5-Azido neuraminic acid thioglycoside as sialylation donor.

Article abstract of DOI:10.1016/S0008-6215(02)00057-5

5-Azido neuraminic acid thioglycoside with O-benzyl protecting groups was synthesized. The sialylations of this new donor type showed good alpha-selectivities for certain primary hydroxyls.

Full text of DOI:10.1016/S0008-6215(02)00057-5

Carbohydrate Research 337 (2002) 755–760
www.elsevier.com/locate/carres
Note
5-Azido neuraminic acid thioglycoside as sialylation donor
Kuo-Cheng Lu,a Sheng-Yuan Tseng,a,b Chun-Cheng Lina,c,*
aInstitute of Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan
bDepartment of Chemistry, National Central Uni6ersity, Chungli 32045, Taiwan
cDepartment of Chemistry, National Changhua Uni6ersity of Education, Changhua 50058, Taiwan
Received 29 October 2001; received in revised form 1 February 2002; accepted 20 February 2002
Abstract
5-Azido neuraminic acid thioglycoside with O-benzyl protecting groups was synthesized. The sialylations of this new donor type
showed good a-selectivities for certain primary hydroxyls. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: 5-Azidoneuraminic acid; Sialic acid; Thioglycoside; Sialylation
N-Acetylneuraminic acid (sialic acid, Neu5Ac) is
found as the terminal sugar of many oligosaccharide
chains on the cell surface and is among the most
important residue for interactions with their receptors.1
Recent findings show that synthetic N-acyl-modified
In order to tackle the importance of different func-
tionalities of sialic acid, its derivatives are needed to
study its interactions with various proteins. Although
many N-substituted sialic acid analogs have been syn-
thesized by either chemoenzymatic or chemical meth-
ods,5,6 they did not serve as good substrates for CMP
sialic acid synthetase. To develop a universal sialic acid
donor which can be easily functionalized at C-5 for
chemical sialylation is of current interest. Modifications
of NHAc of sialic acid donors have been reported to
have influence on the reactivities and selectivities of
sialylations.7 We proposed that the use of 5-azido sialic
acid as donor for the chemical preparation of N-substi-
tuted sialic acid conjugates: In our previous studies on
peracetylated 5-azido sialic acid donor, demonstrated it
to be a good donor.8 In this note, we describe our
studies of benzyl protected 5-azido sialic acid, 1 (Fig.
1), as sialylation donor.
Preparation of azido thioglycoside donor 1.—Synthe-
sis of 5-azido benzyl-protected thioglycoside 1 is shown
in Scheme 1. The readily available compound 2 synthe-
sized as described in Ref. 9 was treated with acetyl
chloride10 to give glycosyl chloride 3. Substitution of
the chlorine atom in compound 3 with p-thiocresol in
the presence of Hunig’s base11 afforded the a-thiogly-
coside 4 (84%) which was then N,O-deacetylated with
methanesulfonic acid in MeOH for 24 h at 60 °C12 to
give compound 5 in 81% yield. Treatment of 5 with
trifluoromethanesulfonyl azide (TfN3)13 as the diazo
transfer reagent furnished azido derivative 6 (84%),
D
-mannosamines could be taken up by cells, efficiently
metabolized in the promiscuous sialic acid biosynthetic
pathway, and incorporated into cell surface sialoglyco-
conjugates.2 Applications of this approach in different
biological systems have revealed important and unex-
pected functions of the N-acyl side chain of sialic acids,
including its crucial roles for the interactions of differ-
ent viruses with their sialylated host cell receptors.3
Also, the immunogenicity of the group B menigococcal
polysaccharide has been successfully improved by sub-
stituting N-acetyl with N-propionyl groups in the
polysialic acid.4 Therefore, engineering of the side chain
of sialic acid offers a new tool to study its biological
relevance and to exploit it as a tag for therapeutic and
diagnostic applications.
Fig. 1. Structure of 1.
* Corresponding author. Fax: +886-2-27831237.
E-mail address: cclin@chem.sinica.edu.tw (C.-C. Lin).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00057-5

756
K.-C. Lu et al. / Carbohydrate Research 337 (2002) 755–760
ents. Acceptors 714–915 and 1216 were obtained by
following reported procedures. Compounds 11 and 13
were synthesized by modification of Demchenko’s7b
and Yu’s8 procedures.
E6aluation of 5-azido thioglycoside donor 1.—Sialyla-
tion reactions were proceeded under NIS/TfOH reac-
tion conditions.17 Acceptors 7–13 (1 equiv) were
reacted with donor 1 (2 equiv) in the presence of NIS (3
equiv to donor), TfOH (3–10 mol% to NIS), and
,
molecular sieve (3 A) in MeCN at −40 °C to give
disaccharides 19–24, as shown in Table 1. The results
showed that the primary hydroxyl acceptors gave better
a-selectivities and yields than those of the secondary
acceptors. For the primary acceptor, the less hindered
one afforded better a-selectivity. The selectivities and
yields decreased dramatically when secondary hydroxyl
acceptors were used, e.g. for acceptor 13, no desired
product was observed except elimination product from
donor. These results may be attributed to the low
reactivity of donor 1. The anomeric configurations of
these disaccharides were determined by NMR spec-
troscopy based on the chemical shifts of H-3e of sialic
acid and empirical rules.18 The chemical shifts of H-3e
of a-glycosides were more downfield than those of
b-glycosides. The a anomers of 19, 20, and 22 were
further confirmed by measurements of the long-range
JC-1, H-3ax coupling constants.19 By selective proton de-
coupled 13C NMR experiments, the coupling pattern of
C-1 of a anomer gave a doublet C-1 signal and the
coupling constants of 19a, 20a, and 22a were 6.4, 6.6,
and 7.3 Hz, respectively.
