Synthesis of Fondaparinux: Modular synthesis investigation for heparin synthesis

Article abstract of DOI:10.1016/j.carres.2013.01.003

The anti-thromboembolic pentasaccharide Fondaparinux was synthesized in 36 steps for the longest linear route, with 0.017% overall yield from d-glucose. Only three kinds of protecting groups were used for hydroxyl protection, Bn, Ac, and Bz, to accomplish this complex synthesis without decreasing the synthetic efficiency. Three l-idopyranosyl donors were investigated. Thioethyl glycoside is an efficient donor for l-idopyranosyl glycosylation with full α-selectivity, while l-idopyranosyl trichloroacetimidate resulted in poor α/β selectivity. A practical synthesis of key intermediate 1,6-anhydro-l-idopyranose 17 by H⁺/β-CD catalyst was developed.

Full text of DOI:10.1016/j.carres.2013.01.003

Carbohydrate Research 371 (2013) 32–39
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis of Fondaparinux: modular synthesis investigation
for heparin synthesis
b
b
Feng Lin a,b, , Gaoyan Lian , Ying Zhou
⇑
a State Key Laboratory of New Drugs & Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 1111 Zhongshanbeiyi Road, Shanghai 200437, China
b State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, CAS, 345 Linlin Road, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The anti-thromboembolic pentasaccharide Fondaparinux was synthesized in 36 steps for the longest lin-
Received 22 November 2012
Received in revised form 4 January 2013
Accepted 6 January 2013
ear route, with 0.017% overall yield from
hydroxyl protection, Bn, Ac, and Bz, to accomplish this complex synthesis without decreasing the syn-
thetic efficiency. Three -idopyranosyl donors were investigated. Thioethyl glycoside is an efficient donor
for -idopyranosyl glycosylation with full -selectivity, while -idopyranosyl trichloroacetimidate
resulted in poor /b selectivity. A practical synthesis of key intermediate 1,6-anhydro- -idopyranose
17 by H+/b-CD catalyst was developed.
D-glucose. Only three kinds of protecting groups were used for
L
Available online 16 January 2013
L
a
L
a
L
Keywords:
Fondaparinux
Modular synthesis
Ó 2013 Elsevier Ltd. All rights reserved.
L-Idopyranosyl donor
1. Introduction
This synthesis of Fondaparinux was investigated as model study
for modular synthesis of heparin/HS. We investigated some strate-
gies and developed an efficient synthesis of GH disaccharide unit,
Heparin and heparin sulfate (HS) are linear polysaccharides
consisting of uronic acid-(1,4)-a-glucosamine repeating disaccha-
using ethylthio–idosyl donor with superior a-stereoselectivity
ride subunits. Variable patterns of substitution (O-sulfate, N-sul-
fate, and N-acetyl) of the disaccharide subunits give rise to a
large number of complex sequences, rendering them the most
complex nature-occurring biopolymers. Heparin and HS play cru-
cial roles in numerous biological systems through their interaction
with diverse proteins.1
than that of corresponding imidate donor; we also applied TEM-
PO–Ca(ClO)2 selective oxidation method9 to introduce uronic acid
structure at disaccharide stage. Our work was reported herein.
2. Results and discussion
Heparin is used in clinic as an anticoagulant drug for over
70 years owing to its high affinity binding with antithrombin III.2
Its synthetic derivative, Fondaparinux (1, trade name: ArixtraÒ) is
the first in a new class of selective factor Xa inhibitors. By selec-
tively binding to antithrombin, it induces a conformational change
that specifically increases by about 340-fold the natural neutraliza-
tion of factor Xa by antithrombin, resulting in dose-dependent
inhibition of factor Xa but no effect on thrombin.3 Unlike unfrac-
tionalized heparin, Fondaparinux does not bind to platelets or
platelet factor 4 and less likely to induce thrombocytopenia.4
The synthetic route of Fondaparinux developed by Sanofi-Aven-
tis compromised more than 50 steps,5 and the annual global sales
of ArixtraÒ were £2.54m in 2009.6 Concerning this complex and
synthetic-challenging molecule, chemical community has devel-
oped some interesting strategies, such as modular synthesis7 and
late-stage oxidation to introduce uronic acid structure.8
Protection strategy plays an important role in heparin-HS syn-
thesis. Though some new protecting groups were introduced for
modular synthesis strategy, we found that the simplest way
worked well and was proved effective in several heparin-HS syn-
theses:3 acyl groups were used for those hydroxyl groups which
will be sulfonated, while benzyl groups for those will not and azido
group was the protecting form for amino group. This simple and
unified design seemed quite limited in applications, but we proved
here that combining sophisticated selective protection–deprotec-
tion techniques, this protection strategy was of great efficiency
and flexible in heparin synthesis, and is valuable in modular syn-
thesis researches.
For the strategy of building blocks, we preferred using disaccha-
rides with glucosamino unit on the reducing end.10 The retro-syn-
thetic analysis is shown in Figure 1. Uronic units were introduced
at the disaccharide level to avoid the glycosylation with the usually
inactive uronate derivatives.
Compounds 511 and 1212 were prepared starting from
D-glu-
cose, and disaccharide 6 was synthesized from cellobiose in 13
⇑
Corresponding author. Tel.: +86 21 65310547; fax: +86 21 65169893.
E-mail address: linfeng@sioc.ac.cn (F. Lin).
steps with 12% yield (Scheme 1).13
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.01.003

F. Lin et al. / Carbohydrate Research 371 (2013) 32–39
33
D
E
F
G
H
-
-
-
-
CO2
OSO3
O
OSO3
O
OSO3
O
O
-O
Fondaparinux (1)
CO2
OH
-
O
O
OSO3
OH
OH
O
OH
O
OMe
NHSO3
OH
-
-
-
-
NHSO3
OSO3
NHSO3
OH
OAc
CO2Me
O
OAc
OBz
O
O
O
O
CO2Me
OBn
2
O
O
OAc
OBn
OBn
O
OBn
O
OMe
N3
BnO
N3
OBz
N3
OBn
OBz
O
OAc
OAc
O
CO2Me
O
NH
CCl3
O
O
CO2Me
O
O
O
OBn
OBn
OAc
O
OBn
OBn
OMe
OMe
BnO
BnO
HO
HO
N3
N3
N3
OBz
OBn
3
4
O
OBz
O
OAc
O
CH2OH
O
NH
CCl3
O
O
CH2OH
OBn
O
OAc
O
+
O
OBn
OBn
OBn
HO
N3
N3
OBn
OBz
N3
7
5
6
OBz
O
O
O
O
O
AcO
AcO
D-Glc
Ph
O
L
O
+
OBn
OBn
O
HO
OMe
OBn
O
OBz
N3
OBn
9
L = OAc
10 L = OC(NH)CCl3
11 L = SEt
12
8
Cellobiose
D-Glc
D-Glc
Figure 1. The retrosynthetic analysis of Fondaparinux.
