Convenient synthesis of an N-glycan octasaccharide of the bisecting type

Article abstract of DOI:10.1021/jo900016j

Convenient synthesis of an N-glycan octasaccharide (A) of the bisecting type was described. The stable and easily prepared orthoester F, 3 -O-chloroacetyl-4 ,6 -O-benzylidene-α-D-glucopyranose 1, 2 (chloromethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-g

Full text of DOI:10.1021/jo900016j

Convenient Synthesis of an N-Glycan Octasaccharide of the
Bisecting Type
Guangfa Wang, Wei Zhang, Zhichao Lu, Peng Wang, Xiuli Zhang, and Yingxia Li*
Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Medicine and Pharmacy,
Ocean UniVersity of China, Qingdao 266003, China
liyx417@ouc.edu.cn
ReceiVed January 5, 2009
Convenient synthesis of an N-glycan octasaccharide (A) of the bisecting type was described. The stable
and easily prepared orthoester F, 3′′-O-chloroacetyl-4′′,6′′-O-benzylidene-R-D-glucopyranose 1′′,2′′-
(chloromethyl 3′,6′-di-O-benzyl-2′-deoxy-2′-phthalimido-ꢀ-D-glucopyranosyl-(1f4)-3,6-di-O-benzyl-2-
deoxy-2-phthalimido-ꢀ-D-glucopyranosylazide-4′-yl orthoacetate), was designed as the key intermediate
with two advantages: (1) distinguishing OH-2′′ from OH-3′′ in ꢀ-D-glucopyranoside to construct the
central ꢀ-D-mannopyranoside by inverting the configuration of OH-2′′ in ꢀ-D-glucopyranoside and (2)
distinguishing OH-4′′ and OH-6′′ from OH-3′′ in the ꢀ-D-mannopyranoside to introduce the bisecting
GlcNAc residue.
Introduction
bisected N-glycans, which are the product of GnT-III, have
unique function in terms of the suppression of cancer metasta-
sis,3 they also have been shown to be involved in cell
development4 and function of receptors.5 Therefore, the bisected
N-glycans are of great significance with regard to cancer therapy.
Because of difficulties in obtaining homogeneous glycans from
natural sources, we decided to synthesize octasaccharide A,
which is a part of the structure of a bisected N-glycan and
Glycoproteins with N-glycans found on cell surfaces and in
blood serum play important roles in many key biological events.
It is widely recognized that the functions of glycoproteins are
influenced by their N-glycans.1 Among the great variety of their
functions, tumor metastasis is a focal point in scientific research
nowadays. ꢀ-1f6 GlcNAc branching, produced by GnT-V,
plays a major role in cancer invasion and metastasis by
stabilizing the proteases and angiogenic releasing factor.2 While
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J. Biol. Chem. 2006, 281, 13038. (c) Zhao, Y.; Sato, Y.; Isaji, T.; Fukuda, T.;
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2508 J. Org. Chem. 2009, 74, 2508–2515
10.1021/jo900016j CCC: $40.75 2009 American Chemical Society
Published on Web 02/26/2009

Synthesis of an N-Glycan Octasaccharide
FIGURE 1. Retrosynthesis of octasaccharide A.
suitable for enzymatic elongation and coupling to proteins for
function study.
on the inversion of carbohydrate hydroxyl groups.11 In the
procedure, the designed protecting groups were exactly available
for the formation of bisected N-glycans, and all the fragments
involved were easily prepared. On the basis of this procedure,
octasaccharide A, an N-glycan oligosaccharide of the bisecting
type, was synthesized conveniently.
However, the synthesis of bisected N-glycans is very difficult
due to the sterically crowded ꢀ-mannosyl center. Only a few
groups have developed synthetic approaches to this important
class of compounds.6 Among them, Unverzagt’s strategy for
introduction of the bisecting GlcNAc residue has been proven
the most effective.6 In fact, the construction of the central ꢀ-D-
mannosidic linkage with proper protection is the other choke
point for the synthesis of bisected N-glycans. As we know,
methods for the construction of ꢀ-D-mannosidic linkage include
the intramolecular aglycon delivery,7 direct ꢀ-D-mannoside
coupling protocol,8 and epimerization of ꢀ-D-glucopyranosides
at the C-2 position by SN2 inversion9 or by sequential oxidation/
reduction routes.10 Some of these methods have been applied
to the synthesis of N-glycan oligosaccharides. However, the
majority of these approaches need some precious glycosyl
donors or tedious protection-deprotection manipulation. In this
paper, we have described a convenient procedure to construct
the central ꢀ-D-mannoside according to Ramstro¨m’s new finding
Results and Discussion
For retrosynthesis of octasaccharide A (Figure 1), it was
disconnected to core trisaccharide B, disaccharide donor C, and
monosaccharide donor D according to Unverzagt’s strategy for
introduction of the bisecting GlcNAc residue.6 R1,3-Arm was
first installed followed by the introduction of the bisecting
GlcNAc residue, and R1,6-arm was installed in the final
glycosylation step. To ensure the introduction sequence, the
central ꢀ-mannoside in building block B was designed as
depicted in Figure 1. The 4,6-O-benzylidene would be removed
at a desired stage for the introduction of bisecting GlcNAc via
regioselective protection of OH-6. The new structure B would
permit good coupling to various building blocks for the synthesis
of bisected N-glycans.
The retrosynthesis of fragment B is depicted in Figure 2. The
intermediate E was designed to prepare B according to
Ramstro¨m’s new finding,11 in which the OH-3′′ was acetylated
as the neighboring ester group to the triflate. The transformation
from E into B was easily performed under the modified
Lattrell-Dax reaction conditions by the inversion of OH-23.
However, distinguishing OH-3′′ from OH-2′′ with two different
ester groups in the ꢀ-D-glucopyranoside seemed to be a
challenge. In fact, the regioselective protection of one of the
two similar secondary 2,3-dihydroxyl groups of ꢀ-D-glucopy-
ranoside was difficult to achieve in oligosaccharide synthesis.12
(6) (a) Eller, S.; Schuberth, R.; Gundel, G.; Seifert, J.; Unverzagt, C. Angew.
Chem., Int. Ed. 2007, 46, 4173. (b) Schuberth, R.; Unverzagt, C. Tetrahedron
Lett. 2005, 46, 4201. (c) Weiss, H.; Unverzagt, C. Angew. Chem., Int. Ed. 2003,
42, 4261. (d) Unverzagt, C.; Seifert, J. Tetrahedron Lett. 2000, 41, 4549.
(7) (a) Barresi, F.; Hindsgaul, O. Can. J. Chem. 1994, 72, 1447. (b) Stork,
G.; La Clair, J. L. J. Am. Chem. Soc. 1996, 118, 247. (c) Ito, Y.; Ohnishi, Y.;
Ogawa, T.; Nakahara, Y. Synlett 1998, 1102.
(8) (a) Dudkin, V. Y.; Crich, D. Tetrahedron Lett. 2003, 44, 1787. (b) Dudkin,
V. Y.; Miller, J. S.; Danishefsky, S. J. Tetrahedron Lett. 2003, 44, 1791. (c)
Dudkin, V. Y.; Miller, J. S.; Danishefsky, S. J. J. Am. Chem. Soc. 2004, 126,
736. (d) Mandai, H.; Mukaiyama, T. Chem. Lett. 2005, 34, 702. (e) Tanaka, S.;
Takashina, M.; Tokimoto, H.; Fujimoto, Y.; Tanaka, K.; Fukase, K. Synlett 2005,
15, 2325.
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1996, 37, 8795. (b) Li, B.; Zeng, Y.; Hauser, S.; Song, H.; Wang, L. X. J. Am.
Chem. Soc. 2005, 127, 9692.
(10) (a) Shaban, M. A.; Jeanloz, R. W. Carbohydr. Res. 1976, 52, 115. (b)
Warren, C. D.; Auge, C.; Laver, M. L.; Suzuki, S.; Power, D.; Jeanloz, R. W.
Carbohydr. Res. 1980, 82, 71. (c) Kerekgyarto, J.; Van der Ven, J. G.; Kamerling,
J. P.; Liptak, A.; Vliegenthart, J. F. Carbohydr. Res. 1993, 238, 135. (d) Hiro,
S.; Usuki, Y.; Iio, H. Carbohydr. Res. 2006, 341, 1796.
(11) (a) Dong, H.; Pei, Z.; Angelin, M.; Bystroem, S.; Ramstro¨m, O. J. Org.
Chem. 2007, 72, 3694. (b) Dong, H.; Pei, Z.; Ramstro¨m, O. J. Org. Chem. 2006,
71, 3306.
(12) Varki, A. Glycobiology 1993, 3, 97.
J. Org. Chem. Vol. 74, No. 6, 2009 2509

Wang et al.
SCHEME 1. Synthesis of Trisaccharide B
FIGURE 2. Retrosynthesis of trisaccharide B.