Scheme 1. Synthesis of donor 1.
which was followed by per-benzylation to obtain azido
thioglycoside donor 1 in 67% yield. The configurational
assignment at C-5 was confirmed by the 1H NMR
spectroscopic data (J4,5 9.2, J5,6 10.4 Hz).
Acceptors used in the present study.—As shown in
Fig. 2, acceptors 7–10 were chosen for the studies on
the steric effects of acceptors in sialylations. The sialyla-
tion products of donor 1 with acceptor 7/8 and 9/10
gave STn and pseudo GM3 precursors, respectively.
Acceptor 11 was used for the synthesis of (a2-9) sialic
acid dimer. Secondary hydroxyl acceptors 12 and 13
were used for testing the sialylation reactions with more
hindered acceptors (primary vs. secondary acceptor).
Their products can serve as precursors for the synthesis
of GM3 and (a2-8) sialic acid dimer. Also, due to the
change of 5-NHAc to N3, the 8 and/or 9-hydroxy
groups could not form intramolecular hydrogen bond
with the azido group. Thus, acceptors 11 and 13 could
be potentially better acceptors than their 5-NHAc par-
In summary, the 5-azido thioglycoside donor 1
showed good a-selectivities for certain primary hy-
droxyl acceptors. The selectivity tendency of donor 1
was the same as its parent per-O-acetylated 5-azido
sialyl donor.8 However, further studies are needed to
understand how the protecting groups affect the reac-
Table 1
Sialylation reactions
Entry Acceptor Promotor
Product
Yield (%)
(a/b)
1
2
3
4
5
6
7
7
8
9
10
11
12
13
NIS/TfOH 14 (only a) 43
NIS/TfOH 15 (9:1)
NIS/TfOH 16 (4:1)
NIS/TfOH 17 (3.3:1)
NIS/TfOH 18 (7:3)
NIS/TfOH 19 (1.4:1)
NIS/TfOH
61
57
64
42
17
a
,
Key: NIS, TfOH, 3 A MS, CH3CN, −40 °C.
Fig. 2. Acceptors for sialylation.

K.-C. Lu et al. / Carbohydrate Research 337 (2002) 755–760
757
1
tivity and selectivity. Use of donor 1 to synthesize
sialo-antigens with modification of sialic acid at C-5 is
in progress.
MeOH); [h]2D3 +2.66° (c 0.019, EtOAc); H NMR (400
MHz, CDCl3): l 1.85 (s, 3 H, NHAc), 2.00–2.07 (m, 1
H, H-3ax), 2.01 (s, 3 H, OAc), 2.05 (s, 3 H, OAc), 2.07
(s, 3 H, OAc), 2.13 (s, 3 H, OAc), 2.35 (s, 3 H, OAc),
2.82 (dd, 1 H, J3eq,4 4.4, Jgem 12.8 Hz, H-3eq), 3.87 (dd,
1 H, J6,7 2.0, J6,5 10.4 Hz, H-6), 3.97 (ddd, 1 H, J5,4
10.4, J5,6 10.4, J5,NH 10.4 Hz, H-5), 4.21 (dd, 1 H, J9a,8
6.4, Jgem12.4 Hz, H-9a), 4.38 (dd, 1 H, J9b,8 2.8,
Jgem12.4 Hz, H-9b), 4.81 (ddd, 1 H, J4,3eq 4.4, J4,5 10.4,
J4,3ax 11.6 Hz, H-4), 5.03 (s, 2 H, Bn), 5.13 (d, 1 H,
JNH,5 10.4 Hz, NH), 5.16 (ddd, 1 H, J8,9b 2.8, J8,9a 6.4,
J8,7 6.4 Hz, H-8), 5.27 (dd, 1 H, J7,6 2.0, J7,8 6.4 Hz,
H-7), 7.08–7.10 (m, 2 H), 7.28–7.39 (m, 7 H); 13C (100
MHz, CDCl3): l 20.73, 20.81, 20.81, 20.93, 21.32,
23.14, 38.12, 49.25, 62.04, 67.80, 67.85, 69.69, 70.54,
75.01, 87.26, 124.96, 128.48, 128.48, 128.48, 128.48,
128.60, 128.60, 129.67, 129.67, 136.94, 136.94, 140.16,
167.43, 170.05, 170.15, 170.16, 170.59, 170.76. HRMS
(FAB) Calcd for C33H40NO12S [M+H]+: 674.2272
Found: 674.2265.
1. Experimental
General methods.—1H and 13C NMR spectra were
recorded on Bruker AM-400 or 500 MHz. Assignment
1
of H NMR spectra was achieved using 2D methods
(COSY). Chemical shifts are expressed in ppm using
residual CHCl3 as reference. High-resolution mass spec-
tra were obtained by means of a Micromass (Autospec)
mass spectrometer. Optical rotation was measured at
22 °C. Analytical thin-layer chromatography (TLC)
was performed on precoated plates (Silica Gel 60 F-
254). Silica gel 60 (E. Merck) was employed for all flash
chromatography. All reactions were carried out in
oven-dried glassware (120 °C) under an atmosphere of
nitrogen unless indicated otherwise. All solvents were
dried and distilled by standard techniques.