O
OR
O
a
c
10 steps, 20%
Ref 13
Ph
O
Cellobiose
O
8
O
O
6
N3
13
BnO
OBn
R= H
14 R= Ac
b
Scheme 1. Preparation of the disaccharide 6. Reagents and conditions: (a) NaN3, NH4Cl, Bu4NI, DMF–H2O, 86%; (b) Ac2O, Py, 90%; (c) TsOH, rt, 82%.
The epoxide 15 was prepared from
56% yield.14 Hung and co-workers reported the key intermediate
1,6-anhydro-
-idopyranose 17,8a but its large scale preparation
was difficult. Because the preparation of expensive -idopyranose
intermediates was the bottle-neck for heparin synthesis, we tried
to develop a practical preparation method for 1,6-anhydro-idose
17 from epoxide 15 in one step.
Hydrolysis of 15 in various acids (HCl, TsOH, HOAc, or H2SO4)
and solvents (EtOH, MeOH, or H2O) at refluxing temperature was
screened. With 0.4 M H2SO4 and EtOH/H2O = 1/5 (v/v) mixed
solvent, the best yield (17%) of 17 was obtained, while the major
D
-glucose in 5 steps with
product was idopyranose 16, accompanied with some black solid
which might come from carbonization of the substrate. Then sev-
eral phase transfer catalysts were screened to avoid the formation
of black solid, in order to improve the yield of 17. Quaternary
ammonium salt (TBAB, Aliquat 336) gave no improvement, while
addition of 0.1 equiv of b-CD improved the yield of 17 to 37%.
The H2SO4 adding process affected the substrate solubility greatly,
which in turn affected the yield of 17. At 60 °C, b-CD in hot 0.4 M
H2SO4 solution was added into the 17/EtOH solution, then the reac-
tion temperature was raised rapidly to 90 °C. By this process, the
black solid did not form, the system kept a clear solution to the
L
L

34
F. Lin et al. / Carbohydrate Research 371 (2013) 32–39
end, and the yield of 17 raised to 65%. The by-product 16 can be
transferred to 17 under a second run of the same condition. Thus,
the total yield of 17 with two runs was improved to 80%. This
method is a good supplement for Hung’s preparation for 17
(Scheme 2).
was 2:1. We proposed that the idosyl trichloroacetimidate 10
was an active donor, so that some pathway without NGP (neighbor
group participation) effect occurred, which resulted in b-isomer. By
lowering the reaction temperature, the reactivity of donor de-
ceased and NGP effect increased, resulting in improved
a-selectiv-
The catalysis mechanism of b-CD was not clear yet. The yield of
17 was around 20% in the absence of b-CD, which must serve more
than PTC reagent, because other PTC reagent (such as quaternary
ammonium salt) showed no improvement. If H2SO4 was absent
while all other conditions were the same, only 50% epoxide open-
ing product 19 was obtained, which indicated that hydrogen bond
(HB) network in the b-CD cage could catalyze epoxide opening. Be-
cause 17 could be prepared from 16, the HB network of b-CD may
induce the conformation change of idosyl ring 16 and stabilize its
4C1 conformer which benefits the formation of 1,6-anhydro-idose.
After selective benzoylation of the 2-OH, the product 18 was
acetolyzed with catalyst TESOTf to provide the ring-opening ace-
ity. Hung and co-workers reported good a-selectivity of idosyl
trichloroacetimidate glycosylation at ꢀ78 °C,8a,14 which was in
accordance with our observation.
Then donor thioglycoside 11 was investigated, which was ex-
pected to be moderate and undergo NGP pathway to ensure high
a-selectivity. Thioglycoside 11 was prepared from 9 in 85% yields,
and the glycosylation of 11 with 12 gave 85% of 21 with full
a
a
selectivity. Comparing with iduronate thioglycosides, which were
poor donors12b due to the electron-withdrawn effect of carboxyl
group at 5-position, L-idopyranosyl thioglycoside 11 was a donor
with good synthetic efficiency and great
(Table 1).
a-glycosylation selectivity
tates 9 (
a
/b mixture, 72% two steps).
To test its donor property, 11 was also reacted with 22 and 24,
the good yield and -glycosylation selectivity proved thioethyl gly-
coside 11 is an excellent -idopyranosyl donor.
a
2.1. Investigation of three
L
-idopyranosyl donors
L
Owing to Petitou and co-workers’ systematic study of various
iduronic acid glycosyl donors, most of the heparin synthesis used
L
L
-
-
2.2. Preparation of uronate at disaccharide stage
by TEMPO–Ca(OCl)2 selective oxidation
iduronate donors. Because
L
-idosyl donor is more active than corre-
-idosyl donors to eval-
sponding -iduronate donor, we studied on
L
L
We employed TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)–
KBr–Ca(OCl)2 system as an effective selective oxidation method
to prepare the uronate disaccharide synthons. The reaction was
very sensitive to pH value. The traditional TEMPO–KBr–NaOCl
method requires strictly in situ pH control of the reaction mixture,
which makes the procedure complex and sometimes less repro-
ducible. We made a change that Ca(OCl)2 was added to Na2CO3–
NaHCO3 buffer to produce NaOCl as a terminal oxidant. Using solid
Ca(OCl)2 could avoid the dilution of the reaction system and
Na2CO3–NaHCO3 buffer also help to maintain pH value at 8–9.5.
This practical improvement showed its convenience in our synthe-
sis. After methylation by CH2N2, uronate 26 was obtained in 73%
yield. The trisaccharide 3 was prepared according to the literature
in 68% yield (three steps)13 (Scheme 4).
uate their application in modular synthesis. Three donors were
investigated: trichloroacetimidate, thioglycoside, and acetate.
The glycosylation of idopyranosyl acetate 9 with glucoside 12
required 0.5 equiv of TMSOTf, (Scheme 3) and the optimized yield
of 21a is only 60%. Although the a-selectivity is great, the strong
acidic condition caused a remarkable decomposition of the sub-
strate. This indicated that the idopyranosyl acetate is not an active
donor, the strong acidic reaction condition limits its application in
modular synthesis.