Herein, we explored a new strategy. The key intermediate
orthoester F, 3′′-O-chloroacetyl-4′′,6′′-O-benzylidene-R-D-glu-
copyranose 1′′,2′′-(chloromethyl 3′,6′-di-O-benzyl-2′-deoxy-2′-
phthalimido-ꢀ-D-glucopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-
2-phthalimido-ꢀ-D-glucopyranosylazide-4′-yl orthoacetate) was
designed on the basis of the following considerations: (1)
Distinguishing OH-3′′ from OH-2′′ with two different ester
groups in the ꢀ-D-glucopyranoside could be easily achieved by
switching chloroacetyl in F to the acetyl group before 1,
2-orthoester was transformed into the corresponding 3-O-acetyl-
2-O-chloroacetyl-ꢀ-D-glucopyranoside.13 (2) The cyclic 4′′,6′′-
O-benzylidene protecting group was selected to distinguish OH-
4′′ and OH-6′′ from OH-3′′ for the introduction of three
substituents to the ꢀ-mannosyl center. (3) The formation of
orthoester F depended critically on the presence of the cyclic
4,6-O-benzylidene protecting groups which stabilized the in-
termediate oxycarbenium ion.14 (4) The preparation of orthoester
F could be conveniently carried out by designing ethyl 4,6-O-
benzylidene-2,3-di-O-chloroacetyl-1-thio-ꢀ-D-glucopyrano-
side 1 as the glycosyl donor, which could be easily synthesized
from the known compound 3.15
The preparation of the core trisaccharide B started from
compound 315 according to the above retrosynthetic analysis
(Scheme 1). The unmasked hydroxyls of compound 3 were
protected with a chloroacetyl group to afford donor 1, which
was treated with disaccharide 216 in the presence of NIS/TfOH
(0.1 equiv), giving the desired orthoester 4 (key intermediate
F) in 82% yield. The chloroacetyl ester of 4 was removed by
using NaOMe, and then the unmasked hydroxyl was acetylated
to afford orthoester 5 (95%). In the following attempt to
transform orthoester 5 into the corresponding trisaccharide 6,
we found that 4,6-O-benzylidene-protected-orthoester 5 was very
stable even in the presence of 0.5 equiv of TMSOTf at -30
°C. Then we tried TfOH as the promoter, as a result, part of
the orthoester 5 was observed to be transformed into trisaccha-
ride 6 in the presence of 0.5 equiv of TfOH at -30 °C after
1 h. When more TfOH was used (1.0 equiv), trisaccharide 6
was afforded in 93% yield. After selective removal of the
chloroacetyl group in 6,17 the free hydroxyl group of 7 was
inverted in a two-step procedure to afford the desired ꢀ-man-
noside.11 First, the 2′′-OH of 7 was converted to a good leaving
group using triflic anhydride-pyridine and then was substituted
by an intramolecular nucleophilic attack of the neighboring
acetyl moiety. The SN2 reaction proceeded smoothly by warming
the solution of the triflate intermediate in H2O/DMF (1:20) to
50 °C, providing the key fragment B and isomer 8 as a mixture
in a very satisfying yield (94%). The ratio of B and 8 was 12.7:1
1
(determined by H NMR analysis), and pure B was obtained
after careful column chromatography separation. The anomeric
configuration of B was unequivocally deduced from the singlet
at 4.85 ppm corresponding to the ꢀ-mannosyl anomeric proton
and the JC1′′,H1′′ ) 161.8 Hz which is a typical range for ꢀ-D-
mannopyranoside. By the strategy of formation rearrangement
4,6-O-benzylidene-protected-1,2-orthoester, the core trisaccha-
ride B was synthesized conveniently (62% for 7 steps).
The synthesis of disaccharide C is summarized in Scheme
2; the OH-6 of monosaccharide 918 was selectively acetylated
to afford 10 (82%). The latter was then treated with donor 1119
in the presence of TMSOTf to afford disaccharide 12 (85%).
(13) (a) Ogawa, T.; Beppu, K.; Nakabayashi, S. Carbohydr. Res. 1981, 93,
C6. (b) Urban, F. J.; Moore, B. S.; Breitenbach, R. Tetrahedron Lett. 1990, 31,
4421. (c) Wang, W.; Kong, F. J. Org. Chem. 1998, 63, 5744.
(14) Yu, H.; Ensley, H. E. Tetrahedron Lett. 2003, 44, 9363.
(15) (a) Hou, S. J.; Xu, P.; Zhou, L.; Yu, De Q.; Lei, P. S.; Zou, C. C. Chin.
Chem. Lett. 2006, 17, 183. (b) Ennis, S. C.; Gridley, J. J.; Osborn, H. M. I.;
Spackman, D. G. Synlett 2000, 11, 1593.
(17) Nicolaou, K. C.; Caulfield, T. J.; Kataoka, H.; Stylianides, N. A. J. Am.
Chem. Soc. 1990, 112, 3693.
(18) Baeschlin, D. K.; Green, L. G.; Hahn, M. G.; Hinzen, B.; Ince, S. J.;
Ley, S. V. Tetrahedron: Asymmetry 2000, 11, 173.
(19) (a) Macmillan, D.; Daines, A. M.; Bayrhuber, M.; Flitsch, S. L. Org.
Lett. 2002, 4, 1467. (b) Grundler, G.; Schmidt, R. R. Carbohydr. Res. 1985,
135, 203.
(16) (a) Unverzagt, C. Chem. Eur. J. 2003, 9, 1369. (b) Pratt, M. R.; Bertozzi,
C. R. J. Am. Chem. Soc. 2003, 125, 6149.
2510 J. Org. Chem. Vol. 74, No. 6, 2009

Synthesis of an N-Glycan Octasaccharide
SCHEME 2. Synthesis of Disaccharide Donor C
SCHEME 3. Synthesis of Octasaccharide A
After cleavage of the biacetal function in 12 (91%), the
unmasked hydroxyl was acetylated to afford disaccharide donor
C in 96% yield.
Conclusions
In summary, we have developed a new and concise method
for the synthesis of bisected N-glycan of the bisecting type based
on the 4,6-O-benzylidene-protected-1,2-orthoesters strategy,
which greatly simplified the procedure for introduction of the
central ꢀ-mannoside and assured the sequence for three sub-
stituents. Notably, all the fragments involved were easily
prepared. Owing to its simplicity and mild reaction conditions,
the results of the present investigation will be widely used in
the synthesis of N-glycans.
Monosaccharide D was prepared in five steps from com-
mercially available glucosamine hydrochloride.20
With the core trisaccharide B, disaccharide donor C and donor
D in hand, the synthesis of octasaccharide A was then carried
out (Scheme 3).6 Treating donor C with trisaccharide B was
first performed to install the disaccharide residue on to the OH-
3′′of central ꢀ-mannoside in the presence of NIS/TfOH to afford
pentasaccharide 14 in 90% yield (path 1). Compared with the
corresponding glycosyl trichloroacetimidate donor,21 thiogly-
coside donor C proved more effective in the glycosylation.
While compound B was not easily separated from the mixture
of B and 8. We then tried another route (path 2): After cleaving
the acetyl esters of B and 8, the product was treated with
disaccharide donor C in the presence of NIS/TfOH. To our
delight, the glycosylation reaction preferred the equatorial OH-
3′′ of benzylidene protected ꢀ-mannoside to provide the only
product R-(1f3)-linked pentasaccharide,21 in which the OH-
2′′ of ꢀ-mannoside was then acetylated. Pentasaccharide 14 was
also provided in 79% yield over 3 steps (Path 2). After cleavage
of the benzylidene with TsOH in CH3CN, the OH-6′′ of the
ꢀ-mannoside was selectively protected with the chloroacetyl
group to afford 16. Monosaccharide donor D was treated with
16 to introduce the bisecting residue in the presence of NIS/
TfOH, providing 17 in 78% yield. After selective removal of
the chloroacetyl group (91%), installation of the other disac-
charide residue onto OH-6′′ of central ꢀ-mannoside was finally
carried out to provide the bisected N-glycan octasaccharide A
in 74% yield. The structure of octasaccharide A was well
characterized by 1D NMR and 2D NMR experiments (1H NMR,
13C NMR, COSY, TOCXY, HMQC, HMBC).