Benzyl (p-tolyl 5-amino-3,5-dideoxy-2-thio-
D-glyc-
Benzyl (5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-
ero-h- -galacto-non-2-ulpyrnosid)onate (5).—To
D
a
3,5-dideoxy-
D
-glycero-h-
D
-galacto-non-2-ulpyranosid)-
stirred solution of compound 4 (1.0 g, 1.5 mmol) in dry
MeOH (50 mL) was added methanesulfonic acid
(MsOH, 0.2 mL, 22.2 mmol). The mixture was stirred
for 24 h at 60 °C, then neutralized with Dowex 1×8
(OH−) resin, after which the suspension was filtered.
The filtrate was concentrated and purified by silica gel
column chromatography (3:7 MeOH–CHCl3) to give
compound 5 (557.3 mg, 81%). Rf 0.3 (3:7 MeOH–
CHCl3); [h]2D3 −30.77° (c 0.004, MeOH); 1H NMR (400
MHz, CDCl3): l 1.85 (dd, 1 H, J3ax,4 11.2, Jgem 12.8 Hz,
H-3ax), 2.33 (s, 3 H, SPhCH3), 2.86 (dd, 1 H, J3eq,4 4.4,
Jgem 12.8 Hz, H-3eq), 3.04 (t, 1 H, J5,4=J5,6 10.0 Hz,
H-5), 3.46–3.52 (m, 1 H, H-4), 3.53 (dd, 1 H, J9a,8 2.0,
Jgem10.0 Hz, H-9a), 3.63–3.66 (m, 1 H, H-6), 3.66 (dd,
1 H, J9b,8 6.4, Jgem10.0 Hz, H-9b), 3.79–3.83 (m, 2 H,
H-7, H-8), 5.12 (s, 2 H, Bn), 7.09 (d, J 7.6 Hz, 2 H),
7.26 (d, J 8.0 Hz, 2 H), 7.34–7.42 (m, 5 H); 13C (100
MHz, CDCl3): l 21.48, 41.72, 54.26, 64.58, 69.24,
69.74, 70.20, 73.51, 77.46, 87.96, 126.45, 129.85, 129.85,
129.90, 129.90, 130.09, 130.09, 130.80, 130.80, 137.94,
137.94, 141.87, 170.43. HRMS (FAB) Calcd for
C23H30NO7S [M+H]+: 464.1744 Found: 464.1737.
onate (3).—AcCl (80 mL) was added to the flask which
contained compound 29 (8.08 g, 13.3 mmol) and then
purged with HCl gas for 10 min. The mixture was
stirred for 2 days at rt and the concentrated, diluted
with EtOAc, washed with water and satd NaHCO3,
and concentrated in vacuo to give compound 3 (6.89 g,
90%) which was used without further purification; Rf
0.3 (1:1 EtOAc–hexane containing 10% MeOH). 1H
NMR (400 MHz, CDCl3): l 1.91 (s, 3 H, NHAc), 2.02
(s, 3 H, OAc), 2.04 (s, 3 H, OAc), 2.04 (s, 3 H, OAc),
2.12 (s, 3 H, OAc), 2.29 (dd, 1 H, J3ax,4 11.2, Jgem 14.0
Hz, H-3ax), 2.78 (dd, 1 H, J3aq,4 4.8, Jgem 14.0 Hz,
H-3eq), 4.08 (dd, 1 H, J9a,8 5.6, Jgem12.4 Hz, H-9a), 4.20
(ddd, 1 H, J5,4 10.8, J5,6 10.8, J5,NH 10.8 Hz, H-5), 4.36
(dd, 1 H, J6,7 2.4, J6,5 10.8 Hz, H-6), 4.40 (dd, 1 H, J9b,8
2.4, Jgem12.4 Hz, H-9b), 5.18 (ddd, 1 H, J8,9b 2.4, J8,9a
5.6, J8,7 6.4 Hz, H-8), 5.23 (d, 1 H, Jgem 12.0 Hz, Bn),
5.36 (d, 1 H, Jgem 12.0 Hz, Bn), 5.37–5.44 (m, 2 H, H-4,
NH), 5.47 (dd, 1 H, J7,6 2.4, J7,8 6.4 Hz, H-7), 7.35–
7.41 (m, 5 H).
Benzyl (p-tolyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-2-thio-
D
-glycero-h-
D
-galacto-non-2-ulpyran-
Benzyl (p-tolyl 5-azido-3,5-dideoxy-2-thio-
D-glycero-
osid)onate (4).—To a solution of sialic acid chloride 3
(6.89 g, 11.8 mmol) and thiocresol (2.20 g, 17.6 mmol)
in CH2Cl2 (75 mL) was added N-ethyldiisopropylamine
(Hunig’s base, 3.1 mL, 17.6 mmol) at rt, and then
stirred for overnight. When the reaction showed to be
complete, the mixture was concentrated and then di-
luted with EtOAc, washed with water, satd NaHCO3,
brine, and then concentrated. The residue was purified
by silica gel column chromatography (1:1 EtOAc–hex-
ane containing 5% MeOH) to give compound 4 (6.65 g,
84%). Rf 0.2 (1:1 EtOAc–hexane containing 5%
h-D-galacto-non-2-ulpyranosid)onate (6).—A solution
of NaN3 (2.3 g, 35.3 mmol) in water (5.7 mL) was
cooled in an ice bath and treated with CH2Cl2 (3 mL).
The resulting biphasic mixture was stirred vigorously
and then treated with Tf2O (2.5 g, 8.8 mmol) over a
period of 5 min. The reaction was stirred in an ice bath
for 2 h, the organic layer was separated and aqueous
phase was washed with CH2Cl2 as less volume as
possible. The organics were washed once with satd
NaHCO3 solution and used without further
purification.

