve substantial amount of the oligosaccharides
and their analogues. Herein, we report a concise chemical synthe-
sis of the unique tetrasaccharide 1 found in the human clotting fac-
q
This work has been carried out in the Medicinal and Process Chemistry Division,
Central Drug Research Institute, Chattar Manzil Palace, Lucknow 226001, India.
CDRI Communication no. 7517.
* Corresponding author. Tel.: +91 33 2569 3240; fax: +91 33 2355 3886.
E-mail address: akmisra69@rediffmail.com (A.K. Misra).
tor IX as its 2-trimethylsilylethyl glycoside in which
a-L-fucose
present at the reducing end and N-acetyl neuraminic acid at the
non-reducing terminus. 2-(Trimethylsilyl) ethyl group (SE) can
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.02.003

474
C. Mukherjee, A. K. Misra / Tetrahedron: Asymmetry 20 (2009) 473–477
act as a temporary anomeric protecting group for removal when-
ever needed.
cinimide
(NIS)–trimethylsilyl
trifluoromethane
sulfonate
(TMSOTf)18 combination furnished disaccharide derivative 8 in
92% yield. The presence of signals at d 5.46 (d, J = 8.3 Hz, H-1B)
and 4.71 (d, J = 3.6 Hz, H-1A) in the 1H NMR and at d 97.7 (C-1A)
and 96.3 (C-1B) in the 13C NMR spectra confirmed the formation
of compound 8. Controlled deacetylation24 of the compound 8
using dilute sodium methoxide gave the disaccharide acceptor 9
2. Results and discussion
In general, L-fucose can be found in the non-reducing position of
the naturally occurring glycoconjugates.14 Although a number of
reports have appeared in the literature for the successful synthesis
in quantitative yield. That the 4-O-acetyl group of the L-fucose moi-
ety remained intact under deacetylation conditions may be due to
the steric crowding around it. Selective glycosylation of disaccha-
ride diol acceptor 9 with thioglycoside donor 625 in the presence
of NIS–TMSOTf afforded the trisaccharide derivative 10 in 92%
yield. The exclusive b-selective glycosylation took place at the 4-
hydroxy position of the disaccharide acceptor 9 due to the pres-
ence of the N-phthalimido group at the C-2 position. Signals at d
5.22 (d, J = 8.4 Hz, H-1B), 4.67 (d, J = 3.6 Hz, H-1A), 4.51 (d,
J = 7.9 Hz, H-1C) in the 1H NMR and at d 101.4 (C-1C), 97.8 (C-1A),
96.2 (C-1B) in the 13C NMR spectra confirmed the formation of
compound 10. Removal of the N-phthalimido group from the tri-
saccharide derivative 10 using ethylenediamine26 followed by
N-acetylation furnished the trisaccharide acceptor 11 in 88% yield.
of oligosaccharides containing an
a-L-fucose moiety, a practical
problem of the glycosylation of L-fucose residue is the formation
of the anomeric mixture.15 Therefore, development of a glycosyla-
tion condition suitable for the stereoselective preparation of
a-
L-fucosides is always welcome.
L
-Fucose moiety present at the
reducing end of the tetrasaccharide 1 in
a
stereochemistry poses
extra challenge for its chemical synthesis. For the preparation of
the tetrasaccharide 1, suitably protected monosaccharide synthons
have been prepared from commercially available reducing sugars
following reported reaction conditions (Fig. 2).
OBn
OSE
O
AcO
O
SEt
O
a-Selective glycosylation of trisaccharide acceptor 11 with sialic
AcO
OBn
acid thioglycoside donor 727 in the presence of NIS–TfOH followed
by conventional acetylation of the reaction mixture furnished tet-
rasaccharide derivative 12 in 42% yield (Scheme 2). Signature sig-
nals at d 5.58–5.51 (m, 1H, H-8D), 5.34 (dd, J = 8.7, 2.4 Hz, H-7D),
4.79 (d, J = 10.0 Hz, H-1B), 4.77 (d, J = 3.8 Hz, H-1A), 4.54 (d,
J = 7.9 Hz, H-1C), 2.58 (dd, J = 12.7, 4.5 Hz, H-3eD) and 1.69 (t,
J = 12.3 Hz, H-3aD) in the 1H NMR and at d 100.6 (C-1A), 97.7
(C-1B), 97.1 (C-1C), 96.9 (C-2D) in the 13C NMR spectra confirmed
the formation of compound 12. Hydrogenolysis of the tetrasaccha-
ride derivative 12 over 20% Pd(OH)2–C28 followed by saponifica-
tion using sodium methoxide afforded pure tetrasaccharide 1 as
its sodium salt of 2-(trimethylsilyl) ethyl glycoside in 74% yield.
The presence of signals at d 4.95 (d, J = 3.6 Hz, H-1A), 4.70 (d,
J = 7.0 Hz, H-1B), 4.57 (d, J = 7.8 Hz, H-1C) in the 1H NMR and at d
103.5 (C-1C), 100.0 (C-2D), 99.7 (C-1B), 98.5 (C-1A) in the 13C NMR
spectrum confirmed the presence of the required glycosyl linkages
in the target tetrasaccharide 1. Structures of all the intermediates
and final tetrasaccharide 1 have been unambiguously established
with the help of 1D and 2D NMR spectral analysis.