Then trichloroacetimidate 10 was investigated. The yields of
selective anomeric Ac deprotecting by benzylamine or hydra-
zine–HOAc were moderate, because 6-O-Ac was likely cleaved un-
der these conditions. By changing THF/MeOH solvent ratio, which
in turn changed the saturated concentration of ammonia in the
mixed solvent at 0 °C, we selectively deprotected the anomeric
Ac to give 20 in 90% yields. Further treatment with CCl3CN and
K2CO3 provided trichloroacetimidate 10. Glycosylation of 10 with
12 under the promotion of 0.1 equiv TMSOTf at 0 °C provided
Selective oxidation of 7a with the same oxidation condition
with Bu4NBr as PTC reagent, provided 54% of 4. When Bu4NBr
was replaced by Aliquat 336 (C8H17)3NCl), the yield was increased
to 93%. This result showed that the choice of PTC reagent should be
considered in TEMPO selective oxidation.
21
a
/b in 96% yield, which was inseparable by TLC or column chro-
Coupling of 3 with 4 under the promotion of TMSOTf provided 2
matography; and the 1H NMR was complicated to tell
a/b ratio.
with a
selectivity in 78% yield (H-1000 at 5.10 ppm with J value of
With 0.2 M anhydrous HCl/methanol, 40 and 60-O-Ac was selec-
tively cleaved in 94% yield, with 6-O-Bz intact. After deacetylation,
3.6 Hz, C-1000 at 97.49 ppm). Then the acyl group and the methyl es-
ter of 2 were deprotected to afford 28 in 65% yields. The free OH
group of 28 was sulfated with triethylamine-sulfur trioxide com-
plex. The azido and the OBn groups were hydrogenolysed with
Pd/C and then the resulting amino groups were selectively sulfated
7a
/b could be separated by column chromatography, and
a/b ratio
was 3:2. By this two-step determination method, we found that
when the glycosylation temperature was ꢀ20 °C, the ratio of
a/b
a
O
OBn
O
OBn
O
HO
BnO
HO
HO
5 steps
a
OH
O
D-Glc
+
Ref 14
HO
O
O
O
OH
16
17
15
OH
OBn
O
HO
AcO
OBn
OR
O
HO
BnO
c
b
O
OAc
OBz
O
BzO
18
O
O
9 R= Ac
20 R= H
19
Scheme 2. Preparation of
L-idopyranose 9. Reagents and conditions: (a) 0.4 M H2SO4, 0.1 equiv b-CD, 90 °C, 80% (combined the 2nd run of 16); (b) BzCl, Py, CH2Cl2, 0 °C, 85%;
(c) TESOTf, Ac2O, 0 °C, 85%.

F. Lin et al. / Carbohydrate Research 371 (2013) 32–39
35
a
c
b
O
BzO
RO
BnO
BzO
RO
20
10
OBz
O
OBn
O
O
12
d
+
O
9
BnO
RO
RO
O
f
e
BnO
11
N3
OMe
N3OMe
BzO
21α
21β R = Ac
7β R = H
R = Ac
g
g
7
α R = H
Scheme 3. Glycosylation investigation of three kinds of donor. Reagents and conditions: (a) NH3, THF/MeOH (v/v, 8:2), 0 °C, 90%; (b) CCl3CN, K2CO3, CH2Cl2, 78%; (c) TMSOTf
(0.1 equiv), CH2Cl2, 96% (
MeOH, 0 °C, 90%.
a
/b mixture); (d) TMSOTf (0.5 equiv), CH2Cl2, 60% (
a
only); (e) HSEt, BF3ꢁEt2O, 0 °C, 85%; (f) NIS, AgOTf, 0 °C, 4 Å MS, 85% (a only); (g) 0.2 M HCl/
Table 1
4. Experimental
Glycosylation with L-idopyranosyl thioglycoside 11
Acceptor
Product
Yield
92%,
4.1. General methods
BzO
HO
BzO
O
OBn
O
O
Reagents and solvents were of reagent grade and used without
further purification. Methylene dichloride was distilled from cal-
cium hydride. All reactions involving air or moisture sensitive re-
agents or intermediates were performed under a nitrogen or
argon atmosphere. Analytical TLC was visualized by irradiation
(254 nm) or by staining with H2SO4/methanol solution. 1H and
13C NMR spectra were obtained using 300 MHz Brucker AM300.
High resolution mass spectra (MALDI/DHB) were performed on
IonSpec 4.7 Tesla FTMS from Varian.
OMe
BnO
O
BnO
AcO
AcO
N3
OMe
a
a
only
N3
22
BzO
23
O
AcO
AcO
OBn
O
OBn
O
OBn
O
O
O
95%,
only15
OH
BzO
N3
N3
24
25
4.2. 3-O-Benzyl-1,6-anhydro-b-L-idopyranose (17)
with pyridine–sulfur trioxide complex to furnish functionalized
Fondaparinux (1) in 56% yield (Scheme 5).
At 60 °C, b-CD (1.1 g, 0.1 equiv) in 0.4 M sulfuric acid solution
(50 mL) was added rapidly into 10 mL ethanol solution of compound
158a (2.74 g, 9.4 mmol), then heated to 90 °C for 9 h. Then it was
cooled to 0 °C and neutralized with Na2CO3, concentrated in vacuo,
and purified by column chromatography (CH2Cl2/EA = 2:1). Compound
17 (1.543 g, 65% yield) and 16 (0.75 g, 30%) as syrup were obtained. By
applying this reaction condition to compound 16, additional 0.349 g of
17 could be obtained. The combined yield of 17 is 80%.
3. Conclusion
The anti-thromboembolic pentasaccharide Fondaparinux (1)
was synthesized in 36 steps for the longest linear route, with
0.017% overall yield from
D-glucose and cellobiose. Only three
Rf = 0.71 (PE/EA = 1:2). 1H NMR (300 MHz, CDCl3): d 7.39–7.26
(m, 5H, ArH), 5.30 (d, 1H, J = 2.1 Hz, H-1), 4.96 (d, 1H, J = 11.7 Hz,
PhCH2), 4.74 (d, 1H, J = 11.7 Hz, PhCH2), 4.43 (t, 1H, J = 4.5 Hz, H-
5), 4.03 (d, 1H, J = 7.2 Hz, H-6a), 3.87 (m, 1H, H-4), 3.73 (m, 1H,
H-6b), 3.66 (dd, 1H, J = 1.8, 7.5 Hz, H-2), 3.39 (t, 1H, J = 7.8 Hz, H-
3). 13C NMR (75 MHz, CDCl3): d 138.4, 128.7, 128.0, 127.9, 101.8,
84.2, 75.4, 75.0, 74.5, 71.1, 65.1. ESI/MS: 275.1 (M+Na+). Anal. Calcd
for C13H16O5: C, 61.90; H, 6.39. Found: C, 61.73; H, 6.47.