758
K.-C. Lu et al. / Carbohydrate Research 337 (2002) 755–760
Compound 5 (1.2 g, 4.06 mmol) was dissolved in
127.83, 127.94, 127.98, 127.98, 128.08, 128.08, 128.26,
128.26, 128.30, 128.30, 128.43, 128.43, 128.58, 128.58,
128.58, 128.58, 129.44, 129.44, 135.09, 136.64, 136.64,
137.26, 138.24, 138.51, 139.32, 139.76, 167.81. HRMS
(FAB) Calcd for C51H51N3O7SNa [M+H]+: 872.3345
Found: 872.3321.
water (13.6 mL) and treated with potassium carbonate
(K2CO3, 731.0 mg, 5.3 mmol) and CuSO4·5 H2O (8.8
mg, 0.04 mmol). To the sugar solution was added
MeOH (20 mL) and TfN3 solution. Then, more MeOH
(8 mL) was added to homogenity. The reaction was
allowed to stirred for 18 h and the solvent was re-
moved. The residue was purified by silica gel column
chromatography (1:4 MeOH–CHCl3) to give azido
product 6 (1.45 g, 84%). Rf 0.4 (1:1 EtOAc–hexane
containing 10% MeOH); [h]2D3 −26.47° (c 0.002,
tert-Butyldiphenylsilyl [benzyl (5-azido-4,7,8,9-tetra-
O-benzyl-3,5-dideoxy-
ranosid)onate]-h-(2,6)-2-azido-3-O-benzyl-2-deoxy-i-
-galactopyranoside (15).—General procedures for the
D-glycero-h-D-galacto-non-2-ulpy-
D
sialylation reaction: NIS (53.1 mg, 0.2 mmol) was
1
MeOH); H NMR (400 MHz, CDCl3): l 1.86 (dd, 1 H,
added into the flask which contained donor 1 (50 mg,
0.06 mmol), acceptor 814 (62.8 mg, 0.12 mmol) and 3 A
,
J3ax,4 11.2, Jgem 12.8 Hz, H-3ax), 2.33 (s, 3 H, SPhCH3),
2.80 (dd, 1 H, J3eq,4 3.6, Jgem 12.8 Hz, H-3eq), 3.27–
3.31 (m, 1 H, H-6), 3.49 (t, 1 H, J5,4=J5,6 9.6 Hz, H-5),
3.51–3.57 (m, 1 H, H-8), 3.60 (dd, 1 H, J9a,8 5.6,
Jgem11.2 Hz, H-9a), 3.66 (dd, 1 H, J7,6 1.2, J7,8 8.8 Hz,
H-7), 3.73–3.77 (m, 1 H, H-4), 3.80 (dd, 1 H, J9b,8 2.4,
Jgem11.2 Hz, H-9b), 5.08 (s, 2 H, Bn), 7.09 (d, J 8.4 Hz,
2 H), 7.26 (d, J 8.0 Hz, 2 H), 7.33–7.42 (m, 5 H); 13C
(100 MHz, CDCl3): l 21.48, 41.45, 64.27, 64.72, 69.17,
70.61, 71.41, 73.62, 77.04, 87.80, 126.54, 129.82, 129.82,
129.88, 129.88, 130.15, 130.15, 130.76, 130.76, 137.92,
137.92, 141.79, 170.38. HRMS (FAB) Calcd for
C23H28N3O7S [M+H]+: 490.1649 Found: 490.1638.
Benzyl (p-tolyl 5-azido-4,7,8,9-tetra-O-benzyl-3,5-
MS (113 mg) in CH3CN (1.5 mL) at −40 °C under Ar
and then stirred for 30 min. TfOH (0.3 L, 0.003 mmol)
was added to the mixture solution at −40 °C. After
stirred for 30 min Et3N (one drop) was added and the
solvent was removed. The mixture was diluted with
EtOAc and washed with Na2S2O3, satd. NaHCO3,
brine, dried over MgSO4, and concentrated. The
residue was purified by silica gel column chromatogra-
phy (1:2 EtOAc–hexane) to give disaccharide 15 (a/=
9:1, 45.0 mg, 61%); Rf 0.33 (1:2 EtOAc–hexane); [h]D23
−19.35° (c 0.012, EtOAc); 1H NMR (400 MHz,
CDCl3): l 1.07 (s, 9 H, 3 CH3), 1.60 (dd, 1 H, J3ax,4
11.6, Jgem 12.8 Hz, H-3ax), 2.44 (br, 1 H, OH), 2.65
(dd, 1 H, J3eq,4 3.6, Jgem 12.8 Hz, H-3eq), 3.07 (dd, 1 H,
J3%,4% 3.2, J3%,2% 10.0 Hz, H-3%), 3.10–3.12 (m, 1 H, H-5%),
3.43–3.48 (m, 2 H, H-4, H-6%a), 3.61–3.79 (m, 8 H,
H-5, H-6, H-8, H-9a, H-9b, H-2%, H-4%, H-6%b), 3.95 (d,
1 H, J7,8 7.6 Hz, H-7), 4.26 (d, 1 H, J1%,2% 7.6 Hz, H-1%),
4.42 (d, 1 H, Jgem 11.2 Hz, Bn), 4.46–4.67 (m, 7 H, Bn),
4.76 (d, 1 H, Jgem 11.2 Hz, Bn), 4.97 (d, 1 H, Jgem 12.0
Hz, Bn), 5.08 (d, 1 H, Jgem 12.0 Hz, Bn), 7.22–7.36 (m,
34 H), 7.68–7.71 (m, 6 H); 13C (100 MHz, CDCl3): l
19.13, 26.78, 26.78, 26.78, 36.57, 60.96, 61.55, 64.26,
65.31, 67.38, 68.59, 71.48, 71.65, 72.25, 72.66, 73.25,
73.76, 74.63, 76.06, 76.73, 77.99, 79.36, 96.71, 98.83,
127.31, 127.31, 127.36, 127.36, 127.51, 127.51, 127.51,
127.69, 127.69, 127.69, 127.69, 127.86, 127.86, 127.92,
127.92, 127.92, 127.96, 127.96, 127.96, 128.01, 128.01,
128.01, 128.20, 128.20, 128.31, 128.31, 128.46, 128.46,
128.46, 128.46, 128.68, 128.68, 129.66, 129.66, 129.87,
129.78, 132.85, 133.04, 134.99, 135.82, 135.82, 135.96,
135.96, 137.30, 137.37, 137.93, 138.34, 138.88, 177.16.