N
HO
AcO
O
4
5
SE: 2-(Trimethylsilyl)ethyl
OBn
AcO
AcO
OAc
COOMe
SPh
O
AcO
AcHN
O
SPh
AcO
OAc
OAc
7
6
Figure 2. Suitably protected monosaccharide intermediates for the synthesis of
tetrasaccharide 1.
The synthesis of 2-(trimethylsilyl) ethyl 4-O-acetyl-2-O-benzyl-
a-L
-fucopyranoside 4 was accomplished from compound 216 in
three steps. The
a-selective glycosylation of the thioglycoside 2
with 2-(trimethylsilyl) ethanol using methyltrifluoromethane sul-
fonate (MeOTf) as a thioglycoside activator17 in dry ether furnished
compound 3 in 70% yield together with its b-isomer (10%). Re-
moval of the isopropylidene group from compound 3 using 80%
aq AcOH followed by selective acetylation via orthoesterification22
using triethyl orthoacetate and p-toluenesulfonic acid furnished
compound 4 in 88% yield (Scheme 1). It is worth noting that a
number of methods have been attempted for the preparation of
compound 3, which include: (a) the use of compound 2 and phe-
nylthioglycoside as a glycosyl donor and NIS/TMSOTf18 or DMTST19
or CuBr2–AgOTf–TBAB20 as an activator; (b) the use of fucosyl tri-
chloroacetimidate derivative instead of thioglycoside with TMSOTf
3. Conclusion
In conclusion, a unique tetrasaccharide 1 present in the human
clotting factor IX and mammalian Notch 1 has been synthesized for
the first time in a concise manner. In the synthetic strategy, the
a-
L-fucose moiety present at the reducing end has been prepared by
tuning the reaction condition after a series of experimentation.
Condensation of N-acetyl neuraminic acid derivative (sialic acid)
at the non-reducing terminus was achieved in moderate yield.
The target compound was achieved with the minimum number
of steps using regioselective glycosylation and functional group
manipulation. All intermediate steps are high yielding, reproduc-
ible, and can be used for a scale-up preparation of the target
compound.
as activator;21 and (c) using a 3,4-di-O-acetyl-
L-fucose thioglycosyl
donor instead of a 3,4-O-isopropylidine-fucose thioglycosyl donor
and the same set of activators used earlier. Unfortunately, none
of them gave satisfactory
a-selectivity in the glycosylation.
OSE
OSE
SEt
OBn
O
a
O
O
b, c
OBn
OBn
O
O
HO
AcO
O
O
4. Experimental
4
3
2
SE: 2-(Trimethylsilyl)ethyl
4.1. General methods
Scheme 1. Reagents and conditions: (a) 2-(trimethylsilyl) ethanol, MeOTf, Et2O,
0 °C, 6 h, 70%; (b) 80% aq AcOH, 80 °C, 1 h; (c) (i) triethyl orthoacetate, p-TsOH, DMF,
rt, 2 h; (ii) 80% aq AcOH, rt, 1 h, 88% over two steps.
All the reactions were monitored by thin layer chromatography
over silica gel-coated TLC plates. The spots on TLC were visualized
by warming ceric sulphate (2% Ce(SO4)2 in 2 N H2SO4) sprayed
plates in hot plate. Silica gel 230–400 mesh was used for column
Coupling of compound 4 with thioglycoside donor 5,23 prepared
from D-glucosamine hydrochloride, in the presence of a N-iodosuc-

C. Mukherjee, A. K. Misra / Tetrahedron: Asymmetry 20 (2009) 473–477
475
OBn
J = 12.6 Hz, 1H, PhCH2), 4.72 (d, J = 3.5 Hz, 1H, H-1), 4.67 (d,
J = 12.6 Hz, 1H, PhCH2), 4.29 (dd, J = 7.9, 5.5 Hz, 1H, H-3), 4.11–
4.04 (m, 1H, H-5), 4.00 (dd, J = 5.5, 2.5 Hz, 1H, H-4), 3.76–3.67
(m, 1H, OCH2a), 3.47 (dd, J = 8.0, 3.5 Hz, 1H, H-2), 3.49–3.39 (m,
1H, OCH2b), 1.38, 1.32 (2s, 6H, C(CH3)2), 1.26 (d, J = 6.7 Hz, 3H,
CCH3), 1.07–0.83 (m, 2H, SiCH2), 0.00 (s, 9H, Si(CH3)3); 13C NMR
O
AcO
O
RO
RO
O
O
OSE
a
N
4 + 5
O
OBn
8: R = Ac
9: R = H
(CDCl3, 75 MHz):
d 138.4–127.5 (aromatic carbons), 108.6
b
(C(CH3)2), 96.4 (C-1), 76.3 (C-2), 76.2 (C-4), 75.9 (C-3), 72.1
(PhCH2), 65.3 (OCH2), 62.9 (C-5), 28.1 (CCH3), 26.3 (CCH3), 17.8
(SiCH2), 16.2 (CCH3), ꢂ1.5 (3C, Si(CH3)3); ESI-MS: m/z = 417.8
[M+Na]+. Anal. Calcd for C21H34O5Si (394.22): C, 63.92; H, 8.69.
Found: C, 63.70; H, 8.95.