kinds of protecting groups were used for hydroxyl protection, Bn,
Ac, and Bz, to accomplish this complex synthesis without decreas-
ing the synthetic efficiency. This work proved that this simple and
unified protection strategy, when combined with sophisticated
selective protection–deprotection techniques, was valuable for
heparin modular synthesis. We also studied the modular synthesis
of disaccharide building blocks, especially
synthesis of key intermediate 1,6-anhydro-idopyranose 17 was im-
proved by H+/b-CD catalyst. Three
-idopyranosyl donors were
investigated. Thioethyl glycoside is an efficient donor for -idopyr-
anosyl glycosylation with full -selectivity, while the /b selectiv-
ity from -idopyranosyl trichloroacetimidate glycosylation should
L-idopyranosides. The
L
4.3. 2-O-Benzoyl-3-O-benzyl-1,6-anhydro-b-L-idopyranose (18)
L
a
a
At 0 °C, to 17 (690 mg, 2.7 mmol) and pyridine (0.65 mL,
8.2 mmol) in 10 mL CH2Cl2, was added BzCl (0.4 mL, 3.56 mmol).
After 4 h, concentrated and purified by column chromatography
(hexane/EtOAc = 2:1) to obtain 18 (0.89 g, 91% yield) as white solid.
Rf = 0.36 (PE/EA = 2:1). 1H NMR (300 MHz, CDCl3): d 8.06 (dd, 2H,
L
be carefully investigated. TEMPO–Ca(ClO)2 selective oxidation is
a practical method for disaccharide uronate building blocks, and
the choice of PTC should be concerned. Thus we investigated and
developed some synthetic methods for heparin modular synthesis.
O
OAc
O
OAc
O
OAc
O
O
BnO
BnO
OMe
O
O
c
a
b
OMe
O
O
6
N3
3
HO
O
N3
BnO
O
O
N3
BnO
OBn
26
OBn
27
a or d
7α
4
Scheme 4. Preparation of uronates through TEMPO oxidation. Reagents and conditions: (a) TEMPO, Ca(ClO)2, KBr, Bu4NBr, NaHCO2–Na2CO3 buffer, CH2Cl2, CH2N2, for 6, 73%;
for 7 , 54%; (b) 5, TBSOTf, toluene, 0 °C, 95%; (c) (1) Ac2O, TESOTf, 0 °C, 30 min, (2) NH3, THF/MeOH = 7:3, 0 °C, 30 min, (3) CCl3CN, K2CO3, CH2Cl2, rt, 68%, three steps; (d)
TEMPO, Ca(ClO)2, KBr, Aliquat 336, NaHCO2–Na2CO3 buffer, CH2Cl2, CH2N2, 83%, two steps.
a

36
F. Lin et al. / Carbohydrate Research 371 (2013) 32–39
OH
O
HO2C
OH
OBn
O
OH
O
O
O
O
N3
a
b
BnO
BnO
O
3
4
+
2
N3
HO
O
O
BnO
N
3 OMe
O
BnO
HO
OH
BnO
28
-
OSO3
O
-O2C
O
OSO3-
O
OBn
O-
O -O3SO
O
N3
O
BnO
O
d
c
BnO
N3 O
BnO
BnO
O
-
O
O3SO
N3
OMe
BnO
OSO3-
29
OSO3-
O
-O2C
O
-
OSO3
O
OH
O
O-
O
H2N
O
e
O
HO
HO
1
O
-O3SO
O
H2N
HO
O
-O3SO
H2N
OMe
HO
-
OSO3
OH
30
Scheme 5. Final elaboration of Fondaparinux (1). Reagents and conditions: (a) TMSOTf, CH2Cl2, 4 Å MS, ꢀ20 °C, 78%; (b) LiOH, H2O2, ꢀ5 °C, 6 h; then NaOH, 6 h, 65%; (c)
SO3ꢁNMe3, 50 °C, 91%; (d) 10% Pd/C, H2, H2O/t-BuOH, 95%; (e) SO3ꢁPy, NaHCO3, 65%.
J = 1.3, 7.4 Hz, BzH), 7.57 (t, 1H, J = 7.4 Hz, BzH), 7.46 (t, 2H, J = 7.6 Hz,
BzH), 7.26 (m, 5H, ArH), 5.53 (d, 1H, J = 1.4 Hz, H-1), 5.07 (dd, 1H,
J = 1.5, 8.1 Hz, H-2), 4.80 (d, 1H, J = 11.5 Hz, PhCH2), 4.65 (d, 1H,
J = 11.5 Hz, PhCH2), 4.51 (t, 1H, J = 4.6 Hz, H-5), 4.15 (d, 1H,
J = 7.5 Hz, H-6a), 3.87 (t, 1H, J = 8.2 Hz, H-3), 3.76 (dd, 1H, J = 4.6,
7.5 Hz, H-6b). 13C NMR (75 MHz, CDCl3): d 165.58, 137.78, 133.24,
129.75, 129.26, 128.25, 128.20, 127.78, 127.64, 99.37, 80.20, 76.68,
75.03, 74.55, 71.28, 65.20. ESI-MS (m/z): 379.25 [M+Na+]. Anal. Calcd
for C20H20O6: C, 67.41; H, 5.66. Found: C, 67.18; H, 5.74.
with CH2Cl2 and washed with aq NaHCO3 and brine, dried and con-
centrated and purified by column chromatography (hexane/
EtOAc = 4:1) to obtain
a
/b mixture 11 (232 mg, 81% yield) as white
solid. Rf = 0.78 (PE/EA = 2:1), 1H NMR (
a/b = 1:1.5) (300 MHz, CDCl3):
d 8.18–8.07 (m, 5H, BzH), 7.57 (m, 2.5H, BzH), 7.47–7.32 (m, 17.5H,
ArH), 5.44 (s, 1H), 5.27 (t, 1H, J = 1.5 Hz), 5.22 (m, 1.5H), 5.13 (d,
1.5H, J = 1.8 Hz), 4.92–4.68 (m, 10H), 4.25 (m, 5H), 3.97 (t, 1.5H,
J = 3.0 Hz), 3.82 (m, 1H), 2.78 (m, 3H), 2.71–2.66 (m, 2H), 2.06 (s,
7.5H, Ac), 1.97 (s, 3H, Ac), 1.79 (s, 4.5H, Ac), 1.34 (m, 7.5H, CH3). 13C
NMR (a/b mixture) (75 MHz, CDCl3): d 170.6, 170.1, 170.0, 165.7,
4.4. 1,4,6-Tri-O-acetyl-2-O-benzoyl-3-O-benzyl-
L
-idopyranose (9)
165.5, 165.1, 137.1, 133.5, 129.9, 129.8, 129.5, 128.4, 128.3, 128.2,
127.9, 127.8, 127.6, 82.6, 81.4, 72.9, 72.7, 72.6, 72.5, 72.0, 71.9, 69.4,
68.9, 67.2, 66.0, 64.0, 62.8, 62.7, 26.7, 25.9, 20.7, 20.5, 15.1, 15.0. HRMS
Under N2 at 0 °C, TMSOTf (0.15 mL, 0.1 equiv) was added into
18 (2.3 g, 6.5 mmol) in 14 mL Ac2O. After 30 min, the reaction
was quenched with NEt3 at 0 °C, then 20 mL MeOH was added
and stirred for 30 min, concentrated and purified by column chro-
(ESI) Calcd for C26H30O8SNa 525.1554. Found 525.1561; IR (cmꢀ1
)
mmax = 2929, 1743, 1724, 1453, 1267, 1111, 1070, 1027, 713.