HRMS (FAB) Calcd for C73H78N6O12SiNa [M+H]+:
1281.5344 Found: 1281.5313.
dideoxy-2-thio-D-glycero-h-D-galacto-non-2-ulpyran-
osid)onate (1).—DMF (4.0 mL) was added to a flask
containing compound 6 ( 200 mg, 0.4 mmol) and
tetrabutyl ammonium iodine (TBAI 10 mg, 0.03 mmol),
then cooled to 0 °C. NaH (80.0 mg, 3.3 mmol) was
added to the solution and stirred for 30 min. BnBr (0.3
mL, 2.4 mmol) was added dropwise and then the
mixture was stirred for overnight. When the reaction
was completed, it was cooled to 0 °C, and quenched by
water. The solvent was removed and then diluted with
EtOAc followed by washing with water and brine, and
then dried over MgSO4. The mixture was purified by
silica gel column chromatography (1:1 EtOAc–hexane)
to afford donor 1 (234 mg, 67%); Rf 0.25 (1:1 EtOAc–
1
hexane); [h]2D3 −8.0° (c 0.006, EtOAc); H NMR (400
MHz, CDCl3): l 1.73 (dd, 1 H, J3ax,4 11.6, Jgem 12.8 Hz,
H-3ax), 2.25 (s, 3 H, SPhCH3), 2.80 (dd, 1 H, J3eq,4 4.4,
Jgem 12.8 Hz, H-3eq), 3.41 (ddd, 1 H, J4,3eq 4.4, J4,5 9.2,
J4,3ax 11.6 Hz, H-4), 3.50–3.58 (m, 1 H, H-5), 3.54 (dd,
1 H, J6,7 9.2, J6,5 10.4 Hz, H-6), 3.73 (dd, 1 H, J9a,8 4.4,
Jgem 10.4 Hz, H-9a), 3.84–3.91 (m, 2 H, H-8, H-9b),
3.85 (dd, 1 H, J7,8 7.2, J7,6 9.2 Hz, H-7), 4.37 (d, 1 H,
Jgem 11.6 Hz, Bn), 4.41 (d, 1 H, Jgem 11.6 Hz, Bn), 4.43
(d, 1 H, Jgem 11.2 Hz, Bn), 4.51–4.56 (m, 4 H, Bn), 4.78
(d, 1 H, Jgem 11.2 Hz, Bn), 4.91 (d, 1 H, J 12.4 Hz, Bn),
4.98 (d, 1 H, J 12.4 Hz, Bn), 6.92–6.94 (m, 2 H),
7.22–7.37 (m, 27); 13C (100 MHz, CDCl3): l 21.20,
37.31, 61.06, 67.36, 69.68, 71.42, 72.31, 73.29, 74.34,
76.00, 76.68, 77.31, 78.46, 86.72, 125.49, 127.15, 127.40,
127.59, 127.59, 127.59, 127.63, 127.63, 127.63, 127.83,
tert-Butyldiphenylsilyl [benzyl (5-azido-4,7,8,9-tetra-
O-benzyl-3,5-dideoxy-
ranosid)onate]-h-(2,6)-2-azido-2-deoxy-i-
D
-glycero-h-
D
-galacto-non-2-ulpy-
-galacto-
D
pyranoside (14).—For a form; Rf 0.20 (1:3 EtOAc–hex-
1
ane); [h]2D3 − 19.35° (c 0.003, EtOAc); H NMR (400
MHz, CDCl3): l 1.25 (br, 1 H, OH), 1.68 (dd, 1 H,
J3ax,4 11.6, Jgem 12.8 Hz, H-3ax), 2.43 (d, 1 H, J 9.6 Hz,
OH), 2.56 (dd, 1 H, J3eq,4 4.8, Jgem 12.8 Hz, H-3eq),
3.08 (m, 1 H, H-3%), 3.19 (ddd, 1 H, J4,3eq 4.8, J4,5 9.6,

K.-C. Lu et al. / Carbohydrate Research 337 (2002) 755–760
759
J4,3ax 11.6 Hz, H-4), 3.30 (t, 1 H, J5,4=J5,6 9.6 Hz,
H-5), 3.40–3.49 (m, 4 H, H-2%, H-5%, H-6%a, H-6%b),
3.63–3.72 (m, 3 H, H-6, H-8, H-4%), 3.77–3.80 (m, 2 H,