6, a
OBn
AcO
OBn
O
AcO
O
N
O
AcO
O
O
O
HO
OAc
OSE
4.1.2. 2-(Trimethylsilyl) ethyl 4-O-acetyl-2-O-benzyl-a-L-
fucopyranoside 4
O
OBn
10
A solution of compound 3 (820 mg, 2.08 mmol) in 80% aq AcOH
(15 mL) was stirred at 80 °C for 1 h. The reaction mixture was con-
centrated under reduced pressure and co-evaporated with toluene
(3 ꢀ 20 mL) to give the crude diol. To the solution of the diol deriv-
ative (730 mg) in dry DMF (5 mL) were added triethyl orthoacetate
c
OBn
HO
OBn
O
HO
O
O
HO
HO
O
O
OH
OSE
(750 lL) and p-TsOH (50 mg), and the reaction mixture was al-
NHAc
lowed to stir at room temperature for 2 h. After complete con-
sumption of the starting material, the reaction mixture was
neutralized with triethylamine (0.5 mL), and solvents were re-
moved under reduced pressure. A solution of the crude mass in
80% aq AcOH (10 mL) was allowed to stir at room temperature
for 1 h. The reaction mixture was concentrated under reduced
pressure and co-evaporated with toluene (3 ꢀ 20 mL) to give the
crude product, which was purified over SiO2 using hexane–EtOAc
(3:1) as eluant to give pure compound 4 (720 mg, 88%) as syrup;
OBn
11
d
7
AcO
O
OBn
AcO
OAc
OBn
MeOOC
O
AcO
O
O
AcO
AcO
AcHN
O
O
O
OAc
OSE
NHAc
OAc
12
OBn
½
a 2D5
ꢁ
¼ ꢂ53 (c 1.0, CHCl3); IR (neat): 2924, 2360, 1736, 1246,
1219, 1096, 1038, 839, 762, 670 cmꢂ1 1H NMR (CDCl3,
300 MHz): 7.34–7.23 (m, 5H, aromatic protons), 5.17 (d,
SE: 2-(Trimethylsilyl) ethyl
e
;
d
1
J = 2.9 Hz, 1H, H-4), 4.75 (d, J = 3.5 Hz, 1H, H-1), 4.67 (d,
J = 11.9 Hz, 1H, PhCH2), 4.59 (d, J = 11.9 Hz, 1H, PhCH2), 4.09 (dd,
J = 10.0, 3.6 Hz, 1H, H-3), 4.01–3.94 (m, 1H, H-5), 3.72–3.64 (m,
1H, OCH2a), 3.62 (dd, J = 9.9, 3.5 Hz, 1H, H-2), 3.41–3.33 (m, 1H,
OCH2b), 2.12 (s, 3H, COCH3), 1.06 (d, J = 6.6 Hz, 3H, CCH3), 0.97–
0.84 (m, 2H, SiCH2), 0.00 (s, 9H, Si(CH3)3); 13C NMR (CDCl3,
75 MHz): d 170.8 (COCH3), 138.3–127.7 (aromatic carbons), 96.3
(C-1), 76.9 (C-2), 73.1 (C-4), 72.7 (PhCH2), 67.9 (C-3), 65.1
(OCH2), 64.6 (C-5), 20.8 (COCH3), 18.0 (SiCH2), 16.1 (CCH3), ꢂ1.3
(3C, Si(CH3)3); ESI-MS: m/z = 414.0 [M+NH4þ]+. Anal. Calcd for
C20H32O6Si (396.20): C, 60.58; H, 8.13. Found: C, 60.35; H, 8.37.
Scheme 2. Reagents and conditions: (a) NIS, TMSOTf, MS 4 Å, CH2Cl2, ꢂ40 °C,
45 min, 92% for 8, 92% for 10; (b) CH3ONa, CH3OH, rt, 2 h, quantitative; (c) (i)
ethylenediamine, n-butanol, 80 °C, 6 h; (ii) acetic anhydride, pyridine, rt, 12 h; (iii)
CH3ONa, CH3OH, rt, 6 h, 88% in three steps; (d) (i) NIS, TfOH, CH3CN, MS 3 Å, ꢂ30 to
ꢂ20 °C, 24 h, 42%; (ii) acetic anhydride, pyridine, rt, 8 h; (e) (i) H2, 20% Pd(OH)2–C,
CH3OH, rt, 24 h; (ii) CH3ONa, CH3OH, rt, 8 h then few drops of H2O, 5 h, 74%.
chromatography. 1H and 13C NMR, 2D COSY, HSQC, and NOESY
spectra were recorded on Brucker Advance DPX 300 MHz using
CDCl3 and D2O as solvents and TMS as internal reference unless
stated otherwise. Chemical shift value is expressed in d ppm. ESI-
MS were recorded on a MICROMASS QUTTRO II triple quadrupole
mass spectrometer. Elementary analysis was carried out on Carlo
ERBA-1108 analyzer. Optical rotations were measured at 25 °C on
a Rudolf Autopol III polarimeter. Commercially available grades
of organic solvents of adequate purity are used in many reactions.