Experimental Section
3′′-O-Chloroacetyl-4′′,6′′-O-benzylidene-r-D-glucopyranose
1′′,2′′-(Chloromethyl 3′,6′-Di-O-benzyl-2′-deoxy-2′-phthalimido-
ꢀ-D-glucopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
ꢀ-D-glucopyranosylazide-4′-yl Orthoacetate) (4). After a mixture
of 1 (8.28 g, 17.80 mmol), 216 (8.78 g, 8.90 mmol), and 4 Å
molecular sieves in freshly distilled CH2Cl2 (100 mL) was stirred
for 3 h at rt and then cooled to -30 °C, NIS (6.03 g, 26.70 mmol)
was added. TfOH (79 µL, 0.89 mmol) was then added dropwise
over a 1 min period. The reaction mixture was stirred for 30 min
at -30 °C and then filtered through Celite. The filtrate was diluted
with CH2Cl2 and washed with aqueousueous NaHCO3, aqueous
Na2S2O3, and brine. The organic layer was dried over Na2SO4,
filtered, and concentrated. The crude mixture was purified by
column chromatography on silica gel (toluene/acetone ) 50:1) to
afford 4 (10.1 g, 82%) as a light yellow foam. [R]20 ) +20.8 (c
0.6, CHCl3). H NMR (600 MHz, CDCl3): δ 7.88-6.75 (m, 33
D
1
H), 6.06 (d, 1 H, J ) 5.5 Hz), 5.48 (s, 1 H), 5.44 (dd, 1 H, J ) 9.3,
4.4 Hz), 5.28 (d, 1 H, J ) 8.2 Hz), 5.14 (d, 1 H, J ) 9.9 Hz),
4.86-4.81 (m, 2 H), 4.64 (d, 1 H, J ) 12.1 Hz), 4.56-4.50 (m, 5
H), 4.41 (dd, 1 H, J ) 11.0, 5.5 Hz), 4.31-4.23 (m, 3 H),
4.16-4.12 (m, 2 H), 4.08-4.03 (m, 2 H), 3.96-3.81 (m, 5 H,),
3.70-3.55 (m, 5 H), 3.43 (dd, 1 H, J ) 11.0, 3.3 Hz), 3.37 (d, 1
H, J ) 9.9 Hz), 3.26 (d, 1 H, J ) 9.4 Hz). 13C NMR (150 MHz,
13C3): δ 166.0, 138.5, 138.3, 138.2, 138.1, 136.5, 129.2, 129.0,
128.3, 128.3, 128.2, 128.1, 128.0, 127.8, 127.7, 127.5, 127.5, 127.4,
(20) (a) Myszka, H.; Bednarczyk, D.; Najder, M.; Kaca, W. Carbohydr. Res.
2003, 338, 133. (b) Silva, D. J.; Wang, H.; Allanson, N. M.; Jain, R. K.; Sofia,
M. J. J. Org. Chem. 1999, 64, 5926.
(21) Seifert, J.; Unverzagt, C. Tetrahedron Lett. 1996, 37, 6527.
J. Org. Chem. Vol. 74, No. 6, 2009 2511

Wang et al.
127.2, 126.9, 126.1, 125.3, 123.2, 119.6, 101.4, 99.3, 96.6, 85.5,
78.5, 77.7, 76.5, 76.3, 75.4, 75.3, 75.1, 74.5, 74.5, 73.6, 73.1, 72.8,
68.5, 67.7, 67.5, 62.7, 56.5, 55.1, 46.8, 40.5. HRMS (ESI) m/z:
calcd for C73H67Cl2N5O19Na [M + Na]+ 1410.3705; found,
1410.3738.
mmol) and thiourea (502 mg, 6.60 mmol). After the reaction mixture
was stirred for 10 h at rt, it was diluted with CH2Cl2 and washed
with aqueous 1 N HCl, aqueous NaHCO3, and brine. The organic
phase was dried over Na2SO4, filtered, and concentrated. The crude
mixture was purified by column chromatography on silica gel
(EtOAc/petroleum ether ) 1:3) to afford 7 (1.55 g, 92%) as a white
foam. [R]20D ) +25.5 (c 0.1, CHCl3). 1H NMR (600 MHz, CDCl3):
δ 7.84-7.55 (m, 8 H), 7.41-7.26 (m, 15 H), 7.02-6.89 (m, 7 H),
6.81-6.76 (m, 3 H), 5.38 (s, 1 H), 5.28 (d, 1 H, J ) 8.4 Hz), 5.17
(d, 1 H, J ) 9.4 Hz), 5.08 (t, 1 H, J ) 9.5 Hz), 4.86 (d, 1 H, J )
12.7 Hz), 4.80 (d, 1 H, J ) 12.2 Hz), 4.69 (d, 1 H, J ) 7.7 Hz),
4.61 (d, 1 H, J ) 12.1 Hz), 4.56-4.49 (m, 4 H), 4.41-4.37 (m, 2
H), 4.28 (dd, 1 H, J ) 9.6, 9.2 Hz), 4.22 (dd, 1 H, J ) 11.0, 8.7
Hz), 4.18-4.09 (m, 3 H), 4.06 (dd, 1 H, J ) 10.6, 9.2 Hz), 3.82
(dd, 1 H, J ) 11.5, 2.3 Hz), 3.66 (dd, 1 H, J ) 11.5, 1.9 Hz), 3.58
(d, 1 H, J ) 11.0 Hz), 3.52-3.37 (m, 6 H), 3.33-3.32 (m, 1 H),
3.23-3.19 (m, 1 H), 2.12 (s, 3 H). 13C NMR (150 MHz, CDCl3):
δ 171.0, 138.3, 138.1, 137.4, 136.9, 133.8, 131.5, 129.1, 128.6,
128.3, 128.2, 128.0, 127.9, 127.5, 127.3, 127.0, 126.2, 103.6, 101.4,
96.9, 85.6, 78.8, 78.4, 78.0, 76.7, 76.3, 74.9, 74.7, 74.5, 74.4, 74.3,
73.8, 73.3, 72.7, 68.5, 67.7, 67.2, 66.3, 56.5, 55.2, 21.0. HRMS
(ESI) m/z: calcd for C71H67N5O18Na [M + Na]+ 1300.4379; found,
1300.4387.
O-(2-O-Acetyl-4,6-O-benzylidene-ꢀ-D-mannopyranosyl)-(1f4)-
O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyranosyl)-
(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyra-
nosylazide (B) and O-(3-O-Acetyl-4,6-O-benzylidene-ꢀ-D-manno-
pyranosyl)-(1f4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-
D-glucopyranosyl)-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
ꢀ-D-glucopyranosylazide (8). To a solution of compound 7 (671
mg, 0.525 mmol) in distilled CH2Cl2 (10 mL) was added pyridine
(170 µL, 0.787 mmol). The reaction was cooled to -15 °C. Tf2O
(133 µL, 0.788 mmol) was then added dropwise over a 2 min
period, and the reaction mixture was stirred for 3 h. The reaction
was concentrated under high vacuum. The crude mixture was
dissolved in 20 mL of DMF, then 1 mL of H2O was added, and
the reaction mixture was stirred for 24 h at 50 °C. Then the reaction
mixture was concentrated under high vacuum and purified by flash
chromatography (EtOAc/petroleum ether ) 1:3) to afford B and 8
(631 mg, 94%) as a white foam. The ratio of B and 8 was 12.7:1
(determined by NMR analysis). Pure B was obtained by column
chromatography on silica gel (300-400 mesh, EtOAc/petroleum
ether ) 1:6) and pure 8 was obtained by HPLC (CH3CN:H2O).
Compound B. [R]20D ) +0.6 (c 0.2, CHCl3). 1H NMR (600 MHz,
DMSO-d6): δ 7.95-6.75 (m, 33 H), 5.61 (s, 1 H), 5.44 (d, 1 H, J
) 6.8 Hz), 5.33 (d, 1 H, J ) 3.7 Hz), 5.30 (d, 1 H, J ) 9.2 Hz),
5.25 (d, 1 H, J ) 8.7 Hz), 4.85 (d, 1 H, J ) 11.4 Hz), 4.85 (s, 1
H), 4.81 (d, 1 H, J ) 12.4 Hz), 4.63 (d, 1 H, J ) 12.4 Hz), 4.56
(d, 1 H, J ) 12.4 Hz), 4.44 (d, 1 H, J ) 11.9 Hz), 4.41 (d, 1 H, J
) 11.9 Hz), 4.38 (d, 1 H, J ) 12.4 Hz), 4.28 (d, 1 H, J ) 12.4
Hz), 4.15-4.01 (m, 5 H), 3.97 (dd, 1 H, J ) 10.6, 8.2 Hz),
3.83-3.74 (m, 3 H), 3.70-3.65 (m, 2 H) 3.63-3.59 (m, 2 H), 3.52
(d, 1 H, J ) 10.5 Hz), 3.39 (dd, 1 H, J ) 11.5, 3.7 Hz), 3.27-3.26
(m, 1 H), 3.20-3.16 (m, 1 H), 2.05 (s, 3 H). 13C NMR (150 MHz,
DMSO-d6): δ 169.6, 168.1, 167.4, 167.2, 167.1, 138.4, 138.1, 138.0,
137.9, 137.7, 134.9, 134.8, 130.7, 130.6, 128.3, 128.2, 128.0, 127.9,
127.8, 127.6, 127.5, 127.4, 127.3, 127.2, 126.3, 123.4, 100.9, 98.3,
96.3, 84.9, 78.4, 77.2, 76.3, 76.2, 75.6, 74.9, 74.1, 73.9, 73.7, 72.0,
71.9, 71.8, 68.1, 67.7, 67.6, 66.6, 56.0, 54.6, 20.9. JC1′′,H1′′ ) 161.8
Hz. HRMS (ESI) m/z: calcd for C71H67N5O18Na [M + Na]+
1300.4379; found, 1300.4337.