H-9a, H-9b), 3.98 (d, 1 H, J7,8 7.6 Hz, H-7), 4.27 (d, 1
H, J1,2 7.6 Hz, H-1%), 4.40 (d, 1 H, Jgem 11.2 Hz, Bn),
4.46 (d, 1 H, Jgem 11.2 Hz, Bn), 4.50–4.58 (m, 2 H, Bn),
4.51 (d, 1 H, Jgem 12.0 Hz, Bn), 4.55 (d, 1 H, Jgem 12.0
Hz, Bn), 4.57 (d, 1 H, Jgem 11.6 Hz, Bn), 4.76 (d, 1 H,
Jgem 11.6 Hz, Bn), 4.94 (d, 1 H, Jgem 12.0 Hz, Bn), 5.04
(d, 1 H, Jgem 12.0 Hz, Bn), 7.20–7.41 (m, 30 H),
7.67–7.71 (m, 5 H); 13C (100 MHz, CDCl3): l 18.70,
26.37, 26.37, 26.37, 34.92, 60.39, 60.58, 66.01, 66.87,
67.13, 67.71, 71.18, 71.52, 71.74, 72.30, 73.01, 74.34,
75.29, 76.35, 76.77, 77.42, 96.41, 98.60, 126.87, 126.87,
127.03, 127.10, 127.10, 127.14, 127.14, 127.24, 127.43,
127.43, 127.48, 127.48, 127.58, 127.83, 127.83, 127.93,
127.93, 127.93, 127.96, 127.96, 127.96, 127.96, 128.05,
128.05, 128.13, 128.13, 128.31, 128.31, 128.35, 129.34,
129.40, 132.34, 132.79, 134.38, 135.41, 135.41, 135.51,
135.51, 136.78, 137.00, 137.67, 138.19, 167.10. HRMS
(FAB) Calcd for C66H72N6O12SiNa [M+H]+:
1191.4874 Found: 1191.4869.
−46.15° (c 0.001, EtOAc); 1H NMR (400 MHz,
CDCl3): l 1.25 (s, 3 H, CH3), 1.30 (s, 3 H, CH3), 1.36
(s, 3 H, CH3), 1.48 (s, 3 H, CH3), 1.73 (dd, 1 H, J3ax,4
12.4, Jgem 12.4 Hz, H-3ax), 2.83 (dd, 1 H, J3eq,4 4.4, Jgem
12.4 Hz, H-3eq), 3.43 (ddd, 1 H, J4,3eq 4.4, J4,5 9.6, J4,3ax
12.4 Hz, H-4), 3.56 (t, 1 H, J5,4=J5,6 10.0 Hz, H-5),
3.69 (dd, 1 H, J6,7 1.6, J6,5 10.0 Hz, H-6), 3.69–3.74 (m,
2 H, H-6%a, H-6%b), 3.89–3.93 (m, 4 H, H-8, H-9a,
H-9b, H-5%), 3.95 (dd, 1 H, J7,6 1.6, J7,8 6.8 Hz, H-7),
4.15 (dd, 1 H, J4%,3% 1.2, J4%,5% 7.6 Hz, H-4%), 4.27 (dd, 1
H, J2%,3% 2.4, J2%,1% 5.6 Hz, H-2%), 4.40 (d, 1 H, Jgem 11.6
Hz, Bn), 4.49–4.59 (m, 5 H, H-3%, Bn), 4.62 (d, 1 H,
Jgem 11.6 Hz, Bn), 4.66 (d, 1 H, Jgem 11.6 Hz, Bn), 4.75
(d, 1 H, Jgem 11.6 Hz, Bn), 4.97 (d, 1 H, Jgem 12.0 Hz,
Bn), 5.18 (d, 1 H, Jgem 12.0 Hz, Bn), 5.48 (d, 1 H, J1%,2%
5.2 Hz, H-1%), 7.24–7.38 (m, 25 H); 13C (100 MHz,
CDCl3): l 24.55, 24.92, 26.01, 26.11, 37.02, 61.02, 62.8,
66.56, 67.21, 69.86, 70.56, 70.67, 71.26, 72.54, 73.31,
74.29, 76.43, 76.79, 76.79, 77.29, 78.68, 94.25, 99.01,
108.59, 109.11, 127.27, 127.40, 127.66, 127.78, 127.78,
127.80, 127.80, 127.89, 127.96, 127.96, 128.00, 128.00,
128.17, 128.17, 128.25, 128.25, 128.30, 128.30, 128.34,
128.34, 128.43, 128.43, 128.55, 128.68, 128.68, 135.16,
137.38, 138.10, 138.59, 139.17, 167.52. HRMS (FAB)
Calcd for C56H63N3O13Na [M+H]+: 1008.4258
Found: 1008.4284.