matography (hexane/EtOAc = 4:1) to obtain
88%yield) as white syrup.
a
/b mixture 9 (2.86 g,
4.6. 4,6-Di-O-acetyl-2-O-benzoyl-3-O-benzyl- /b- -idopyranose (20)
a
L
9a
: Rf = 0.79 (PE/EA = 2:1) 1H NMR (300 MHz, CDCl3): d 8.12 (m,
At 0 °C, 9 (472 mg) in THF–MeOH mixed solvent (8:2, v/v) was
bubbled with ammonia till saturation, and stirred for 30 min at
0 °C. TLC showed most of 9 converted. Concentrated and purified
2H, BzH), 7.60 (t, 1H, J = 7.5 Hz, BzH), 7.46 (t, 2H, J = 7.8 Hz, BzH),
7.37–7.26 (m, 5H, ArH), 6.20 (d, 1H, J = 1.2 Hz, H-1), 5.30 (m, 1H,
H-2), 4.93 (t, 1H, J = 2.1 Hz, H-4), 4.78 (dd, 2H, J = 12.3 Hz,
PhCH2–), 4.47 (m, 1H, H-5), 4.27 (m, 2H, H-6), 4.00 (t, 1H,
J = 3.0 Hz, H-3), 2.07, 1.92 (s each, 3H each for acetyl).
by column chromatography (hexane/EtOAc = 4:1) to obtain
a/b mix-
ture 20 (389 mg, 90% yield) as white syrup. 1H NMR (
a/b mixture,
300 MHz, CDCl3): d 8.17–7.27 (m, 10H, Ar), 5.28 (s, 1H), 5.13 and
4.95 (AB, 2H, CH2Ph), 4.82 (br s, 1H), 4.78 (m, 1H), 4.62 (m, 1H),
4.35 (m, 1H), 4.30–4.19 (m, 2H), 4.05 (br, 1H), 2.09 (s, 3H, OMe),
9b: Rf = 0.82 (PE/EA = 2:1) 1H NMR (300 MHz, CDCl3): d 8.07 (m,
2H, BzH), 7.59 (t, 1H, J = 7.5 Hz, BzH), 7.46 (t, 2H, J = 7.8 Hz, BzH),
6.22 (s, 1H, H-1), 5.21 (m, 1H, H-2), 4.99 (br, 1H, H-4), 4.81 (dd,
2H, J = 12.3 Hz, PhCH2–), 4.61 (m, 1H, H-5), 4.31 (m, 2H, H-6),
3.90 (br, 1H, H-3), 2.09 (s, 6H, Ac), 1.94 (s, 3H, Ac).
1.89 and 1.83 (2s, 3H total, OMe). 13C NMR (
a/b mixture, 75 MHz,
CDCl3) anomer A: d 92.69, 73.64, 71.07, 66.41, 65.78, 62.90, 62.73;
anomer B: d 91.81, 73.02, 72.0, 68.53, 66.01, 62.65; 170.75, 170.10,
165.60, 165.12, 20.80, 20.60. ESI-MS (m/z): 481.30 [M+Na+]. HRMS
13C NMR (/ mixture) (75 MHz, CDCl3): d 170.6, 170.0, 168.9, 168.8,
165.5, 136.9, 136.6, 130.0, 129.9, 128.6, 128.5, 127.8, 127.5, 91.5,
90.5, 73.7, 73.2, 72.5, 72.0, 71.8, 66.9, 66.5, 66.2, 66.0, 65.7, 62.4,
62.3, 21.0, 20.9, 20.8, 20.7. ESI-MS (m/z): 518.4 [M+NH4+], 523.3
[M+Na+]. Anal. Calcd for C26H28O10: C, 62.39; H, 5.64. Found: C,
(ESI) Calcd for C24H26O9Na 481.1469. Found 481.1467; IR (cmꢀ1
3345, 1745, 1365, 1227, 1111, 1070, 715.
) m
4.7. Methyl O-(2-O-benzoyl-3-O-benzyl-
(1?4)-2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-
glucopyranoside (7 ) and methyl O-(2-O-benzoyl-3-O-benzyl-b-
-idopyranosyl)-(1?4)-2-azido-6-O-benzoyl-2-O-benzyl-2-
deoxy- -glucopyranoside (7b)
a-L-idopyranosyl)-
62.33; H, 5.74. IR (cmꢀ1
) mmax = 1745, 1371, 1224, 1114, 715.
a-D-
a
4.5. Ethyl 4,6-di-O-acetyl-2-O-benzoyl-3-O-benzyl-1-thio-a/b-L-
idopyranoside (11)
L
a
-D
Under N2 at 0 °C, BF3ꢁEt2O (0.09 mL, 1.1 equiv) was added into 9
(281 mg,0.56 mmol) and EtSH (0.063 mL,0.84 mmol) in dry 4 mL
CH2Cl2, after 3 h, quenched with NEt3 at 0 °C. The solution was diluted
100 mg K2CO3, 20 (156 mg, 0.34 mmol), and Cl3CCN (0.1 mL,
1 mmol) in 1 mL CH2Cl2 were stirred for 2 h. Concentrated and
purified by column chromatography (hexane/EtOAc = 4:1) to

F. Lin et al. / Carbohydrate Research 371 (2013) 32–39
37
obtain
(hexane/EtOAc = 3:1).
a
/b mixture 10 (166 mg, 81% yield) as white syrup. Rf = 0.5
TLC showed the reaction was completed (CH2Cl2/MeOH = 20:1).
