A straightforward α-selective aromatic glycosylation and its application for stereospecific synthesis of 4-methylumbelliferyl α-T-antigen

Article abstract of DOI:10.1016/j.carres.2008.12.013

A practical and efficient α-selective aromatic glycosylation with simple per-O-acetyl glycopyranosyl trichloroacetimidates is reported. The method is particularly effective for L-fucosyl and 2-azido-2-deoxy-d-galatosaminyl imidates, with which exclusive α-selectivity was achieved. The synthetic utility of this method was demonstrated in the stereoselective synthesis of 4-methylumbelliferyl α-T-antigen.

Full text of DOI:10.1016/j.carres.2008.12.013

Carbohydrate Research 344 (2009) 432–438
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
A straightforward
a-selective aromatic glycosylation and its application
for stereospecific synthesis of 4-methylumbelliferyl
a-T-antigen
a,
Shih-Sheng Chang a, Chun-Cheng Lin b, Yaw-Kuen Li a, , Kwok-Kong Tony Mong
*
*
a Department of Applied Chemistry, National Chiao Tung University, Hsinchu, 1001, Ta Hsueh Rd, Taiwan, ROC
b Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 October 2008
Received in revised form 7 December 2008
Accepted 11 December 2008
Available online 25 December 2008
A practical and efficient a-selective aromatic glycosylation with simple per-O-acetyl glycopyranosyl tri-
chloroacetimidates is reported. The method is particularly effective for L-fucosyl and 2-azido-2-deoxy-
galatosaminyl imidates, with which exclusive -selectivity was achieved. The synthetic utility of this
method was demonstrated in the stereoselective synthesis of 4-methylumbelliferyl -T-antigen.
D-
a
a
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Stereoselective
Aromatic
Glycosylation
Imidate
a-T-Antigen
1. Introduction
chromatography purification, and a large excess of phenol is
sometimes required to boost the glycosylation yield.28–31
Because of the important role of synthetic aryl glycosides as en-
zyme substrates or inhibitors, a simple preparatory method is highly
desired. Herein, we describe a straightforward synthesis of O-aryl
A wide variety of natural products contain glycosidic linkages
to phenols as exemplified by the structures of the potent antibi-
otic vancomycin and anti-diabetic phlorizin.1–4 More importantly,
the presence of such phenyl aglycons is found to be essential for
their biological functions.5–8 Synthetic O-aryl glycosides with
chromogenic or fluorogenic aglycons are invaluable substrates
for the study of enzyme kinetics;9–13 therefore, it comes as no
surprise that the first synthesis of O-aryl glycoside dates back
to 1879.14 A distinct feature of aromatic glycosylation is the poor
nucleophilicity of the phenolic oxygen in comparison to an ali-
phatic oxygen, rendering the glycosylation less efficient in the
former cases. To combat this problem, different synthetic
measures have been taken that include (1) SN2 substitution of
an anomeric leaving group with the phenolate under basic condi-
tions;15,16 (2) a Helferich glycosylation procedure with glycosyl
acetates;17–22 (3) KoenigꢀKnorr glycosylation with glycosyl
halides;23–27 and (4) oxidative condensation of glycosyl hemiace-
tals and phenol by the Mitsunobu reaction.28–31 However, most of
these methods suffer from some drawbacks that limit their wider
application. For example, the harsh reaction conditions in Helfe-
rich glycosylation are not suitable for glycosides with fragile aryl
glycosidic bonds,17–22 inadequate selectivity demands tedious
a
-glycosides from per-O-acetyl glycosyl imidates, and their applica-
tion for the synthesis of fluorogenic 4-methylumbelliferyl -T-anti-
gen 1 (4-MU -T-antigen). 4-MU -T-antigen is widely used as
synthetic substrate for N-acetyl
-galactosaminidases;32–34 it com-
a
a
a
a
prises the cancer-related b-Galp-(1?3)-GalpNAc disaccharide
(known as T-antigen)35,36 connected with a 4-methylumbelliferyl
aglycon (4-MU) via an
2. Results and discussion
2.1. Optimization of -selective aromatic glycosylation
a-glycosidic linkage (Fig. 1).
a
In a project connected with the kinetic study of -fucosidases,
a
-L
we required chromogenic p-nitrophenyl -L-fucopyranosyl
a
OH
OH
HO
HO
HO
O
O
O
OH
AcHN
O
O
O
1 4(MU) α-T-antigen
* Corresponding authors. Tel.: +886 3 5131204; fax: +886 3 5723764 (K.-K.T.M.).