Methyl [benzyl (5-azido-4,7,8,9-tetra-O-benzyl-3,5-
dideoxy -
D
- glycero - h -
D
- galacto - non - 2 - ulpyranosid)-
-galactopyranoside
onate]-h-(2,6)-2,3-di-O-benzyl-h-
D
(16).—For a form; Rf 0.34 (1:3 EtOAc–hexane); [h]D23
+48.08° (c 0.010, EtOAc); 1H NMR (400 MHz,
CDCl3): l 1.71 (dd, 1 H, J3ax,4 11.6, Jgem 12.8 Hz,
H-3ax), 2.44 (br, 1 H, OH), 2.73 (dd, 1 H, J3eq,4 4.0,
Jgem 12.8 Hz, H-3eq), 3.29 (s, 3 H, OMe), 3.43–3.47 (m,
1 H, H-4), 3.51 (t, 1 H, J5,4=J5,6 10.0 Hz, H-5),
3.66–3.88 (m, 9 H, H-6, H-8, H-9a, H-9b, H-2%, H-3%,
H-5%, H-6%a, H-6%b), 3.95–3.97 (m, 2 H, H-7, H-4%), 4.42
(d, 1 H, Jgem 11.6 Hz, Bn), 4.50 (d, 1 H, Jgem 11.6 Hz,
Bn), 4.50 (s, 2 H, H-1%, Bn), 4.57 (d, 1 H, Jgem 11.6 Hz,
Bn), 4.61 (d, 1 H, Jgem 12.0 Hz, Bn), 4.60–4.63 (m, 2 H,
Bn), 4.63 (d, 1 H, Jgem 12.0 Hz, Bn), 4.65 (d, 1 H, Jgem
11.2 Hz, Bn), 4.70 (d, 1 H, Jgem 11.6 Hz, Bn), 4.75 (d,
1 H, Jgem 12.0 Hz, Bn), 4.78 (d, 1 H, Jgem 12.0 Hz, Bn),
5.01 (d, 1 H, Jgem 12.4 Hz, Bn), 5.11 (d, 1 H, Jgem 12.4
Hz, Bn), 7.24–7.37 (m, 35 H); 13C (100 MHz, CDCl3):
l 36.83, 55.32, 60.96, 62.79, 66.89, 67.39, 68.03, 69.32,
71.40, 72.41, 72.51, 73.28, 73.48, 74.07, 74.41, 75.57,
76.22, 77.21, 77.52, 78.26, 98.55, 99.06, 126.89, 127.33,
127.46, 127.65, 127.65, 127.73, 127.73, 127.73, 127.73,
127.79, 127.79, 127.95, 127.95, 127.95, 128.01, 128.01,
128.01, 128.01, 128.19, 128.19, 128.28, 128.28, 128.37,
128.37, 128.37, 128.37, 128.41, 128.41, 128.41, 128.45,
128.45, 128.45, 128.50, 128.50, 128.68, 128.68, 135.04,
137.32, 137.97, 138.28, 138.41, 138.98, 167.61. HRMS
(FAB) Calcd for C65H69N3O13Na [M+H]+: 1122.4727
Found: 1122.4733.
Methyl {methyl 5-azido-4,7,8-tri-O-benzoyl-9-O-
[benzyl (5-azido-4,7,8,9-tetra-O-benzyl-3,5-dideoxy-D-
glycero -
deoxy-
D
- galacto - non - 2 - ulpyra - nosid)onate] - 3,5-di-
D
-glycero-h-D-galacto-non-2-ulpyranosid}onate
1
(18).—For a form; Rf 0.23 (1:3 EtOAc–hexane). H
NMR (400 MHz, CDCl3): l 1.51–1.57 (m, 1 H, H-
3ax), 1.77 (dd, 1 H, J3%ax,4% 11.6, Jgem 12.8 Hz, H-3%ax),
2.59 (dd, 1 H, J3eq,4 4.0, Jgem 12.8 Hz, H-3eq), 2.66 (dd,
1 H, J3%eq,4% 5.2, Jgem 12.8 Hz, H-3%eq), 3.05 (s, 3 H,
OMe), 3.31–3.39 (m, 2 H, H-4, H-9a), 3.41 (t, 1 H,
J
5,4=J5,6 10.0 Hz, H-5), 3.52 (t, 1 H, J5%,4%=J5%,6% 10.0
Hz, H-5%), 3.54–3.58 (m, 2 H, H-6, H-9b), 3.62–3.65
(m, 1 H, H-8), 3.76 (s, 3 H, CO2Me), 3.83 (dd, 1 H, J7,6
1.6, J7,8 7.2 Hz, H-7), 3.87 (dd, 1 H, J6%,7% 0.8, J6%,5% 10.0
Hz, H-6%), 4.02 (dd, 1 H, J9%a%,8% 3.6, Jgem 11.2 Hz, H-9%a),
4.08 (dd, 1 H, J9%b,8% 3.2, Jgem 11.2 Hz, H-9%b), 4.24 (d, 1
H, Jgem 11.6 Hz, Bn), 4.33 (m, 4 H, Bn), 4.37 (d, 1 H,
Jgem 11.6 Hz, Bn), 4.48 (d, 1 H, Jgem 11.6 Hz, Bn), 4.60
(d, 1 H, Jgem 11.6 Hz, Bn), 4.66 (d, 1 H, Jgem 12.0 Hz,
Bn), 5.02 (d, 1 H, Jgem 12.0 Hz, Bn), 5.46 (ddd, 1 H,
J4%,3%eq 5.2, J4%,5% 10.0, J4%,3%ax 11.6 Hz, H-4%), 5.68 (ddd, 1
H, J8%,9%b 3.2, J8%,9%a 3.6, J8%,7% 8.8 Hz, H-8%), 6.09 (dd, 1 H,
J7%,6% 0.8, J7%,8% 8.8 Hz, H-7%), 7.13–7.43 (m, 31 H),
7.47–7.57 (m, 3 H), 7.97–8.00 (m, 4 H), 8.08–8.10 (m,
2 H); 13C (100 MHz, CDCl3): l 36.19, 36.93, 51.18,
52.59, 60.67, 60.81, 62.10, 67.10, 68.75, 68.96, 70.18,
70.22, 70.98, 71.53, 72.23, 73.19, 73.99, 74.05, 76.19,
77.21, 78.06, 98.70, 98.81, 127.23, 127.47, 127.53,
127.72, 127.72, 127.72, 127.72, 127.72, 127.72, 127.72,
127.72, 127.80, 127.80, 127.86, 127.86, 128.10, 128.10,
1,2,3,4-Di-O-isopropylidene-6-O-[benzyl
4,7,8,9-tera-O-benzyl-3,5-dideoxy- -glycero-h-
acto-non-2-ulpyranosid)onate]-h- -galactopyranoside
(17).—For a form; Rf 0.20 (1:4 EtOAc–hexane); [h]D23
(5-azido-
D
D
-gal-
D

760
K.-C. Lu et al. / Carbohydrate Research 337 (2002) 755–760
2. (a) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007–
2010;
128.23, 128.23, 128.29, 128.29, 128.29, 128.29, 128.29,
128.36, 128.36, 128.45, 128.45, 128.50, 128.50, 128.50,
128.58, 128.58, 128.58, 129.31, 129.40, 129.58, 129.76,
129.76, 130.19, 130.19 133.37, 133.37, 135.04, 137.33,
138.25, 138.46, 139.04, 165.16, 165.29, 165.46, 167.27,
167.36. HRMS (FAB) Calcd for C76H75N6O18Na [M+
H]+: 1381.4955 Found: 1381.4922.