3′′-O-Acetyl-4′′,6′′-O-benzylidene-r-D-glucopyranose 1′′,2′′-
(Chloromethyl 3′,6′-Di-O-benzyl-2′-deoxy-2′-phthalimido-ꢀ-D-
glucopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
ꢀ-D-glucopyranosylazide-4′-yl Orthoacetate) (5). To a solution
of 4 (8.33 g, 6.00 mmol) in 1:3 CH2Cl2/CH3OH (400 mL) was
added a 1 M solution of NaOMe in MeOH until a pH of 10 was
reached. The reaction was stirred for 1 h at rt. Then the solution
was neutralized with ion-exchange resin, filtered, and concentrated.
The residue was dissolved in CH2Cl2 (100 mL). Pyridine (5 mL),
Ac2O (1.2 mL, 12.00 mmol), and DMAP (100 mg) were added.
The mixture was stirred for 5 h at rt. One milliliter of methanol
was injected into the solution, and the solution was stirred for
another 0.5 h at rt. The solution was diluted with CH2Cl2 and
washed with aqueous 1 N HCl, saturated aqueous NaHCO3,
and brine. The organic phase was dried over Na2SO4, filtered, and
concentrated. The crude mixture was purified by column chroma-
tography on silica gel (EtOAc/petroleum ether ) 1:2) to yield 5
(7.69 g, 95%) as a white foam. [R]20D ) +22.5 (c 0.5, CHCl3). 1H
NMR (600 MHz, CDCl3): δ 7.83-6.76 (m, 33 H), 6.02 (d, 1H, J
) 5.5 Hz), 5.48 (s, 1 H), 5.34 (dd, 1 H, J ) 9.7, 4.1 Hz), 5.27 (d,
1 H, J ) 8.7 Hz), 5.13 (d, 1 H, J ) 9.2 Hz), 4.86-4.83 (m, 2 H),
4.64 (d, 1 H, J ) 11.9 Hz), 4.56-4.53 (m, 2 H), 4.51-4.48 (m, 3
H), 4.41 (dd, 1 H, J ) 11.0, 5.5 Hz), 4.31-4.27 (m, 2 H), 4.23
(dd, 1 H, J ) 9.6, 8.7 Hz), 4.15-4.12 (m, 2 H), 4.05-4.00 (m, 2
H), 3.87-3.81 (m, 3 H), 3.72-3.65 (m, 3 H), 3.62 (dd, 1 H, J )
9.5, 9.1 Hz), 3.55 (d, 1 H, J ) 10.6 Hz), 3.42 (dd, 1 H, J ) 11.5,
3.7 Hz), 3.37-3.35 (m, 1 H), 3.29-3.27 (m, 1 H), 1.98 (s, 3 H).
13C NMR (150 MHz, CDCl3): δ 169.5, 167.7, 138.5, 138.4, 138.3,
138.1, 137.9, 136.8, 134.0, 133.9, 133.7, 131.8, 131.5, 131.2, 129.2,
129.0, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 127.6, 127.5, 127.4,
127.1, 126.9, 126.1, 125.3, 123.6, 123.3, 119.5, 101.4, 99.2, 96.6,
85.5, 78.6, 78.2, 76.4, 75.5, 75.1, 74.6, 74.5, 73.6, 73.24, 73.1, 72.8,
68.6, 67.7, 67.6, 56.6, 55.2, 46.9, 20.9. HRMS (ESI) m/z: calcd
for C73H68ClN5O19Na [M + Na]+ 1376.4095; found, 1376.4105.
O-(3-O-Acetyl-4,6-O-benzylidene-2-chloroacetyl-ꢀ-D-glucopy-
ranosyl)-(1f4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-
glucopyranosyl)-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
ꢀ-D-glucopyranosylazide (6). After a mixture of 5 (1.25 g, 0.92
mmol) and 4 Å molecular sieves in freshly distilled CH2Cl2 (100
mL) was stirred at rt for 3 h and then cooled to -30 °C, TfOH (82
µL, 0.92 mmol) was added dropwise over a 5 min period. The
mixture was stirred for 1 h at -30 °C. The reaction mixture was
neutralized with Et3N and then filtered through Celite; the filtrate
was concentrated and purified by column chromatography on silica
gel (toluene/acetone ) 50:1) to afford 6 (1.16 g, 93%) as a white
foam. [R]20D ) -15.6 (c 0.5, CHCl3). 1H NMR (600 MHz, CDCl3):
δ 7.90-6.77 (m, 33 H), 5.38 (s, 1 H), 5.27 (d, 1 H, J ) 8.2 Hz),
5.18 (t, 1 H, J ) 9.6 Hz), 5.17 (d, 1 H, J ) 9.2 Hz), 4.96 (dd, 1 H,
J ) 9.2, 8.2 Hz), 4.88 (d, 1 H, J ) 12.8 Hz), 4.74 (d, 2 H, J ) 7.7
Hz), 4.66 (d, 1 H, J ) 11.9 Hz), 4.56-4.45 (m, 4 H), 4.39 (d, 1H,
J ) 11.9 Hz), 4.28-4.12 (m, 5 H), 4.05 (dd, 1 H, J ) 10.1, 9.6
Hz), 3.93 (d, 1H, J ) 14.7 Hz), 3.89 (d, 1H, J ) 14.6 Hz), 3.65 (d,
1 H, J ) 10.5 Hz), 3.59-3.55 (m, 3 H), 3.44-3.38 (m, 3 H),
3.25-3.20 (m, 3 H), 2.03 (s, 3 H). 13C NMR (150 MHz, CDCl3):
δ 170.1, 166.2, 138.4, 138.1, 137.7, 128.7, 128.2, 128.1, 128.0,
127.9, 127.4, 101.5, 99.8, 96.8, 85.5, 78.3, 77.7, 76.4, 75.1, 74.6,
74.5, 73.4, 72.7, 71.8, 68.4, 67.7, 66.8, 66.0, 56.4, 55.2, 40.4, 20.7.
HRMS (ESI) m/z: calcd for C73H68ClN5O19Na [M + Na]+ 1376.4095;
found, 1376.4034.
Compound 8. [R]20D ) +5.3 (c 0.4, CHCl3). 1H NMR (600 MHz,
CDCl3): δ 7.88 (d, 1 H, J ) 5.5 Hz), 7.79-7.56 (m, 7 H),
7.44-7.41 (m, 2 H), 7.38-7.27 (m, 13 H), 7.30 (d, 2 H, J ) 7.3
Hz), 6.98-6.95 (m, 4 H), 6.93 (d, 1 H, J ) 6.9 Hz), 6.81-6.76
(m, 3 H), 5.43 (s, 1 H), 5.29 (d, 1 H, J ) 8.7 Hz), 5.18 (d, 1 H, J
) 9.6 Hz), 4.92 (dd, 1 H, J ) 10.1, 3.2 Hz), 4.86 (d, 1 H, J ) 12.8
Hz), 4.82 (d, 1 H, J ) 11.9 Hz), 4.79 (s, 1 H), 4.61 (d, 1 H, J )
11.9 Hz), 4.58-4.50 (m, 4 H), 4.41 (d, 1 H, J ) 12.4 Hz), 4.38
O-(3-O-Acetyl-4,6-O-benzylidene-ꢀ-D-glucopyranosyl)-(1f4)-
O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyranosyl)-
(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyra-
nosylazide (7). To a solution of 6 (1.79 g, 1.32 mmol) in 80 mL
of CH2Cl2/CH3OH (1:3) was added 2,6-lutidine (0.77 mL, 6.60
2512 J. Org. Chem. Vol. 74, No. 6, 2009

Synthesis of an N-Glycan Octasaccharide
(dd, 1 H, J ) 8.7, 1.8 Hz), 4.29 (dd, 1 H, J ) 9.6, 8.7 Hz), 4.25
(dd, 1 H, J ) 10.5, 8.2 Hz), 4.19-4.10 (m, 4 H), 4.06 (dd, 1 H, J
) 10.6, 9.6 Hz), 3.99 (dd, 1 H, J ) 10.1, 9.6 Hz), 3.68 (d, 1 H, J
) 10.1 Hz), 3.60-3.57 (m, 2 H), 3.53 (dd, 1 H, J ) 10.5, 10.1
Hz), 3.44 (d, 1 H, J ) 11.0, 3.2 Hz), 3.40 (dd, 1 H, J ) 10.1, 2.3
Hz), 3.28 (d, 1 H, J ) 9.6 Hz), 3.22-3.18 (m, 1 H), 2.14 (s, 3 H).