E-mail addresses: ykl@cc.nctu.edu.tw (Y.-K. Li), tmong@mail.nctu.edu.tw (K.-K.T.
Mong).
Figure 1. 4-MU a-T-antigen.
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.12.013

S.-S. Chang et al. / Carbohydrate Research 344 (2009) 432–438
433
substrates. Based on a Helferich glycosylation procedure,17,22 cou-
pling of p-nitrophenol (PNP) with per-O-acetyl -fucopyranosyl
acetate in the presence of ZnCl2 was attempted. Disappointingly,
this reaction failed to provide a detectable product under various
conditions. We attributed the failure to the harsh conditions,
Table 1
Glycosylation of PNP with 2,3,4-tri-O-acetyl-L-fucopyranosyl imidate 3 under differ-
ent reaction conditions
L
2 h, CH2Cl2
OPNP
TMSOTf
3
+
p-nitrophenol
which would be likely damaging to the fragile
a-fucosidic bond.
(PNP)
A further literature survey revealed that various per-O-acetyl glu-
OPNP
OAc
+
O
O
copyranosyl donors have been employed for the preparation of
OAc
O-aryl
-fucopyranosyl imidate for such purposes had not been exploited.
Given its simplicity of preparation and reactive nature, it was
worth exploring the use of 2,3,4-tri-O-acetyl- -fucopyranosyl imi-
date as glycosyl donor for the synthesis of p-nitrophenyl 2,3,4-
tri-O-acetyl- -fucopyranoside. Thus, -fucopyranose was con-
verted to 1,2,3,4-tetra-O-acetyl-
-fucopyranose 2,37 which was
a
-glucopyranosides,19,25,26 but the use of per-O-acetyl
OAc
OAc
AcO
AcO
L
α-anomer 4a
β-anomer 4b
L
a
-L
L
Entry
TMSOTf (mol equiv)
T (°C)
4aa (%)
4ba (%)
L
1
2
3
4
5
6
0.05
0.5
1.0
1.0
1.0
1.0
ꢀ20
ꢀ20
ꢀ20
ꢀ40
0
40
22
56
54
26
22
30
30
0
subsequently transformed to imidate donor 3 by standard proto-
cols (Scheme 1).38 Imidate 3 (1 mol equiv) was then coupled with
PNP (1.2 mol equiv). In contemplating a choice of glycosylation
promoter, TMSOTf was preferred over BF3ꢁEt2O because the use
0
26
22
25
of the former was found to favor 1,2-cis
a-glycosidic bond
formation.26
a
Isolated yield.
To delineate the optimal conditions for reaction, p-nitrophenol
was first glycosylated with imidate 3 using different amounts of
TMSOTf (Table 1, entries 1ꢀ3). Glycosylation with either 0.05 or
0.5 mol equiv of TMSOTf at ꢀ20 °C resulted in a random anomeric
OH
OH
R'
R'
TfN3, K2CO3,
cat. CuSO4
mixture of p-nitrophenyl L
-fucopyranosides 4a39 and 4b40 (Table 1,
O
O
R
R
OH
OH
HO
entries 1 and 2). Intriguingly, when 1.0 mol equiv of TMSOTf was
used, 4a was obtained as a single anomer in acceptable 56% yield
HO
N3
NH3+ Cl−
75%
R = OH; R' = H
1 1 R = OH; R' =
(Table 1, entry 3). Assignment of 1,2-cis
was based on the J1,2 coupling constant of the anomeric proton
a-fucosidic configuration
R = H ; R' = OH
H
3
1 2 R = H ; R' =
(3.6 Hz for 4a and 9.0 Hz for 4b).