Then at 0 °C, 104 mg NaHSO3 (1 mmol) was added, and HOAc
was added to adjust the pH value to 3. After diluting with 20 mL
EtOAc, the aqueous phase was extracted with EtOAc, and the com-
bined organic phase was washed with brine, dried, filtered, and
concentrated. The syrup dissolved in 3 mL ether, and CH2N2 in
ether was added till no bubble came out. Then a few drops of HOAc
were added to consume the excess CH2N2, concentrated and puri-
fied by column chromatography (PE/EA/CH2Cl2 = 2:1:1). 0.451 g
syrup was obtained (73% yields).
Under Argon, 4 Å MS, donor 10 (190 mg, 0.32 mmol, 1.2 equiv)
and acceptor 12 (108 mg, 0.26 mmol) in 2 mL dry CH2Cl2 were
cooled to ꢀ20 °C and 0.09 M TMSOTf/CH2Cl2 (0.3 mL, 0.1 equiv)
was added. After 30 min, moved to RT and stirred till the reaction
completed. Quenched with NEt3, filtered and concentrated, and
purified by column chromatography (hexane/EtOAc = 4:1) to ob-
tain
a
/b mixture 21 (216 mg, 96% yield) as white syrup.
/b mixture 21 (94 mg, 0.11 mmol) in 3 mL
Under Argon at 0 °C,
a
MeOH was added AcCl 0.07 mL (to prepare 0.2 M HCl/MeOH), then
stirred at RT for 3 h. Neutralized with NaHCO3, filtered and concen-
trated, and purified by column chromatography (hexane/
Rf = 0.33 (CH2Cl2/ace = 18:1). 1H NMR (300 MHz, CDCl3): d
5.49 (H-1, br), 5.29 (H-3, dd, J = 1.5 Hz), 4.99 (1H, AB,
J = 10.8 Hz), 4.87 (2H, AB, J = 11.4 Hz), 4.77 (1H, AB, J = 10.8 Hz),
4.64 (H-10, d, J = 7.5 Hz), 4.57 (H-5, d, J = 5.1 Hz), 4.02 (H-4, dd,
J = 7.5 Hz, 0.9 Hz), 3.88 (1H, dd, J = 8 Hz), 3.84 (H-50, d,
J = 9.6 Hz), 3.79 (s, 3H, OMe), 3.76 (m, 1H), 3.65 (1H, br), 3.55
(m, 2H), 3.20 (H-2, br), 2.12 (s, 3H, OAc); ESI-MS (m/z): 622.2
[M+Na+]. Anal. Calcd for C29H33N3O11: C, 58.09; H, 5.55; N,
EtOAc = 2:1) to obtain 7a (53 mg, 63% yield) and 7b (26 mg, 31%
yield) as white syrup.
7
a
Rf = 0.48 (hexane/EtOAc/CH2Cl2 = 1:1:1). ½a D25
ꢂ
+45.8 (c 1.0,
CHCl3), 1H NMR (300 MHz, CDCl3): d 7.97–7.25 (m, 20H, Ar), 5.54
(d, J = 2.1 Hz, 1H), 5.40 (br s, 1H), 5.02 (t, J = 3 Hz, 1H), 4.90–4.75
(m, 4H), 4.59–4.46 (m, 2H), 4.16 (m, 1H), 4.03–3.97 (m, 4H),
3.57–3.44 (m, 5H), 3.25 (m, 1H), 3.05 (m, 1H). 13C NMR (75 MHz,
CDCl3), d 165.86, 165.22, 137.50, 137.37, 133.47, 133.06, 129.74–
7.01. Found: C, 58.19; H, 5.35; N, 6.88. IR (cmꢀ1
1748, 1228, 1070.
) m 3485, 2107,
127.83 (Ar), 98.34 (C-1), 97.76 (C-10
a), 79.09, 75.57, 75.32, 73.87,
4.10. O-(6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-
glucopyranosyl)-(1?4)-O-(methyl 2,3-di-O-benzyl-b-
glucopyranosyluronate)-(1?4)-2-O-acetyl-1,6-anhydro-2-
azido-2-deoxy-b- -glucopyranose (27)
a-D-
72.43, 69.40, 68.21, 67.83, 67.21, 64.08, 62.75, 62.55, 55.34.
ESI-MS (m/z): 792.274 [M+Na+] (Calcd 792.274). Anal. Calcd
for C41H43N3O12: C, 63.97; H, 5.63; N, 5.46. Found: C, 63.74; H,
D-
D
5.39; N, 5.25. IR (cmꢀ1
1027.
) mmax = 3480, 2103, 1722, 1270, 1101,
Under Argon, 4 Å MS, donor 5 (386 mg, 0.68 mmol) and accep-
tor 26 (270 mg, 0.45 mmol) in 6 mL dry toluene were cooled to
ꢀ20 °C and 0.5 M TMSOTf/CH2Cl2 (0.3 mL) was added. After
30 min, moved to RT and stirred till the reaction completed.
Quenched with NEt3, filtered and concentrated, and purified by
column chromatography (hexane/EtOAc = 4:1) to obtain 27
(432 mg, 95% yield) as white syrup.
7b Rf = 0.26 (hexane/EtOAc/CH2Cl2 = 1:1:1). 13C NMR (75 MHz,
CDCl3), d 166.30, 165.16, 137.81ꢃ127.75(Ar), 107.78 (C-10 b),
98.35(C-1), 81.58, 80.40, 80.26, 79.08, 76.37, 75.85, 75.07, 71.73,
70.28, 68.79, 63.54, 60.31, 55.22. ESI-MS (m/z): 792.27 [M+Na+].
IR (cmꢀ1
) mmax 3465, 2101, 1722, 1270, 1101, 1027.
4.8. Methyl O-(4,6-di-O-acetyl-2-O-benzoyl-3-O-benzyl-
idopyranosyl)-(1?4)-2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-
-glucopyranoside (21
a
-
L
-
1H NMR (400 MHz, CDCl3): d 7.34–7.25 (20H, Ar), 5.53 (d, 1H,
J = 3.6 Hz, H-100), 5.47 (s, 1H, H-1), 5.22 (m, 1H, H-3), 5.00 (m,
1H), 4.84–4.73 (m, 4H), 4.67 (d, 1H, J = 7.57 Hz, H-10), 4.56 (m,
2H), 4.15 (t, 1H, J = 8.79 Hz, H-40), 3.96 (d, 1H, J = 6.86 Hz, H-6a),
3.88 (dd, 1H, J = 10.3 and 8.79 Hz, H-300), 3.75 (s, 3H, CO2Me), 3.64
(m, 1H, H-4), 3.51 (t, 1H, J = 8.79 Hz, H-400), 3.26 (dd, 1H, J = 10.5
and 3.60 Hz, H-200), 3.21 (s, 1H, H-2), 2.10 (s, 3H, Ac), 2.03 (s, 3H,
a-
D
a)
Under Argon, 4Å MS, donor 11 (0.74 g,1.46 mmol, 1.2 equiv)
and acceptor 12 (0.50 g, 1.22 mmol) in 20 mL dry CH2Cl2 were
cooled to ꢀ15 °C, NIS (384 mg, 1.2 equiv) and AgOTf/dry toluene
(43 mg, 0.14 equiv) solution (2 mL) were added successively. After
30 min, moved to RT and stirred till the reaction completed.