13C NMR (150 MHz, CDCl3): δ 170.2, 138.3, 138.2, 137.5, 137.1,
129.0, 128.6, 128.3, 128.2, 128.0, 127.9, 127.8, 127.5, 127.4, 127.3,
127.2, 127.0, 126.1, 101.7, 99.8, 96.8, 85.5, 78.3, 77.5, 76.3, 76.2,
75.2, 75.0, 74.6, 74.5, 74.4, 73.4, 72.7, 71.8, 69.3, 68.3, 67.7, 66.8,
56.4, 55.1, 21.0. HRMS (ESI) m/z: calcd for C71H67N5O18Na [M
+ Na]+ 1300.4379; found, 1300.4340.
CDCl3): δ 7.84 (dd, 2 H, J ) 4.6, 3.7 Hz), 7.74 (dd, 2 H, J ) 5.5,
2.7 Hz), 5.81 (dd, 1 H, J ) 10.6, 9.2 Hz), 5.46 (d, 1 H, J ) 8.7
Hz), 5.17 (t, 1 H, J ) 9.6 Hz), 4.91 (s, 1 H), 4.36 (dd, 1 H, J )
11.0, 8.7 Hz), 4.31 (dd, 1 H, J ) 12.4, 5.0 Hz), 4.19 (dd, 1 H, J )
12.4, 1.9 Hz), 4.05 (dd, 1 H, J ) 2.3, 0.9 Hz), 4.01 (d, 1 H, J )
11.4 Hz), 3.93-3.90 (m, 2 H, H-5), 3.86 (dd, 1 H, J ) 11.5, 6.0
Hz), 3.66 (dd, 1 H, J ) 8.7, 6.4 Hz), 3.43 (t, 1 H, J ) 9.6 Hz),
2.82 (d, 1 H, J ) 9.7 Hz), 2.73 (s, 1 H), 2.48-2.40 (m, 2 H), 2.13,
2.05, 2.02, 1.88 (s, 3 H each), 1.14 (t, 3 H, J ) 7.3 Hz). 13C NMR
(150 MHz, CDCl3): δ 171.2, 170.7, 170.1, 169.4, 134.3, 131.3,
97.1, 82.0, 80.4, 72.3, 70.9, 70.4, 70.3, 68.7, 68.5, 63.5, 61.7, 60.4,
54.4, 25.3, 21.0, 20.7, 20.6, 20.4, 14.5, 14.2. HRMS (ESI) m/z:
calcd for C30H37NO15SNa [M + Na]+ 706.1782; found, 706.1792.
Ethyl 3,4,6-Tri-O-acetyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-ph-
thalimido-ꢀ-D-glucopyranosyl)-1-thio-r-D-mannopyranoside (C).
Compound 13 (1.62 g, 2.37 mmol) and pyridine (1.9 mL, 23.70
mmol) were dissolved in CH2Cl2 (40 mL), and the solution was
cooled to 0 °C. Ac2O (1.1 mL, 11.85 mmol) and 100 mg of DMAP
were added, and the reaction was stirred for 2 h at rt. Subsequently,
the solution was diluted with CH2Cl2, washed with aqueous 1 N
HCl, aqueous NaHCO3, and brine. The organic phase was dried
over Na2SO4, concentrated, and purified by column chromatography
(2′S,3′S)-Ethyl 3-O,4-O-[2′,3′-Dimethoxybutan-2′,3′-diyl]-6-
O-acetyl-1-thio-r-D-mannopyranoside (10). To a solution of 918
(13.54 g, 40.0 mmol) in CH2Cl2 (300 mL) was added pyridine (13
mL, 160.0 mmol), and the resulting mixture was cooled to -15
°C. Ac2O (4.7 mL, 50 mmol) was added dropwise over 5 min. The
reaction was stirred for 24 h. Subsequently, the solution was diluted
with CH2Cl2 and washed with aqueous 1 N HCl, aqueous NaHCO3,
and brine. The organic phase was dried over Na2SO4, concentrated,
and purified by column chromatography on silica gel (EtOAc/
petroleum ether ) 1:1) to afford 10 (12.42 g, 82%) as an oil. [R]20
D
) +287.5 (c 0.4, CHCl3). 1H NMR (600 MHz, CDCl3): δ 5.33 (s,
1 H), 4.35-4.31 (m, 2 H), 4.26 (dd, 1 H, J ) 11.9, 5.9 Hz), 4.09
(t, 1 H, J ) 10.1 Hz), 4.02 (dd, 1 H, J ) 3.2, 1.4 Hz), 3.97 (dd, 1
H, J ) 10.4, 2.8 Hz), 3.27 (s, 3 H), 3.22 (s, 3 H), 2.71-2.56 (m,
2 H), 2.07 (s, 3 H), 1.32 (s, 3 H), 1.30 (t, 3 H, J ) 7.3 Hz), 1.28
(s, 3 H). 13C NMR (150 MHz, CDCl3): δ 170.8, 100.4, 100.0, 84.5,
71.0, 68.8, 68.6, 63.5, 62.6, 48.0, 47.8, 25.1, 20.8, 17.7, 17.6, 14.9.
HRMS (ESI) m/z: calcd for C16H28O8SNa [M + Na]+ 403.1403;
found, 403.1405.
on silica gel (EtOAc/petroleum ether ) 1:1) to afford C (1.74 g,
1
96%) as a white foam. [R]20 ) +23.3 (c 0.6, CHCl3). H NMR
D
(600 MHz, CDCl3): δ 7.82 (dd, 2 H, J ) 5.5, 3.2 Hz), 7.72 (dd, 2
H, J ) 5.5, 3.2 Hz), 5.80 (dd, 1 H, J ) 10.6, 9.2 Hz), 5.40 (d, 1
H, J ) 8.7 Hz), 5.16 (dd, 1 H, J ) 10.1, 9.1 Hz), 5.12 (dd, 1 H,
J ) 11.0, 10.1 Hz), 4.95 (d, 1 H, J ) 1.4 Hz), 4.89 (dd, 1 H, J )
10.1, 3.2 Hz), 4.43 (dd, 1 H, J ) 10.6, 8.7 Hz), 4.31 (dd, 1 H, J )
12.4, 5.0 Hz), 4.27 (dd, 1 H, J ) 3.2, 1.9 Hz), 4.09 (dd, 1 H, J )
11.9, 2.3 Hz), 4.07-4.04 (m, 1 H), 3.85-3.83 (m, 1 H), 3.76 (dd,
1 H, J ) 12.4, 5.5 Hz), 3.64 (dd, 1 H, J ) 11.9, 2.3 Hz), 2.52-2.42
(m, 2 H), 2.12, 2.04, 2.03, 2.00, 1.99, 1.89 (s, 3 H each), 1.19 (t,
3 H, J ) 7.3 Hz). 13C NMR (150 MHz, CDCl3): δ 170.7, 170.6,
170.4, 170.2, 169.4, 169.2, 134.2, 131.5, 123.4, 96.5, 81.4, 75.9,
72.0, 70.5, 70.4, 68.8, 68.5, 65.9, 62.4, 61.9, 54.3, 25.4, 20.7, 20.6,
20.5, 14.5. HRMS (ESI) m/z: calcd for C34H41NO17SNa [M + Na]+
790.1993; found, 790.1978.
(2′S,3′S)-Ethyl 3-O,4-O-[2′,3′-Dimethoxybutan-2′,3′-diyl]-6-
O-acetyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-glu-
copyranosyl)-1-thio-r-D-mannopyranoside (12). A solution of
1119 (3.57 g, 6.16 mmol), 10 (1.95 g, 5.13 mmol), and flame-
activated 4 Å molecular sieves were stirred in freshly distilled
CH2Cl2 (50 mL) under an Ar atmosphere for 0.5 h at rt. The reaction
mixture was then cooled to -30 °C and stirred for 5 min, and then
TMSOTf (134 µL, 0.77 mmol) was injected into the solution. The
reaction mixture was stirred for 30 min at -30 °C; then the solution
was quenched with triethylamine, stirred at rt for 0.5 h, and then
filtered through Celite and concentrated. The crude mixture was
purified by column chromatography on silica gel (EtOAc/petroleum
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyra-
nosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-mannopyranosyl)-(1f3)-
O-(2-O-acetyl-4,6-O-benzylidene-ꢀ-D-mannopyranosyl)-(1f4)-
O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyranosyl)-
(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-
glucopyranosylazide (14) Path 1: After a mixture of B (311 mg,
0.243 mmol), C (280 mg, 0.365 mmol), and 4 Å molecular sieves
in freshly distilled CH2Cl2 (15 mL) was stirred at rt for 3.0 h and
then cooled to -30 °C, NIS (110 mg, 0.486 mmol) was added.