OH
Ac2O, DMAP
After exploring the optimal concentration of TMSOTf, we next
investigated the effect of temperature on the stereochemical out-
come of glycosylation (Table 1, entries 3ꢀ6). Glycosylation at 0
or 25 °C gave rise to a random anomeric mixture (Table 1, entries
pyridine, 90%
OAc
R'
OAc
1. N2H4.AcOH
R'
2. DBU, CCl3CN
O
O
R
R
OAc
AcO
AcO
5 and 6), while exclusive
a-selectivity was obtained at either
80%
N3
N3
OC(= NH)CCl3
ꢀ20 or ꢀ40 °C (Table 1, entries 3 and 4). Thus, 1 mol equiv of
6 R = OAc ; R' = H
1 3 R = OAc; R' = H
1 4 R = H ; R' =
OAc
TMSOTf and ꢀ20 °C reaction temperature were employed in subse-
8 R = H
; R' =
quent glycosylations. It has been argued that the observed a-selec-
OAc
tivity could be attributed to b?
a anomerization under acidic
conditions.41,42 However, such rationale was ruled out in our case
as anomerization to 4a was not observed when pure b-anomer 4b
was exposed to the same reaction conditions.
Scheme 2. Preparation of 2-azido-2-deoxy-glycopyranosyl imidates 6 and 8.
12,47,48 which underwent standard procedures furnishing the
desired glycosyl imidates 649 and 8.50
2.2.
a-Aromatic glycosylation with different per-O-acetyl
glycosyl imidates
Glycosylation of PNP with the anomeric mixtures of glycosyl
imidates 5ꢀ10 was examined (Table 2). As a comparison,
a-glyco-
With the appropriate conditions for a-selective aromatic glyco-
syl imidates 5a, 6a, 7a, and b-glycosyl imidates 5b, 6b, 7b were iso-
sylation in hand, we were prompted to examine the scope of
glycosylation with different glycosyl imidates 5ꢀ10. Thus, known
per-O-acetyl glycosyl imidates 5, 7, 9, and 10 were prepared
according to literature procedures.43–46 Syntheses of 2-azido-2-
deoxy glycopyranosyl imidates 6 and 8 are outlined in Scheme 2.
lated by chromatography and were used for glycosylation studies
(Table 2, entries 2, 3, 5, 6, 8, and 9). The
tion with imidates in the -gluco and -galacto series spanned from
modest (3.4:1 - to b-anomer ratio for donor 5) to -exclusive (for
donor 8) (Table 2, entries 1ꢀ10). No significant difference in
selectivity was noted using either the - or b-glycosyl imidate as
a-selectivity of glycosyla-
D
D
a
a
a-
Hydrogen chloride salts of
D-glucosamine and D-galactosamine
a
were converted to 2-azido-2-deoxy glycosyl derivatives 11 and
donor, although the use of b-anomer severely reduced the glyco-
sylation yield (Table 2, entries 2 vs 3, 5 vs 6, and 8 vs 9). The
a
-selectivity obtained for glycosyl imidates bearing an axial C-4
OR
OH
OH
Ref 11
90%
acetyl function (3, 7, and 8) was generally higher than that
obtained from donors with an equatorial C-4 acetyl function (5
and 6) (Table 2, entries 1 vs 7 and 4 vs 10). To aim at higher selec-
tivity, the known
to form a co-solvent mixture for glycosylation, but no significant
improvement was obtained (data not shown).51 Exclusive
-selec-
tivity observed for -rhamnopyranosyl and -mannopyranosyl
imidates 9 and 10 was expected owing to the joined forces of
O
O
OAc
OAc
OH
AcO
HO
L-fucopyranose
1. N2H4.AcOH
2. DBU, CCl3CN
a-directing solvent diethyl ether was introduced
2
R = Ac
93%
a
3
R = C(=NH)CCl3
L
D
Scheme 1. Preparation of 2,3,4-tri-O-acetyl L-fucopyranosyl imidate 3.

434
S.-S. Chang et al. / Carbohydrate Research 344 (2009) 432–438
Table 2
Glycosylation of p-nitrophenol (PNP) with per-O-acetyl glycosyl imidates 5ꢀ10
Entry
Glycosyl imidate R = C(@NH)CCl3
Product
Yield (%)
51
a
:ba
Ref.