4.1.3. 2-(Trimethylsilyl) ethyl 3,4-di-O-acetyl-6-O-benzyl-2-deoxy-
2-N-phthalimido-b-
-fucopyranoside 8
To a solution of compound 4 (650 mg, 1.64 mmol) and com-
D-glucopyranosyl-(1?3)-4-O-acetyl-2-O-benzyl-
a-L
pound 5 (1.0 g, 1.90 mmol) in anhydrous CH2Cl2 (15 mL) was
added freshly activated powdered MS 4 Å (1.0 g). The reaction mix-
ture was allowed to stir under argon at room temperature for 1 h.
After cooling the reaction mixture to ꢂ40 °C, N-iodosuccinimide
(NIS; 530 mg, 2.36 mmol) was added to it followed by TMSOTf
4.1.1. 2-(Trimethylsilyl) ethyl 2-O-benzyl-3,4-O-isopropylidine-
a-
L
-fucopyranoside 3
To a solution of 2 (1.3 g, 3.84 mmol), 2-(trimethylsilyl) ethanol
(600 L, 4.2 mmol), and freshly activated powdered MS 4 Å (1.5 g)
in anhydrous Et2O (60 mL) was added MeOTf (870 L, 7.7 mmol) at
(5
45 min. The reaction mixture was quenched by the addition of tri-
ethylamine (10
L), filtered through a CeliteÒ bed, and washed
lL), and the reaction mixture was allowed to stir at ꢂ40 °C for
l
l
l
0 °C, and the reaction mixture was allowed to stir at 0 °C for 6 h.
The reaction mixture was quenched by the addition of Et3N
(1 mL) and diluted with CH2Cl2 (40 mL). The organic layer was
washed with water (2 ꢀ 40 mL), dried (Na2SO4), and concentrated
under reduced pressure. The crude product was purified over SiO2
using hexane–EtOAc (19:1) as eluant to give compound 3 (1.05 g,
with CH2Cl2 (3 ꢀ 25 mL). The organic layer was washed succes-
sively with 10% aq Na2S2O3 and water, dried over Na2SO4, and con-
centrated under reduced pressure. The crude product was purified
over SiO2 using hexane–EtOAc (7:2) as eluant to afford pure com-
pound 8 (1.3 g, 92%) as a white solid; mp 129–30 °C; ½a D25
¼ ꢂ37 (c
ꢁ
1.0, CHCl3); IR (KBr): 2950, 2895, 1748, 1718, 1386, 1238, 1054,
70%); ½a 2D5
ꢁ
¼ ꢂ78 (c 1.0, CHCl3); IR (neat): 2361, 1454, 1379,
1H NMR (CDCl3,
7.36–7.22 (m, 5H, aromatic protons), 4.77 (d,
861, 836, 745, 722, 697 cmꢂ1 1H NMR (CDCl3, 300 MHz): d 7.88–
;
1247, 1216, 1080, 863, 838, 760, 698, 668 cmꢂ1
;
7.86 (m, 2H, aromatic protons), 7.76–7.72 (m, 2H, aromatic pro-
tons), 7.40–7.23 (m, 10H, aromatic protons), 5.83 (dd, J = 10.4,
300 MHz):
d

476
C. Mukherjee, A. K. Misra / Tetrahedron: Asymmetry 20 (2009) 473–477
10.4 Hz, 1H, H-3B), 5.46 (d, J = 8.3 Hz, 1H, H-1B), 5.16 (t, J = 9.6 Hz,
1H, H-4B), 5.03 (d, J = 2.6 Hz, 1H, H-4A), 4.88 (d, J = 12.4 Hz, 1H,
PhCH2), 4.71 (d, J = 3.6 Hz, 1H, H-1A), 4.62 (d, J = 12.4 Hz, 1H,
PhCH2), 4.51 (br s, 2H, PhCH2), 4.38–4.28 (m, 2H, H-3A, H-2B),
1H, H-3C), 4.88 (d, J = 12.5 Hz, 1H, PhCH2), 4.67 (d, J = 3.6 Hz, 1H,
H-1A), 4.65–4.48 (m, 4H, H-3B, PhCH2), 4.51 (d, J = 7.9 Hz, 1H, H-
1C), 4.45–4.25 (m, 3H, H-3A, PhCH2), 4.20 (dd, J = 10.7, 8.4 Hz, 1H,
H-2B), 3.95–3.87 (m, 1H, H-5A), 3.84–3.57 (m, 7H, H-2A, H-4B, H-
5B, H-6abB, H-5C, OCH2a), 3.51–3.42 (m, 2H, H-6aC, OCH2b), 3.38–
3.32 (m, 1H, H-6bC), 2.02, 1.98, 1.95, 1.43 (4s, 12H, 4 COCH3),
0.92 (d, J = 6.4 Hz, 3H, CCH3), 1.05–0.80 (m, 2H, SiCH2), 0.00 (br s,
9H, Si(CH3)3); 13C NMR (CDCl3, 75 MHz): d 170.0, 169.9, 169.7,
169.1 (4 COCH3), 167.8 (2C, 2COPhth), 139.2–123.1 (aromatic car-
bons), 101.4 (C-1C), 97.8 (C-1A), 96.2 (C-1B), 81.7 (C-2A), 74.9 (C-5C),
74.4 (C-3A), 73.7 (C-5B), 73.6 (PhCH2), 73.5 (PhCH2), 73.4 (PhCH2),
72.2 (C-4B), 71.0 (C-3C), 70.9 (C-4A), 69.3 (C-3B), 68.9 (C-2C), 67.5
(C-6C), 67.4 (C-6B), 67.2 (C-4C), 65.4 (OCH2), 64.5 (C-5A), 56.4 (C-
2B), 20.7, 20.6, 20.5, 19.7 (4C, 4COCH3), 17.9 (SiCH2), 16.0 (CCH3),
ꢂ1.3 (3C, Si(CH3)3); ESI-MS: m/z = 1178.6 [M+Na]+. Anal. Calcd for
C60H73NO20Si (1155.45): C, 62.32; H, 6.36. Found: C, 62.10; H, 6.60.