TfOH (4 µL, 0.049 mmol) was then added dropwise. The reaction
mixture was stirred for 30 min at -30 °C. The reaction mixture
was then filtered through Celite, and the filtrate was diluted with
CH2Cl2 and washed with aqueous NaHCO3, aqueous Na2S2O3, and
brine. The organic layer was dried over Na2SO4, filtered, and
concentrated. The crude mixture was purified by column chroma-
tography on silica gel (EtOAc/petroleum ether ) 1:1) to afford 14
ether ) 1:1) to afford 12 (3.48 g, 85%) as a white foam. [R]20
)
D
+45.2 (c 0.5, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.81 (dd, 2
H, J ) 5.0, 2.7 Hz), 7.70 (dd, 2 H, J ) 5.5, 2.8 Hz), 5.83 (dd, 1
H, J ) 10.6, 9.2 Hz), 5.55 (d, 1 H, J ) 8.3 Hz), 5.23 (dd, 1 H, J
) 10.1, 9.2 Hz), 5.12 (s, 1 H), 4.49 (dd, 1 H, J ) 10.6, 8.2 Hz),
4.36 (dd, 1 H, J ) 12.4, 4.1 Hz), 4.21 (dd, 1 H, J ) 12.4, 2.3 Hz),
4.08-4.04 (m, 1 H), 4.03 (dd, 1 H, J ) 2.8, 1.4 Hz), 3.99 (dd, 1
H, J ) 11.9, 1.8 Hz), 3.87-3.84 (m, 2 H), 3.73 (t, 1 H, J ) 10.1
Hz), 3.46 (dd, 1 H, J ) 11.9, 7.3 Hz), 3.20 (s, 3 H), 3.12 (s, 3 H),
2.55-2.44 (m, 2 H), 2.12, 2.03, 1.94, 1.87 (s, 3 H each), 1.19 (t,
3 H, J ) 3.2 Hz), 1.18, 1.14 (s, 3 H each). 13C NMR (150 MHz,
CDCl3): δ 170.8, 170.6, 170.2, 169.4, 134.1, 131.6, 129.0, 128.2,
125.3, 123.3, 100.2, 99.5, 96.0, 82.4, 76.3, 72.1, 70.6, 68.7, 67.4,
63.6, 63.0, 61.8, 54.0, 47.9, 47.6, 25.8, 21.4, 20.8, 20.7, 20.6, 20.5,
17.6, 17.4, 14.8. HRMS (ESI) m/z: calcd for C36H47NO17SNa [M
+ Na]+ 820.2462; found, 820.2487.
(434 mg, 90%) as a white foam. [R]20 ) +23.4 (c 0.1, CHCl3).
D
1H NMR (600 MHz, CDCl3): δ 7.87-6.78 (m, 37 H), 5.46 (dd, 2
H, J ) 10.3, 9.9 Hz), 5.26 (d, 1 H, J ) 8.0 Hz), 5.20 (d, 1 H, J )
3.7 Hz), 5.16 (d, 1 H, J ) 9.2 Hz), 5.02 (dd, 1 H, J ) 10.3, 9.9
Hz), 4.97 (dd, 1 H, J ) 9.9, 9.1 Hz), 4.94 (s, 1 H), 4.90-4.88 (m,
2 H), 4.84 (dd, 1 H, J ) 10.3, 3.3 Hz), 4.82 (d, 1 H, J ) 11.7 Hz),
4.66 (d, 1 H, J ) 12.1 Hz), 4.53-4.47 (m, 4 H), 4.38 (d, 1 H, J )
12.1 Hz), 4.34 (d, 1 H, J ) 12.1 Hz), 4.27-4.16 (m, 8 H), 4.04
(dd, 1 H, J ) 10.6, 9.5 Hz), 4.01 (dd, 1 H, J ) 3.3, 1.9 Hz), 3.95
(dd, 1 H, J ) 12.5, 3.7 Hz), 3.85-3.82 (m, 1 H), 3.74 (t, 1 H, J )
9.5 Hz), 3.71-3.64 (m, 4 H), 3.61-3.55 (m 3 H), 3.49 (t, 1 H, J
) 10.3 Hz), 3.42-3.39 (m, 2 H), 3.21-3.19 (m, 1 H), 3.04-2.99
Ethyl 6-O-Acetyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
ꢀ-D-glucopyranosyl)-1-thio-r-D-mannopyranoside (13). To a
solution of 12 (3.48 g, 4.36 mmol) in CH2Cl2 (10 mL) were added
9 mL of TFA and 1 mL of H2O. The reaction mixture was stirred
at rt until the starting materials were consumed. Subsequently, the
solution was diluted with CH2Cl2 and washed with H2O, aqueous
NaHCO3, and brine. The organic phase was dried over Na2SO4,
concentrated, and purified by column chromatography on silica gel
(m, 1 H), 2.15, 2.05, 2.04, 2.00, 1.98, 1.87, 1.85 (s, 3 H each). 13
C
(EtOAc/petroleum ether ) 2:1) to afford 13 (2.70 g, 91%) as a
NMR (150 MHz, CDCl3): δ 170.6, 170.5, 170.2, 170.1, 169.4,
169.2, 168.5, 167.5, 138.6, 138.5, 138.1, 137.8, 137.3, 134.2, 134.1,
1
white foam. [R]20 ) +59.4 (c 0.6, CHCl3). H NMR (600 MHz,
D
J. Org. Chem. Vol. 74, No. 6, 2009 2513

Wang et al.
133.9, 133.8, 131.7, 131.5, 131.4, 130.2, 128.9, 128.7, 128.6, 128.5,
128.3, 128.0, 127.9, 127.7, 127.6, 127.5, 127.2, 127.0, 126.9, 102.4,
98.4, 97.9, 97.1, 95.7, 85.5, 78.7, 78.1, 76.8, 76.6, 76.4, 75.3, 75.2,
74.6, 74.4, 74.3, 73.4, 72.9, 72.8, 71.1, 70.5, 70.4, 69.3, 68.8, 68.5,
68.4, 67.7, 67.4, 66.1, 65.4, 62.8, 61.0, 60.4, 56.4, 55.2, 54.0, 20.8,
20.7, 20.6, 20.5. HRMS (MALDI) m/z: calcd for C103H102N4O35Na
[M - N2 + Na]+ 1977.6217; found, 1977.6260.
74.3, 73.5, 73.2, 72.8, 71.9, 70.6, 70.3, 69.8, 69.0, 68.8, 67.8, 67.7,
67.3, 65.4, 64.4, 62.4, 61.9, 60.4, 56.4, 55.2, 54.3, 40.6, 29.7, 20.8,
20.7, 20.6, 20.4. HRMS (MALDI) m/z: calcd for C98H99ClN4O36Na
[M - N2 + Na]+ 1965.5620; found, 1965.5612.
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyra-
nosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-mannopyranosyl)-(1f3)-
[O-(3,4,6-tri-O-acetyl-2-deoxy-2-trifluoroacetamido-ꢀ-D-glucopy-
ranosyl)-(1f4)]-O-(2-O-acetyl-6-O-chloroacetyl-ꢀ-D-
mannopyranosyl)-(1f4)-O-(3,6-di-O-benzyl-2-deoxy-2-
phthalimido-ꢀ-D-glucopyranosyl)-(1f4)-3,6-di-O-benzyl-2-deoxy-
2-phthalimido-ꢀ-D-glucopyranosylazide (17). After a mixture of
16 (118 mg, 0.060 mmol), D (135 mg, 0.239 mmol), and 4 Å
molecular sieves in freshly distilled CH2Cl2 (10 mL) was stirred at
rt for 3 h and then cooled to -30 °C, NIS (68 mg, 0.299 mmol)
was added. TfOH (27 µL, 0.299 mmol) was then added dropwise
over 1 min. The reaction mixture was stirred for 2 h at -30 °C.