Quenched with NEt3, filtered and concentrated, and purified by
Ac). 13C NMR (100 MHz, CDCl3):
d 170.66, 168.16, 164.76,
138.19–127.39 (Ar), 103.34, 100.28, 97.69, 83.77, 81.52, 80.03,
77.52, 75.84, 75.44, 75.13, 74.91, 74.48, 73.84, 70.58, 69.61,
65.00, 63.32, 62.28, 58.88, 52.72, 20.80. ESI-MS (m/z): 1031.9
[M+Na+].
column chromatography (hexane/EtOAc = 4:1) to obtain 21
a
(910 mg, 86% yield) as colorless oil. ½a D25
ꢂ
+49.1 (c 1.1, CHCl3), 1H
NMR (300 MHz, CDCl3): d 8.10–7.97 (m, 4H), 7.57–7.25 (m, 16H,
Ar), 5.28 (d, J = 2.5 Hz, 1H), 5.19 (br s, 1H), 4.91–4.67 (m, 7H),
4.49 (dd, 1H), 4.03–3.91 (m, 4H), 3.85 (m, 1H), 3.53–3.47 (2s, 3H,
OMe), 3.47–3.41 (m, 1H), 1.99, 1.92 (s, 3H each, OAc). 13C NMR
4.11. Methyl O-(methyl 2-O-benzoyl-3-O-benzyl-
idopyranosyluronate)-(1?4)-2-azido-6-O-benzoyl-3-O-benzyl-
2-deoxy- -glucopyranoside (4)
a-L-
a-D
(75 MHz, CDCl3),
d 170.33, 169.97, 165.82, 165.07, 137.72–
127.49(Ar), 98.32, 97.10, 78.99, 75.02, 74.43, 72.70, 72.57, 69.08,
67.64, 66.68, 64.32, 64.14, 62.65, 62.17, 55.27, 20.65, 20.63. ESI-
MS (m/z): 876.296 [M+Na+] (Calcd 876.295). Anal. Calcd for
With the similar procedure of 26, except PCT reagent was Ali-
quat 336 (0.2 equiv), 0.327 g white foam 4 was obtained (93%
yields). Rf = 0.34 (hexane/EtOAc = 2:1) ½a D25
ꢂ
+31.8 (c 0.98, CHCl3),
C
45H47N3O14: C, 63.30; H, 5.55; N, 4.92. Found: C, 63.49; H, 5.35;
1H NMR (300 MHz, CDCl3): d 8.10–7.25 (20H, aromatic), 5.37 (d,
1H, J = 2.5 Hz, H-10), 5.21 (br s, 1H, H-20), 4.97 (d, H-50, J = 3 Hz),
4.76 (AB, 2H, J = 11.4 Hz), 4.81–4.67 (m, 3H), 4.78 (d, 1H,
J = 3.5 Hz, H-1), 4.48 (AB, 1H, J = 12 Hz), 4.02 (d, 1H, J = 7.8 Hz),
4.12–3.96 (m, 2H), 3.92–3.86 (m, 2H), 3.48 (s, CO2CH3), 3.45 (s,
3H, OMe), 3.44 (m, 1H), 2.66 (d, OH, J = 9 Hz); 13C NMR
N, 4.72. IR (cmꢀ1
)
m
max = 2110, 1744, 1732, 1272, 1052.
4.9. O-(Methyl 2,3-di-O-benzyl-b-D-glucopyranosyluronate)-
(1?4)-2-O-acetyl-1,6-anhydro-2-azido-2-deoxy-b-D-
glucopyranose (26)
(100 MHz, CDCl3):
d 169.44, 165.84, 164.95, 137.60–127.32,
24 mg KBr (0.2 mmol) and 24 mg Bu4NBr (0.07 mmol) in 2.2 mL
NaHCO3–Na2CO3 buffer (pH 9.5) were added into (0.59 g,
98.31, 97.93, 78.59, 75.25, 74.97, 74.73, 72.56, 69.14, 68.82, 68.08,
6
67.83, 63.65, 62.59, 55.29, 51.92. ESI-MS (m/z): 820.3 [M+Na+];
1.0 mmol) and TEMPO (3 mg, 0.01 mmol) in 3.3 mL CH2Cl2 solu-
tion, then at 0 °C with rapid stirring, Ca(ClO)2 (263 mg, 1.7 equiv)
was added in portions. The mixture was stirred at 0 °C for 1 h,
HRMS (ESI) Calcd for
C
42H43N3O13Na 820.2688. Found
820.2691. IR (cmꢀ1
1109, 711.
) m 3488, 3071, 1728, 1648, 1602, 1452, 1278,

38
F. Lin et al. / Carbohydrate Research 371 (2013) 32–39
4.12. Methyl O-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-
-glucopyranosyl)-(1?4)-O-(methyl 2,3-di-O-benzyl-b-
glucopyranosyluronate)-(1?4)-O-(3,6-di-O-acetyl-2-azido-2-
deoxy- -glucopyranosyl)-(1?4)-O-(Methyl 2-O-benzoyl-3-O-
benzyl- -idopyranosyluronate)-(1?4)-2-azido-6-O-benzoyl-
3-O-benzyl-2-deoxy- -glucopyranoside (2)
a
-
2); G 99.92 (C-1); H 94.19 (C-1), 79.78 (C-3), 64.70 (C-2). ESI-MS
(m/z): (negative) 627.0 [(Mꢀ3H)/3] (Calcd 627.4).
D
D-
48 mg pentasaccharide 29 in 1 mL H2O and 2 mL MeOH, was
hydrogenated in the presence of 10% Pd/C (30 mg) at 1 bar. After
10 h, Pd/C was filtered and the filtrate was concentrated and puri-
fied by reverse phase C18 column chromatography (MeOH/H2O 1:9
to 9:1) to obtain pentasaccharide 30 (26 mg, 98% yield). ESI-MS (m/
z): (negative) 601.5 [(Mꢀ3H)/3] (Calcd 601.5); 913.9 [(M+Naꢀ3H)/
2] (Calcd 913.7).
a
a
-D
-L
a-D
Under Argon, 4 Å MS, donor 3 (80 mg, 0.1 mmol) and acceptor 4
(158 mg, 0.12 mmol) in 2 mL dry CH2Cl2 were cooled to ꢀ20 °C and
0.1 M TMSOTf/CH2Cl2 (0.07 mL, 0.06 equiv) was added. After
30 min, moved to RT and stirred till the reaction completed.