3.97–3.81 (m, 2H, H-5A, H-5B), 3.74–3.41 (m, 5H, H-2A, H-6abB
,
OCH2ab), 1.91, 1.85, 1.53 (3s, 9H, 3 COCH3), 0.94 (d, J = 6.6 Hz, 3H,
CCH3), 1.02–0.79 (m, 2H, SiCH2), 0.00 (s, 9H, Si(CH3)3); 13C NMR
(CDCl3, 75 MHz): d 169.9 (2 C, COCH3), 169.4 (COCH3), 167.6 (2
COPhth), 139.1–123.3 (aromatic carbons), 97.7 (C-1A), 96.3 (C-1B),
75.5 (C-3A), 74.0 (C-2A), 73.6 (2 C, 2 PhCH2), 73.2 (C-5B), 71.1 (C-
4A), 70.9 (C-3B), 70.1 (C-4B), 70.0 (C-6B), 65.5 (OCH2), 64.5 (C-5A),
55.1 (C-2B), 20.6, 20.4, 19.8 (3C, 3 COCH3), 17.9 (SiCH2), 15.9
(CCH3), ꢂ1.3 (3C, Si(CH3)3); ESI-MS: m/z = 880.2 [M+NH4]+. Anal.
Calcd for C45H55NO14Si (861.34): C, 62.70; H, 6.43. Found: C,
62.52; H, 6.68.
4.1.4. 2-(Trimethylsilyl) ethyl 6-O-benzyl-2-deoxy-2-N-phtha-
limido-b-
fucopyranoside 9
D
-glucopyranosyl-(1?3)-4-O-acetyl-2-O-benzyl-
a
-
L
-
4.1.6. 2-(Trimethylsilyl) ethyl 6-O-benzyl-b-
(1?4)-2-acetamido-6-O-benzyl-2-deoxy-b- -glucopyranosyl-
(1?3)-2-O-benzyl- -fucopyranoside 11
D-galactopyranosyl-
D
A solution of 8 (1.0 g, 1.16 mmol) in 0.1 M CH3ONa in CH3OH
(25 mL) was allowed to stir at room temperature for 2 h and neu-
tralized with Dowex-50W X8 (H+). The reaction mixture was fil-
tered and evaporated to dryness to give compound 9 (900 mg,
a-L
To a solution of compound 10 (760 mg, 0.66 mmol) in n-butanol
(15 mL) was added ethylene diamine (1 mL), and the reaction mix-
ture was allowed to stir at 80 °C for 6 h. The solvents were evapo-
rated off under reduced pressure and co-evaporated with toluene
(3 ꢀ 10 mL). A solution of the crude product in Ac2O and pyridine
(5 mL, 1:1, v/v) was stirred at room temperature for 12 h. The sol-
vents were removed under reduced pressure and co-evaporated
with toluene (3 ꢀ 10 mL). A solution of the acetylated product in
0.1 M CH3ONa in CH3OH (10 mL) was allowed to stir at room tem-
perature for 6 h and neutralized with Dowex 50W-X8 (H+) and fil-
tered. The reaction mixture was concentrated under reduced
pressure to give the crude product, which was purified over SiO2
using EtOAc–methanol (19:1) as eluant to give pure compound
quantitative) as a white solid; mp 58–60 °C; ½a D25
¼ ꢂ70 (c 1.0,
ꢁ
CHCl3); IR (KBr): 2361, 1714, 1389, 1216, 1077, 761, 670 cmꢂ1
;
1H NMR (CDCl3, 300 MHz): d 7.86 (dd, J = 5.5, 3.1 Hz, 2H, aromatic
protons), 7.71 (dd, J = 5.4, 3.1 Hz, 2H, aromatic protons), 7.37–7.19
(m, 10H, aromatic protons), 5.26 (d, J = 8.2 Hz, 1H, H-1B), 5.04 (d,
J = 2.6 Hz, 1H, H-4A), 4.86 (d, J = 12.4 Hz, 1H, PhCH2), 4.68 (d,
J = 3.6 Hz, 1H, H-1A), 4.60 (d, J = 12.5 Hz, 1H, PhCH2), 4.56 (br s,
2H, PhCH2), 4.43 (dd, J = 10.5, 7.9 Hz, 1H, H-3B), 4.32 (dd, J = 10.1,
3.3 Hz, 1H, H-3A), 4.13 (dd, J = 10.8, 8.3 Hz, 1H, H-2B), 3.91 (dd,
J = 12.9, 6.3 Hz, 1H, H-5A), 3.79–3.53 (m, 5H, H-2A, H-4B, H-6abB
,
OCH2a), 3.52–3.42 (m, 2H, H-5B, OCH2b), 1.46 (s, 3H, COCH3),
1.04–0.81 (m, 2H, SiCH2), 0.92 (d, J = 6.4 Hz, 3H, CCH3), 0.00 (s,
9H, Si(CH3)3); 13C NMR (CDCl3, 75 MHz): d 170.1 (COCH3), 168.2
(2C, 2COPhth), 139.2–123.3 (aromatic carbons), 97.7 (C-1A), 96.0
(C-1B), 74.5 (C-3A), 73.9 (C-2A), 73.8 (C-4B), 73.7 (2 C, C-5B, PhCH2),
73.4 (PhCH2), 71.0 (2C, C-4A, C-3B), 70.3 (C-6B), 65.4 (OCH2), 64.5
(C-5A), 56.6 (C-2B), 19.7 (COCH3), 17.8 (SiCH2), 16.0 (CCH3), ꢂ1.3
(3C, Si(CH3)3); ESI-MS: m/z = 796.3 [M+NH4]+. Anal. Calcd for
C41H51NO12Si (777.32): C, 63.30; H, 6.61. Found: C, 63.10; H, 6.86.