The reaction mixture was then filtered through Celite, and the filtrate
was diluted with CH2Cl2 and washed with aqueous NaHCO3,
aqueous Na2S2O3, and brine. The organic layer was dried over
Na2SO4, filtered, and concentrated. The crude mixture was purified
by column chromatography on silica gel (EtOAc/petroleum ether
) 3:2) to afford 17 (110 mg, 78%) as a white foam. [R]20D ) -22.9
(c 0.9, CHCl3).1H NMR (600 MHz, CDCl3): δ 8.03 (s, 1 H),
7.87-7.81 (m, 2 H), 7.73-7.66 (m, 7 H), 7.59 (d, 1 H, J ) 7.3
Hz), 7.31-7.24 (m, 7 H), 7.18 (d, 2 H, J ) 6.8 Hz), 7.13 (t, 2 H,
J ) 7.8 Hz), 6.94-6.92 (m, 5 H), 6.86-6.85 (m, 3 H), 6.78-6.73
(m, 3 H), 5.76 (dd, 1 H, J ) 11.0, 9.2 Hz), 5.46 (d, 1 H, J ) 8.3
Hz), 5.35 (dd, 1 H, J ) 10.1, 9.6 Hz), 5.28-5.25 (m, 2 H), 5.22
(d, 1 H, J ) 7.7 Hz), 5.13 (d, 1 H, J ) 9.6 Hz), 5.06 (d, 1 H, J )
3.2 Hz), 4.85-4.79 (m, 3 H), 4.69 (s, 1 H), 4.59 (d, 1 H, J ) 2.8
Hz), 4.57 (s, 1 H), 4.53 (dd, 1 H, J ) 12.4, 3.2 Hz), 4.51-4.41
(m, 6 H), 4.33 (d, 1 H, J ) 12.4 Hz), 4.29 (d, 1 H, J ) 11.9 Hz),
4.27-4.06 (m, 12 H), 4.03-3.97 (m, 2 H), 3.95-3.79 (m, 6 H),
3.73-3.70 (m, 1 H), 3.60 (d, 1 H, J ) 10.5 Hz), 3.53-3.48 (m, 2
H), 3.39-3.33 (m, 3 H), 3.18 (d, 1 H, J ) 9.6 Hz), 3.05-3.04 (m,
1 H), 2.13, 2.09, 2.09, 2.08, 2.05, 2.05, 2.03, 2.01, 1.99, 1.87 (s, 3
H each). 13C NMR (150 MHz, CDCl3): δ 171.0, 170.9, 170.8, 170.5,
170.3, 170.0, 169.6, 169.5, 169.4, 167.3, 138.7, 138.3, 138.0, 137.5,
133.7, 128.5, 128.3, 128.2, 128.0, 127.9, 127.8, 127.5, 127.4, 127.1,
127.0, 100.0, 98.7, 97.3, 97.2, 96.9, 85.5, 76.7, 76.5, 75.2, 74.6,
74.5, 74.3, 74.1, 74.0, 73.2, 72.9, 72.7, 72.5, 72.1, 71.4, 71.3, 70.7,
70.3, 69.4, 69.2, 69.0, 68.1, 67.6, 67.0, 65.5, 63.1, 62.4, 61.5, 56.3,
55.4, 55.1, 54.6, 40.5, 20.8, 20.7, 20.6, 20.5. HRMS (MALDI) m/z:
calcd for C112H115ClF3N5O44Na [M - N2 + Na]+ 2348.6448; found,
2348.6418.
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyra-
nosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-mannopyranosyl)-(1f3)-
[O-(3,4,6-tri-O-acetyl-2-deoxy-2-trifluoroacetamido-ꢀ-D-glucopy-
ranosyl)-(1f4)]-O-(2-O-acetyl-ꢀ-D-mannopyranosyl)-(1f4)-O-
(3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyranosyl)-
(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-
glucopyranosylazide (18). To a solution of 17 (78 mg, 0.033 mmol)
in methanol (10 mL) were added thiourea (50 mg, 0.66 mmol) and
2,6-lutidine (78 µL, 0.66 mmol). The reaction mixture was stirred
for 6 h at 64 °C. Then the solution was concentrated and diluted
with CH2Cl2, washed with aqueous 1 N HCl, aqueous NaHCO3,
and brine. The organic phase was dried over Na2SO4, filtered, and
concentrated. The crude mixture was purified by column chroma-
tography on silica gel (EtOAc/petroleum ether ) 2:1) to afford 18
(68 mg, 91%) as a white foam. [R]20D ) -23.3 (c 0.1, CHCl3). 1H
NMR (600 MHz, CDCl3): δ 7.88-6.76 (33 H), 5.78 (dd, 1 H, J )
10.5, 9.1 Hz), 5.51 (d, 1 H, J ) 8.7 Hz), 5.47 (t, 1 H, J ) 10.1
Hz), 5.30-5.25 (m, 3 H), 5.19 (dd, 1 H, J ) 9.7, 9.6 Hz), 5.15 (d,
1 H, J ) 9.6 Hz), 5.01 (d, 1 H, J ) 3.7 Hz), 4.88 (dd, 2 H, J )
12.4, 11.0 Hz), 4.81 (dd, 1 H, J ) 10.1, 3.2 Hz), 4.75 (t, 1 H, J )
9.2 Hz), 4.72 (s, 1 H), 4.61-4.46 (m, 7 H), 4.38 (d, 1 H, J ) 11.9
Hz), 4.34 (s, 1 H), 4.25-3.99 (m, 12 H), 3.85-3.73 (m, 5 H), 3.62
(dd, 2 H, J ) 9.6, 9.2 Hz), 3.55-3.50 (m, 2 H), 3.43-3.38 (m, 3
H), 3.30 (dd, 1 H, J ) 9.6, 3.2 Hz), 3.20 (d, 1 H, J ) 10.1 Hz),
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyra-
nosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-mannopyranosyl)-(1f3)-
O-(2-O-acetyl-ꢀ-D-mannopyranosyl)-(1f4)-O-(3,6-di-O-benzyl-
2-deoxy-2-phthalimido-ꢀ-D-glucopyranosyl)-(1f4)-3,6-di-O-
benzyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyranosylazide (15). To
a solution of 14 (722 mg, 0.364 mmol) in CH3CN (10 mL) was
added TsOH ·H2O (346 mg, 1.820 mmol). The reaction mixture
was stirred for 10 h at rt. Then the solution was neutralized with
concentrated Et3N. The crude mixture was diluted with CH2Cl2 and
washed with brine. The organic phase was dried over Na2SO4,
filtered, and concentrated. The crude mixture was purified by
column chromatography on silica gel (EtOAc/petroleum ether )
1:1) to afford 15 (574 mg, 83%) as a white foam. [R]20 ) -1.7
D
(c 0.5, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.89-6.74 (m, 32
H), 5.73 (dd, 1 H, J ) 10.6, 9.1 Hz), 5.37 (d, 1 H, J ) 8.4 Hz),
5.27 (d, 1 H, J ) 8.5 Hz), 5.16-5.13 (m, 4 H), 4.94 (d, 1 H, J )
1.8 Hz), 4.89-4.85 (m, 3 H), 4.61 (d, 1 H, J ) 12.1 Hz), 4.54-4.48
(m, 4 H), 4.42-4.38 (m, 3 H), 4.29 (dd, 1 H, J ) 12.5, 5.2 Hz),
4.25-4.09 (m, 7 H), 4.04 (dd, 1 H, J ) 10.6, 9.5 Hz), 3.84-3.78
(m, 4 H), 3.74 (dd, 1 H, J ) 9.9, 9.5 Hz), 3.71 (dd, 1 H, J ) 11.7,
2.9 Hz), 3.63 (d, 1 H, J ) 9.9 Hz), 3.57-3.53 (m, 3 H), 3.43-3.38
(m, 2 H), 3.35 (dd, 1 H, J ) 9.2, 3.7 Hz), 3.23-3.21 (m, 1 H),
3.02-2.99 (m, 1 H), 2.11 (s, 6 H), 2.03, 2.02, 2.02, 1.98, 1.86 (s,
3 H each). 13C NMR (150 MHz, CDCl3): δ 170.8, 170.7, 170.7,
170.2, 170.1, 169.4, 168.5, 167.6, 138.4, 138.1, 137.7, 134.4, 134.2,
134.0, 133.8, 131.7, 131.5, 131.3, 128.7, 128.4, 128.2, 128.1, 127.9,
127.6, 127.5, 127.4, 127.3, 127.0, 123.6, 123.4, 123.2, 98.4, 97.6,
97.1, 97.0, 85.5, 77.6, 77.4, 76.6, 75.4, 75.3, 74.7, 74.5, 74.4, 73.3,
72.9, 71.9, 70.6, 70.4, 69.9, 68.9, 68.3, 67.7, 67.2, 65.4, 62.4, 62.0,
56.4, 55.2, 54.4, 20.9, 20.8, 20.7, 20.6, 20.4. HRMS (MALDI) m/z
calcd for C96H98N4O35Na [M - N2 + Na]+ 1889.5904; found,
1889.5905.
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyra-
nosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-mannopyranosyl)-(1f3)-
O-(2-O-acetyl-6-O-chloroacetyl-ꢀ-D-mannopyranosyl)-(1f4)-O-
(3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyranosyl)-
(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-
glucopyranosylazide (16). Compound 15 (512 mg, 0.270 mmol)
and pyridine (0.12 mL, 1.35 mmol) were dissolved in CH2Cl2 (50
mL) and cooled to 0 °C. Chloroacetic anhydride (90%) (61 mg,
0.324 mmol) was added, and the reaction was stirred at 0 °C until
compound 15 was consumed. Subsequently, 0.1 mL of methanol
was injected into the solution and stirred for 0.5 h at rt. The solution
was washed with aqueous 1 N HCl, aqueous NaHCO3, and brine.