Quenched with NEt3, filtered and concentrated, and purified by
column chromatography (hexane/EtOAc/CH2Cl2 = 4:1:1) to obtain
pentasaccharide 2 (135 mg, 70% yield) as white syrup. 1H NMR
(400 MHz, CDCl3): d D 5.36 (d, 1H, J = 3.6 Hz, H-1), 3.23 (dd, 1H,
J = 7.6 and 3.6 Hz, H-2); E 4.40 (d, 1H, J = 7.8 Hz, H-1), 3.33 (H-2);
F 5.10 (d, 1H, J = 3.6 Hz, H-1), 3.64 (H-2); G 5.47 (d, 1H, J = 4.4 Hz,
H-1), 5.00 (H-2); H 4.76 (d, 1H, J = 3.2 Hz, H-1), 3.42 (H-2). 13C
NMR (75 MHz, CDCl3): d 170.55, 170.03, 170.0, 169.62, 168.27,
165.98, 165.27, 137.95, 137.47, 137.27, 133.37, 132.91, 129.80–
125.45 (Ar), 103.14, 98.34, 98.19, 97.49 (2 C), 83.65, 81.63, 80.04,
78.40, 76.77, 75.41, 74.89, 72.95, 71.40, 69.62, 69.05, 62.20,
61.26, 60.62, 55.30, 52.16. HRMS (MALDI/DHB) Calcd for
14 mg SO3ꢁpy and 14 mg NaHCO3 were added into pentasaccha-
ride 30 (26 mg) in 2 mL H2O, the same amount of SO3ꢁpy and NaH-
CO3 was added at the end of 2, 4, and 6 h. then stirred for another
2 h, concentrated and exchanged to sodium salt with DOWEX 50-
WX4-Na, then purified by reverse phase C18 column chromatogra-
phy (CH3CN/H2O 1:9 to 4:1). The fraction contained 1 was com-
bined and desalted on
a Sephadex G-25 column to obtain
pentasaccharide 1 (24 mg, 65% yield).
1H NMR (600 MHz, CDCl3): d D 5.60 (H-1), 3.24 (H-2), 3.61 (H-
3), 3.88 (H-4), 3.56 (H-5); E 4.62 (H-1), 3.40 (H-2), 3.82 (H-3), 3.76
(H-4), 4.16 (H-5); F 5.50 (H-1), 3.44 (H-2), 4.36 (H-3), 3.96 (H-4),
4.14 (H-5); G 5.19 (H-1), 4.30 (H-2), 4.14 (H-3); H 5.00 (H-1),
3.27 (H-2), 3.66 (H-3), 3.94 (H-4), 3.77 (H-5). 13C NMR (150 MHz,
CDCl3): d D 100.98 (C-1), 69.53 (C-6), 60.71 (C-2); E 103.89 (C-1);
F 98.77 (C-1), 68.86 (C-6), 59.39 (C-2); G 102.45 (C-1); H 100.3
(C-1), 69.06 (C-6), 60.42 (C-2), 58.42 (OMe). HiRes ESI-MS (m/z):
(negative) 818.46 [(M+6Naꢀ8H)/2] (Calcd 818.42), 829.45
[(M+7Naꢀ9H)/2] (Calcd 829.41), 840.44 [(M+8Naꢀ10H)/2] (Calcd
840.40).
C95H101N9O30Na 1870.6547. Found 1870.6578.
4.13. Methyl O-(2-azido-3,4-di-O-benzyl-2-deoxy-
glucopyranosyl)-(1?4)-O-(2,3-di-O-benzyl-b-
glucopyranosyluronic acid)-(1?4)-O-(2-azido-2-deoxy-
glucopyranosyl)-(1?4)-O-(3-O-benzyl- -idopyranosyluronic
-glucopyranoside
a-D-
D
-
a-D-
a-L
acid)-(1?4)-2-azido-3-O-benzyl-2-deoxy-
a-
D
Acknowledgments
(28)
This work was supported by the Committee of Science and
Technology of Shanghai (10QB1403900), the National Natural Sci-
ence Foundation of China (20902059), and the National Science
Grand Projects of New Drug R&D (2011ZX09202-101-01), we wish
to thank Professor Biao Yu for his instruction of this work.
At ꢀ5 °C, 2 mL 1.25 M LiOH solution and 4.5 mL 30% H2O2 were
added into the THF solution of 2 (162 mg) and stirred for 6 h at
0 °C, then 5 mL MeOH and 2.2 mL 4 N NaOH were added and stir-
red for 6 h. Quenched with 4 mL 5% Na2SO3 , adjusted pH to 2–3
with 6 M HCl. Extracted with CH2Cl2 3 times, combined the organic
phase and dried over Na2SO4 filtered and concentrated, and puri-
fied by reverse phase C18 column chromatography (CH3CN/H2O
1:9 to 7:3) to obtain pentasaccharide 28 (84 mg, 65% yield). 1H
NMR (400 MHz, CD3OD): d D 5.55 (d, 1H, J = 3.6 Hz, H-1), 3.27
(H-2); E 4.74 (d, 1H, J = 8 Hz, H-1), 3.50 (H-2); F 5.13 (d, 1H,
J = 3.6 Hz, H-1), 3.29 (H-2); G 5.31 (d, 1H, J = 4.4 Hz, H-1), 3.65
(H-2); H 4.78 (d, 1H, J = 3.2 Hz, H-1), 3.36 (H-2). 13C NMR
(75 MHz, CD3OD): d 172.50, 171.97, 139.66–139.31 (Ar), 129.56–
128.78 (Ar), 103.93, 102.27, 99.99, 99.12, 98.86, 85.20, 83.09,
80.87, 79.68, 79.06, 78.38, 77.35, 76.23, 75.89, 75.41, 74.80,
73.13, 72.72, 71.84, 64.67, 64.49, 61.58, 60.58, 55.54. ESI-MS:
1508.70 (M+Na+), 1524.60 (M+K+).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2013.01.
003.
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40 mg 28 and 188 mg SO3ꢁNMe3 in 1.7 mL DMF were stirred at
50 °C for 10 h, concentrated in vacuo, exchanged to sodium salt
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39
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