(b) Mahal, K. L.; Yarema, K. J.; Bertozzi, C. R. Science
1997, 276, 1125–1128.
3. Keppler, O. T.; Horstkorte, R.; Pawlita, M.; Schmidt, C.;
Reutter, W. Glycobiology 2001, 11, 11R–18R.
4. Pon, R. A.; Lussier, M.; Yang, Q.-L.; Jennings, H. J. J.
Exp. Med. 1997, 185, 1929–1938.
5. Lin, C.-C.; Lin, C.-H.; Wong, C.-H. Tetrahedron Lett.
1997, 38, 2649–2652.
6. (a) Schneider, R.; Freyhardt, C. C.; Schmidt, R. R. Eur.
J. Org. Chem. 2001, 1655–1661;
Methyl [benzyl (5-azido-4,7,8,9-tetra-O-benzyl-3,5-
dideoxy - D - glycero - h - D - galacto - non - 2 - ulpyranosid)-
onate]-h-(2,3)-2,6-di-O-benzyl-h-D-galactopyranoside
(19).—For a form; Rf 0.2 (1:3 EtOAc–hexane); [h]D23
(b) Choi, S.-K.; Lee, S.; Whitesides, G. M. J. Org. Chem.
1996, 61, 8739–8745;
(c) Schreiner, E.; Zbiral, E. Liebigs Ann. Chem. 1990,
581–586.
1
+3.03° (c 3.3×10−3, EtOAc); H NMR (400 MHz,
CDCl3): l 1.81 (dd, 1 H, J3ax,4 11.6, Jgem 13.6 Hz,
H-3ax), 2.71 (dd, 1 H, J3eq,4 4.6, Jgem 13.6 Hz, H-3eq),
3.08 (d, 1 H, J 2.4 Hz, OH), 3.27 (s, 3 H, OMe), 3.35
(dd, 1 H, J6%a,5% 4.4, J6%a,6%b 10.4 Hz, H-6%a), 3.41 (dd, 1
H, J6%b,5% 5.6, J6%b,6%a 10.4 Hz, H-6%b), 3.49 (m, 1 H, H-5%),
3.62 (t, J5,4=J5,6 9.6 Hz, H-5), 3.67 (dd, 1 H, J9a,8 4.8,
Jgem 10.4 Hz, H-9a), 3.73 (dd, 1 H, J 3.6, J 10.0 Hz,
H-3%), 3.88–3.93 (m, 3 H, H-6, H-9a, H-4%), 4.04–4.13
(m, 4 H, H-4, H-7, H-8, H-2%), 4.37 (d, 1 H, Jgem 12.0
Hz, Bn), 4.36–4.66 (m, 7 H, H-1%, Bn), 4.43 (d, Jgem
12.0 Hz, Bn), 4.49 (d, Jgem 11.2 Hz, Bn), 4.53 (d, Jgem
11.2 Hz, Bn), 4.65 (d, Jgem 12.0 Hz, Bn), 4.74 (d, Jgem
11.6 Hz, Bn), 5.07 (d, Jgem 12.0 Hz, Bn), 5.17 (d, Jgem
12.0 Hz, Bn), 7.22–7.38 (m, 35 H); 13C (100 MHz,
CDCl3): l 36.03, 55.19, 61.37, 67.77, 67.93, 69.38,
70.38, 70.55, 71.68, 72.41, 72.98, 73.25, 73.54, 73.99,
74.21, 74.84, 75.90, 76.29, 77.20, 79.80, 98.65, 99.74,
127.33, 127.33, 127.41, 127.41, 127.41, 127.47, 127.47,
127.47, 127.63, 127.63, 127.77, 127.77, 127.77, 127.88,
127.88, 128.13, 128.13, 128.13, 128.26, 128.26, 128.26,
128.26, 128.26, 128.26, 128.35, 128.35, 128.35, 128.35,
128.42, 128.42, 128.42, 128.55, 128.55, 128.77, 128.77,
134.97, 137.78, 137.79, 137.92, 138.44, 138.55, 138.95,
167.48. HRMS (FAB) Calcd for C65H69N3O13Na [M+
H]+: 1122.4727 Found: 1122.4704.
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Acknowledgements
This research was supported by Academia Sinica and
the National Science Council of Taiwan (NSC 90-2323-
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