11 (520 mg, 88%) as a white solid; mp 136 °C; ½a D25
¼ ꢂ38 (c 1.0,
ꢁ
CHCl3); IR (KBr): 2924, 2360, 1644, 1216, 1057, 766, 670 cmꢂ1
;
1H NMR (CDCl3, 300 MHz): d 7.34–7.19 (m, 15H, aromatic protons),
4.78–4.71 (m, 3H, H-1A, H-1B, PhCH2), 4.59 (d, J = 12.3 Hz, 1H,
PhCH2), 4.55–4.44 (m, 4H, PhCH2), 4.29 (d, J = 7.7 Hz, 1H, H-1C),
4.13 (dd, J = 10.1, 3.3 Hz, 1H, H-3A), 3.98–3.79 (m, 5H, H-2A, H-5A,
H-3B, H-4C, H-6aC), 3.77–3.53 (m, 10H, H-4A, H-2B, H-4B, H-5B, H-
6abB, H-2C, H-3C, H-6bC, OCH2a), 3.49–3.38 (m, 2H, H-5C, OCH2b),
1.83 (s, 3H, COCH3), 1.19 (d, J = 6.4 Hz, 3H, CCH3), 1.04–0.95 (m,
1H, SiCH2a), 0.92–0.82 (m, 1H, SiCH2b), 0.00 (br s, 9H, Si(CH3)3);
13C NMR (CDCl3, 75 MHz): d 172.7 (NHCOCH3), 138.6–127.6 (aro-
matic carbons), 103.0 (C-1C), 98.4 (C-1A), 96.7 (C-1B), 79.1 (C-4B),
77.5 (C-3A), 74.4 (C-3C), 73.8 (C-3B), 73.6 (C-5B), 73.5 (PhCH2),
73.5 (C-5C), 73.4 (PhCH2), 72.7 (PhCH2), 72.4 (C-2A), 70.5 (C-2C),
70.2 (C-4A), 69.3 (C-6C), 69.1 (C-6B), 68.8 (C-4C), 65.5 (C-5A), 65.2
(OCH2), 57.5 (C-2B), 23.2 (NHCOCH3), 18.0 (SiCH2), 16.2 (CCH3),
ꢂ1.5 (3 C, Si(CH3)3); ESI-MS: m/z = 899.9 [M+1]+. Anal. Calcd for
C46H65NO15Si (899.41): C, 61.38; H, 7.28. Found: C, 61.20; H, 7.52.
4.1.5. 2-(Trimethylsilyl)ethyl 2,3,4-tri-O-acetyl-6-O-benzyl-b-
galactopyranosyl-(1?4)-6-O-benzyl-2-deoxy-2-N-phthalimido-b-
glucopyranosyl-(1?3)-4-O-acetyl-2-O-benzyl- -fucopyranoside 10
D-
D
-
a-L
To a solution of compound 9 (720 mg, 0.93 mmol) and com-
pound 6 (540 mg, 1.10 mmol) in anhydrous CH2Cl2 (15 mL) was
added freshly activated powdered MS 4 Å (1.0 g), and the reaction
mixture was allowed to stir under argon at room temperature for
1 h. After cooling the reaction mixture to ꢂ40 °C, NIS (300 mg,
1.33 mmol) was added to it followed by TMSOTf (5 lL), and the
reaction mixture was allowed to stir at ꢂ40 °C for 45 min. The
4.1.7. 2-(Trimethylsilyl) ethyl (methyl 5-acetamido-4,7,8,9-tetra-
reaction mixture was quenched by the addition of triethylamine
O-acetyl-3,5-dideoxy-
lonate)-(2?3)-2,4-di-O-acetyl-6-O-benzyl-b-
(1?4)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-b-
anosyl-(1?3)-4-O-acetyl-2-O-benzyl- -fucopyranoside 12
D
-glycero-
a
-
D
-galacto-2-nonulopyranosy-
-galactopyranosyl-
-glucopyr
(10
l
L), filtered through a CeliteÒ bed, and washed with CH2Cl2
D
(50 mL). The organic layer was washed successively with 10% aq
Na2S2O3 and water, dried over Na2SO4, and concentrated under re-
duced pressure. The crude product was purified over SiO2 using
hexane–EtOAc (3:2) as eluant to afford pure compound 10
D
a-L
To a solution of compound 11 (500 mg, 0.55 mmol) and com-
pound 7 (650 mg, 1.11 mmol) in anhydrous CH3CN (10 mL) was
added freshly activated powdered MS 3 Å (500 mg), and the reac-