The organic phase was dried over Na2SO4, filtered, and concen-
trated. The crude mixture was purified by column chromatography
on silica gel (EtOAc/petroleum ether ) 2:3) to afford 16 (447 mg,
1
84%) as a white foam. [R]20 ) -7.6 (c 0.8, CHCl3). H NMR
(600 MHz, CDCl3): δ 7.85-D6.74 (m, 32 H), 5.72 (dd, 1 H, J )
10.6, 9.2 Hz), 5.38 (d, 1 H, J ) 8.4 Hz), 5.24 (d, 1 H, J ) 8.0 Hz),
5.18-5.13 (m, 4 H), 4.93 (d, 1 H, J ) 1.8 Hz), 4.87-4.84 (m, 3
H), 4.62 (d, 1 H, J ) 12.1 Hz), 4.57-4.46 (m, 5 H), 4.42-4.39
(m, 2 H), 4.36 (d, 1 H, J ) 12.5 Hz), 4.31 (dd, 1 H, J ) 12.1, 4.4
Hz), 4.23-4.08 (m, 8 H), 4.03 (dd, 1 H, J ) 10.6, 9.5 Hz), 3.99
(d, 1 H, J ) 15.0 Hz), 3.96 (d, 1 H, J ) 15.0 Hz), 3.84-3.79 (m,
4 H), 3.64-3.53 (m, 4 H), 3.41-3.34 (m, 3 H), 3.21-3.19 (m, 1
H), 3.15-3.12 (m, 1 H), 3.05 (brs, 1 H), 2.11, 2.10, 2.03, 2.02,
2.02, 1.97, 1.86 (s, 3H each). 13C NMR (150 MHz, CDCl3): δ 170.8,
170.7, 170.2, 170.1, 169.4, 169.4, 168.3, 168.2, 167.6, 138.7, 138.4,
138.1, 137.8, 134.4, 134.0, 133.8, 131.7, 131.5, 131.3, 128.6, 128.3,
128.0, 127.9, 127.6, 127.5, 127.1, 127.0, 126.0, 123.6, 123.3, 123.2,
98.6, 98.1, 97.0, 96.9, 85.5, 78.0, 76.8, 76.7, 76.5, 75.1, 74.6, 74.4,
2514 J. Org. Chem. Vol. 74, No. 6, 2009

Synthesis of an N-Glycan Octasaccharide
2.67 (d, 1 H, J ) 9.6 Hz), 2.13, 2.12, 2.07, 2.06, 2.05, 2.01, 2.01,
1.99, 1.98, 1.87 (s, 3H each). 13C NMR (150 MHz, CDCl3): δ 172.4,
171.2, 171.0, 170.8, 170.4, 170.3, 169.8, 169.6, 169.5, 168.5, 167.6,
167.3, 157.2, 157.0, 138.6, 138.4, 138.1, 137.3, 134.2, 134.1, 133.8,
131.6, 131.5, 131.3, 128.6, 128.4, 128.3, 128.0, 127.9, 127.6, 127.5,
127.0, 126.4, 123.8, 123.4, 123.2, 116.8, 114.9, 101.0, 98.4, 97.1,
97.0, 96.9, 85.5, 76.8, 76.6, 76.5, 76.3, 75.4, 74.7, 74.5, 74.3, 74.2,
73.6, 73.4, 72.9, 72.5, 72.4, 72.0, 71.3, 71.0, 70.5, 21.0, 20.9, 20.7,
20.6, 20.5, 20.3. HRMS (MALDI) m/z: calcd for C110H114F3N5O43Na
[M - N2 + Na]+ 2272.6732; found, 2272.6703.
Hz), 5.11 (d, 1 H, J ) 9.6 Hz), 5.01 (d, 1 H, J ) 3.2 Hz), 4.96-4.93
(m, 2 H), 4.89-4.84 (m, 3 H), 4.74 (s, 1 H), 4.61-4.53 (m, 6 H),
4.49 (dd, 1 H, J ) 10.5, 8.2 Hz), 4.46 (s, 1 H), 4.44 (d, 1 H, J )
10.5, 8.2 Hz), 4.40 (d, 1 H, J ) 12.8 Hz), 4.35 (d, 1 H, J ) 12.4
Hz), 4.31 (s, 1 H), 4.26 (dd, 1 H, J ) 10.6, 8.3 Hz), 4.21 (d, 1 H,
J ) 11.9 Hz), 4.16-4.09 (m, 9 H), 4.06-3.99 (m, 6 H), 3.92-3.86
(m, 3 H), 3.82-3.77 (m, 3 H), 3.74 (dd, 1 H, J ) 12.4, 2.8 Hz),
3.67 (d, 1 H, J ) 11.0 Hz), 3.57-3.55 (m, 2 H), 3.46-3.42 (m, 3
H), 3.34-3.26 (m, 3 H), 3.20 (d, 1 H, J ) 10.1 Hz), 2.91 (dd, 1 H,
J ) 9.6, 2.8 Hz), 2.14, 2.14, 2.09, 2.08, 2.06, 2.05, 2.02, 2.02,
2.01, 2.00, 1.99, 1.88, 1.88, 1.87, 1.85 (s, 3 H each). 13C NMR
(150 MHz, CDCl3): δ 171.7, 170.9, 170.9, 170.8, 170.6, 170.3,
170.2, 169.9, 169.6, 169.4, 169.2, 169.2, 167.9, 167.3, 167.0, 138.5,
138.4, 138.0, 137.5, 134.3, 134.0, 133.8, 133.6, 131.8, 131.5, 131.3,
128.9, 128.6, 128.4, 128.3, 128.2, 127.9, 127.8, 127.6, 127.5, 127.4,
127.0, 123.8, 123.5, 123.1, 100.5, 98.5, 98.0, 97.7, 97.0, 97.0, 96.5,
85.4, 78.3, 76.7, 75.5, 75.3, 74.5, 74.4, 74.2, 73.8, 73.5, 73.4, 73.2,
72.7, 72.2, 71.4, 71.2, 71.0, 70.8, 70.4, 70.3, 69.9, 69.8, 69.1, 68.9,
68.4, 68.2, 67.4, 67.3, 66.8, 66.6, 65.7, 63.8, 62.9, 62.5, 61.4, 61.4,
56.3, 55.1, 54.9, 54.7, 54.2, 20.9, 20.7, 20.6, 20.5, 20.4. HRMS
(MALDI) m/z: calcd for C142H149F3N6O60Na [M - N2 + Na]+
2977.8637; found, 2977.8623.
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyra-
nosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-mannopyranosyl)-(1f3)-
[O-(3,4,6-tri-O-acetyl-2-deoxy-2-trifluoroacetamido-ꢀ-D-glucopy-
ranosyl)-(1f4)]-[O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-
D-glucopyranosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-
mannopyranosyl)-(1f6)]-O-(2-O-acetyl-ꢀ-D-mannopyranosyl)-
(1f4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-
glucopyranosyl)-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
ꢀ-D-glucopyranosylazide (A). After a mixture of 18 (80 mg, 0.035
mmol), B (54 mg, 0.070 mmol), and 4 Å molecular sieves in freshly
distilled CH2Cl2 (10 mL) was stirred at rt for 3 h and then cooled
to -30 °C, NIS (24 mg, 0.105 mmol) was added. TfOH (3 µL,
0.035 mmol) was then added. The reaction mixture was stirred for
1 h at -30 °C. The reaction mixture was then filtered through Celite,
and the filtrate was diluted with CH2Cl2 and washed with aqueous
NaHCO3, aqueous Na2S2O3, and brine. The organic layer was dried
over Na2SO4, filtered, and concentrated. The crude mixture was
purified by column chromatography on silica gel (EtOAc/petroleum
Acknowledgment. This work is supported by the National
Basic Research Program of China (2003CB716403).
Supporting Information Available: General methods, the
1
ether ) 2:1) to afford A (78 mg, 74%) as a white foam. [R]20
)
synthesis of compounds 1, D, and 14 (path 2), H NMR and
D
1
13C NMR spectra for compounds 1, 4-8, 10, 12-18, and A-D.
This material is available free of charge via the Internet at
http://pubs.acs.org.
-13.3 (c 0.1, CHCl3). H NMR (600 MHz, CDCl3): δ 8.32 (d, 1
H), 7.88-6.72 (m, 36 H), 5.79 (dd, 1 H, J ) 10.5, 9.1 Hz), 5.50
(d, 1 H, J ) 8.2 Hz), 5.42 (dd, 1 H, J ) 10.6, 9.2 Hz), 5.35 (dd,
1 H, J ) 10.1, 9.7 Hz), 5.30-5.25 (m, 2 H), 5.19 (d, 1 H, J ) 8.2
Hz), 5.16 (dd, 1 H, J ) 9.7, 9.6 Hz), 5.12 (dd, 1 H, J ) 10.1, 9.6
JO900016J
J. Org. Chem. Vol. 74, No. 6, 2009 2515