OAc
OAc
O
O
AcO
AcO
AcO
54,55
1
3.4:1
AcO
OR
OPNP
OAc
5
OAc
α:β = 14:1
15
OAc
O
AcO
2
3
15
15
50
20
3.4:1
3.8:1
—
—
AcO
AcO
OR
5a
OAc
O
AcO
OR
AcO
OAc
5b
OAc
O
OAc
O
AcO
AcO
AcO
AcO
4
49
4.5:1
—
N3 OPNP
N3 OR
α:β = 18:1
6
b
16
OAc
O
AcO
AcO
5
6
7
16
16
50
15
60
4.8:1
4.5:1
5.4:1
—
N
3OR
6a
OAc
O
AcO
AcO
—
OR
N3
6b
7
OAc
OAc
O
AcO
AcO
AcO
AcO
O
55
OR
OAc
OPNP
OAc
α:β = 3.6:1
18
OAc
AcO
AcO
O
8
18
18
62
5.0:1
5.6:1
—
AcO
OR
7a
OAc
O
AcO
AcO
9
30
60
—
—
OR
OAc
7b
OAc
O
AcO
AcO
OAc
O
AcO
10
a
a
a
nly
nly
nly
AcO
OR
OR
N
3OPNP
N3
8
9
19
OPNP
O
O
AcO
11
45
55
56
57
AcO
AcO
AcO
OAc
OAc
20
OAc
OAc
OAc
OAc
O
AcO
AcO
O
12
AcO
AcO
OPNP
OR
10
21
a
Anomeric ratio was determined by HPLC analysis of crude anomeric mixture (elution with 7:3 hexane–EtOAc at 1.2 mL/min over Mightysil Si 60 250ꢀ4.6).
b
Anomeric mixture 16 was deacetylated to give a-anomer 17 for NMR characterization.

S.-S. Chang et al. / Carbohydrate Research 344 (2009) 432–438
435
anchimeric assistance and anomeric effect (Table 2, entries 11 and
12).52,53
was also prepared by a known phase-transfer catalyzed glycosyla-
3
tion (For b-anomer of 23: 1H, d = 5.02, J1,2 = 7.8 Hz; 13C,
d = 100.5).60
2.3. Mechanistic aspect
Upon deacetylation and benzylidenation, derivative 23 was
transformed into galactosaminyl acceptor 24, which was then gly-
cosylated with galactopyranosyl imidate 7 to give the fully protected
Based on literature findings and the current experimental data,
we propose a mechanism to account for the
a
-selective aromatic
4-MU
MU -T-antigen 1 was accomplished by standard deprotection. The
overall yield of the fully protected 4-MU
a-T-antigen 25 in 75% yield. Conversion of 25 to the target 4-
glycosylation (Fig. 2).25 In the presence of molar equiv of TMSOTf,
the glycosyl imidate is completely converted to oxocarbenium ion,
which is in association with triflate counterion to form an
oxocarbenium-triflate ion pair. For oxocarbenium ions having a
C2-acetyl function, neighboring-group participation resulted in
an equilibrium mixture of oxocarbenium and acetoxonium ions.
It is generally accepted that nucleophilic attack of ordinary accep-
tor at acetoxonium ion produces the b-glycoside. Nevertheless, for
weakly nucleophilic phenols (especially those having electron-
withdrawing groups), nucleophilic attack occurred predominantly
on the more reactive oxocarbenium–triflate ion pair furnishing a
a
a-T-antigen 25 from galac-
tosamineꢁHCl is 21%, which is comparable to that reported by Kiso.31
However, the present method offers the attractiveness in that only a
simple per-O-acetyl glycosyl imidate donor is required to react with
near stoichiometric amount of a phenol and yet exclusive formation
of an aryl
In summary, we have developed a
sylation for direct access of O-aryl -glycopyranosides. The attrac-
tive feature of this method is the use of simple per-O-acetyl
glycosyl imidates to achieve a respectable level of -selectivity
a-glycosidic bond is realized.
a
-selective aromatic glyco-
a
a
mixture of
the anomeric effect, the
a
- and b-glycosyl oxonium intermediates.25 Owing to
-glycosyl oxonium ion is more stable
without resorting to special protecting functions. The synthetic
utility of the method was demonstrated by a concise and stereose-
a
than b-glycosyl oxonium ion; thus, formation of the former would
lective synthesis of a cancer-related fluorogenic 4-MU a-T-antigen
1. Owing to the prevalent use of aryl glycosides as enzyme sub-
be favored based on a product-development control mechanism.58
In addition, the higher
a-selectivity obtained from imidate donor 3,
strates in biological studies, the described method should be valu-
7, or 8 compared to that obtained from imidate donor 5 or 6 should
be attributed to the extra stability arising from through-space elec-
trostatic interaction between the axially disposed C-4 acetyl func-
able for their synthesis. Work on the preparation of aryl
a-L-
fucosides and their subsequent use in the study of human -fuco-
a-L
sidase is underway and the results will be reported in due course.
tion and ring oxygen atom of the corresponding
a-glycosyl
oxonium ion.59 However, validation of the mechanism demands
elaborative studies and molecular simulation, which is beyond
the scope of present study.