tion mixture was allowed to stir under argon at room temperature
for 1 h. After cooling the reaction mixture to ꢂ30 °C, NIS (300 mg,
(980 mg, 92%) as a white solid; mp 150–52 °C; ½a D25
¼ ꢂ37 (c 1.0,
ꢁ
CHCl3); IR (KBr): 3021, 2360, 1745, 1384, 1216, 1086, 761,
670 cmꢂ1 1H NMR (CDCl3, 300 MHz): d 7.86 (dd, J = 5.0, 3.4 Hz,
;
2H, aromatic protons), 7.70 (dd, J = 5.4, 3.0 Hz, 2H, aromatic pro-
tons), 7.37–7.12 (m, 15H, aromatic protons), 5.33 (d, J = 3.1 Hz,
1H, H-4C), 5.22 (d, J = 8.4 Hz, 1H, H-1B), 5.13 (dd, J = 10.4, 8.0 Hz,
1H, H-2C), 5.04 (d, J = 2.7 Hz, 1H, H-4A), 4.91 (dd, J = 10.3, 3.3 Hz,
1.33 mmol) was added to it followed by TfOH (5 lL), and the reac-
tion mixture was allowed to stir at ꢂ20 °C for 24 h. The reaction
mixture was quenched by the addition of triethylamine (10 L), fil-
tered through a CeliteÒ bed, and washed with CH2Cl2 (50 mL). The
l

C. Mukherjee, A. K. Misra / Tetrahedron: Asymmetry 20 (2009) 473–477
477
organic layer was washed successively with 10% aq Na2S2O3 and
water, dried (Na2SO4), and concentrated under reduced pressure.
A solution of the crude product in Ac2O and pyridine (5 mL, 1:1,
v/v) was stirred at room temperature for 8 h. The solvents were re-
moved under reduced pressure and co-evaporated with toluene
(3 ꢀ 15 mL), and the crude product was purified over SiO2 using
CHCl3–CH3OH (49:1) as eluant to afford pure compound 12
(COONa), 103.5 (C-1C), 100.0 (C-2D), 99.7 (C-1B), 98.5 (C-1A), 79.2
(C-4A), 78.1 (C-4C) 76.4 (C-3A), 76.0 (C-4B), 75.7 (C-2C), 74.0 (C-
3C), 73.3 (C-4D), 72.2 (C-2A), 70.3 (C-6D), 70.0 (C-7D), 69.1 (C-5B),
68.8 (C-3B), 68.5 (C-8D), 67.5 (C-5A), 67.1 (2 C, C-5C, OCH2), 63.7
(C-6C), 61.9 (C-9D), 60.9 (C-6B), 56.2 (C-2B), 52.6 (C-5D), 40.1 (C-
3D), 23.2 (NHCOCH3), 23.0 (NHCOCH3), 18.2 (SiCH2), 16.2 (CCH3),
ꢂ1.4 (3C, Si(CH3)3); ESI-MS: m/z = 943.1 [M+1]+. Anal. Calcd for
C36H63N2NaO23Si (942.35): C, 45.85; H, 6.73. Found: C, 45.63; H,
6.98.
(360 g, 42%) as a white solid; mp 94–95 °C; ½a D25
¼ ꢂ64 (c 1.0,
ꢁ
CHCl3); IR (KBr): 3020, 2361, 1740, 1651, 1217, 766, 670 cmꢂ1
;
1H NMR (CDCl3, 300 MHz): d 7.34–7.19 (m, 15H, aromatic protons),
5.69 (d, J = 9.6 Hz, 1H, NHCOCH3D), 5.58–5.51 (m, 1H, H-8D), 5.34
(dd, J = 8.7, 2.4 Hz, 1H, H-7D), 5.21–5.14 (m, 2H, H-4C, NHCOCH3B),
5.07–4.99 (m, 2H, H-4A, H-3B), 4.96–4.77 (m, 3H, H-2C, H-4D,
PhCH2), 4.79 (d, J = 10.0 Hz, 1H, H-1B), 4.77 (d, J = 3.8 Hz, 1H, H-
Acknowledgments
Instrumentation facilities from SAIF, CDRI are gratefully
acknowledged. C.M. thanks CSIR, New Delhi for providing a Senior
Research Fellowship. This work was supported by Ramanna Fellow-
ship (A.K.M.), Department of Science and Technology, New Delhi
(SR/S1/RFPC-06/2006).
1A), 4.54 (d, J = 7.9 Hz, 1H, H-1C), 4.64–4.35 (m, 7H, H-3A, H-9aD
,
PhCH2), 4.17–4.10 (m, 1H, H-5D), 4.08–3.90 (m, 4H, H-5A, H-2B,
H-4B, H-9bD), 3.84 (br s, 3H, COOCH3), 3.81–3.55 (m, 8H, H-2A, H-
5B, H-3C, H-5C, H-6abC, H-6D, OCH2a), 3.52–3.42 (m, 2H, H-6aB
,
OCH2b), 3.37–3.31 (m, 1H, H-6bB), 2.58 (dd, J = 12.7, 4.5 Hz, 1H,
H-3eD), 2.12, 2.11, 2.10, 2.01, 1.99, 1.95, 1.84 (7s, 30H, 10 COCH3),
1.69 (t, J = 12.3 Hz, 1H, H-3aD), 1.10 (d, J = 6.4 Hz, 3H, CCH3),
0.90–0.82 (m, 2H, SiCH2), 0.00 (br s, 9H, Si(CH3)3); 13C NMR (CDCl3,
75 MHz): d 171.7, 170.8, 170.5, 170.3, 170.0, 169.9, 169.8, 169.6
(10 COCH3), 167.8 (COOCH3), 139.0–127.2 (aromatic carbons),
100.6 (C-1A), 97.7 (C-1B), 97.1 (C-1C), 96.9 (C-2D), 75.6 (C-4A),
75.2 (C-3B), 75.1 (C-4B), 73.6 (C-2A), 73.5 (C-4D), 73.4 (PhCH2),
73.3 (PhCH2), 73.2 (PhCH2), 72.1 (C-3C), 71.7 (C-5C), 71.5 (C-6D),
70.6 (C-4C), 70.5 (C-3A), 69.3 (C-2C), 68.0 (2 C, C-5B, C-8D), 67.6
(C-6C), 67.3 (2 C, C-6B, C-7D), 65.5 (OCH2), 64.1 (C-5A), 62.4 (C-
9D), 53.9 (C-5D), 53.1 (COOCH3), 49.1 (C-2B), 37.5 (C-3D), 23.3,
23.1, 21.4, 21.0, 20.9, 20.7, 20.6 (10C, 10COCH3), 17.9 (SiCH2),
16.0 (CCH3), ꢂ1.4 (3 C, Si(CH3)3); ESI-MS: m/z = 1540.9 [M]+. Anal.
Calcd for C74H100N2O31Si (1540.61): C, 57.65; H, 6.54. Found: C,
57.42; H, 6.80.
References
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4.1.8. 2-(Trimethylsilyl) ethyl (sodium 5-acetamido-3,5-dideoxy-
D
-glycero-
pyranosyl-(1?4)-2-acetamido-2-deoxy-b-
-fucopyranoside 12
To the solution of compound 12 (300 mg, 0.19 mmol) in CH3OH
a
-
D
-galacto-2-nonulopyranosylonate)-(2?3)-b-
D-galacto-
D-glucopyranosyl-(1?3)-
a
-L
(10 mL) was added 20% Pd(OH)2/C (100 mg), and the reaction mix-
ture was allowed to stir at room temperature under a positive
pressure of hydrogen for 24 h. The reaction mixture was filtered
through a CeliteÒ bed and evaporated to dryness. A solution of
the crude product in 0.1 M CH3ONa in CH3OH (5 mL) was allowed
to stir at room temperature for 8 h, then few drops of water was
added to the reaction mixture and it was stirred for another 5 h.
The reaction mixture was neutralized with Dowex 50W-X8 (H+),
filtered, and treated with Dowex 50W-X8 (Na+). The reaction mix-
ture was filtered, concentrated, and purified through a column of
Sephadex LH-20 using CH3OH–H2O (4:1) as eluant to give pure 1
(130 mg, 74%) as a white powder; ½a D25
¼ ꢂ49 (c 1.0, H2O); IR
ꢁ
(KBr): 2935, 2362, 2340, 1725, 1641, 1565, 1378, 1316, 1249,
1074, 861, 838, 771, 671 cmꢂ1 1H NMR (CDCl3, 300 MHz): d 4.95
;
(d, J = 3.6 Hz, 1H, H-1A), 4.70 (d, J = 7.0 Hz, 1H, H-1B), 4.57 (d,
J = 7.8 Hz, 1H, H-1C), 4.21–4.15 (m, 1H, H-3A), 4.09–3.97 (m, 4H,
H-5A, H-6aB, H-4C, H-8D), 3.98–3.84 (m, 6H, H-2A, H-6bB, H-5C, H-
6aC, H-5D, H-7D), 3.83–3.71 (m, 9H, H-4A, H-2B, H-3B, H-4B, H-3C,
H-4D, H-9abD, OCH2a), 3.70–3.56 (m, 5H, H-5B, H-2C, H-6bC, H-6D,
OCH2b), 2.78 (dd, J = 12.4, 3.8 Hz, 1H, H-3eD), 2.07 (br s, 6H, 2
NHCOCH3), 1.90 (t, J = 12.0 Hz, 1H, H-3aD), 1.24 (d, J = 6.4 Hz, 3H,
CCH3), 1.10–0.94 (m, 2H, SiCH2), 0.07 (s, 9H, Si(CH3)3); 13C NMR
(CDCl3, 75 MHz): d 175.9 (NHCOCH3), 175.8 (NHCOCH3), 173.5
27. Marra, A.; Sinaÿ, P. Carbohydr. Res. 1989, 187, 35–42.
28. Perlman, W. M. Tetrahedron Lett. 1967, 8, 1663–1664.