3. Experimental
3.1. General experimental
Chemicals used in experiments were purchased as ACS re-
agent grade and were used without further purification. Hexane
and EtOAc for HPLC elution were purchased as HPLC reagent
grade and was degassed by ultrasonication before use. Molecular
sieves (MS-4 Å) were activated by microwave irradiation and
flame dried intermediately before use. ACS reagent grade CH2Cl2
was distillated over calcium hydride under N2. Amberlite IR-120
H+ was washed sequentially with deionized H2O and MeOH, fol-
lowed by drying in vacuo for 18 h before use. Normal phase and
reverse phase flash column chromatography were performed on
2.4. Synthesis of 4-MU a-T-antigen
To demonstrate the synthetic utility of this
glycosylation, the synthesis of 4-MU -T-antigen 1 was attempted.
A synthetic challenge for preparation of 4-MU -T-antigen 1 is the
construction of
-aryl glycosidic linkage.23 Recently, Kiso reported
a-selective aromatic
a
a
a
the synthesis of 1 by employing Mitsunobu condensation of an ex-
cess of 4-MU (8 equiv) with a 4,6-O-di-(t-butylsilylidene)-pro-
tected disaccharide donor to obtain a less perfect 9:1
a- to b-
anomeric mixture of the desired aryl glycoside.31 With the present
glycosylation method in hand, we were confident to tackle this
synthetic challenge. Thus, 1.2 mol equiv of 4-MU 22 was coupled
Silica Gel 60 (70ꢀ230 mesh) and 75
lM RP-C18 silica gel, respec-
tively. 1H and 13C NMR spectra of the prepared compounds were
recorded either with Bruker 300 MHz and 75 MHz or with Inova
500 MHz and 125 MHz spectroscopies. Chemical shifts (d ppm)
are measured against TMS, generated from the residual CHCl3
lock signal at d 7.26 ppm, against the residual proton signal of
deuterated chloroform, and the 13C resonance signal is calibrated
against the 13C signal of deuterated chloroform. HPLC analysis
was performed with gradient pump of Hitachi L-2130 and UV-
detector L-2400.
with glycosyl imidate 8 to afford 4-MU 2-azido-2-deoxy-a-D-galac-
topyranoside 23 as a single anomer in 70% yield (Scheme 3). It
should be noted that Lemieux reported the synthesis of 23 from
the corresponding chloride donor in 33% yield along with 10% b-
anomer; thus, our method gave far better result than the previous
protocol.23 Support for the
a-glycosidic configuration in 23 was
provided by NMR spectroscopy (a
-anomer of 23: 1H, d = 5.70,
3J1,2 = 3.6 Hz; 13C, d = 97.4). For comparison, the b-anomer of 2323
O
− H+
O
O
AcO
AcO
aromatic
AcO
OC(NH)CCl3
O
+
AcO
O
OAr
H
Ar
ArOH
OTf-
1 eq TMSOTf
α
-lycoside
OC(NH)CCl3
α-glycosyl oxonium
AcO
O
O
Through space
stabilization for α-
AcO
oxonium with C-4
O
axial acetyl function
oxocarbenium triflate ion pair
H
Ar
O
ArOH
AcO
H
O
− H+
AcO
acetoxonium
O
O
ArOH
OAr
AcO
aromatic
Ar
O
O
aromatic
β
-glycoside
β-glycosyl oxonium
β-glycoside
Figure 2. Proposed mechanism for
a-selective aromatic glycosylation.

436
S.-S. Chang et al. / Carbohydrate Research 344 (2009) 432–438
Ph
(a) Na, MeOH, CH2Cl2
(b) C6H5CH(OMe)2,
cat. TsOH, CH3CN
O
1 mol equiv TMSOTf,
OAc
O
O
O
OH
AcO
AcO
O
8 , CH2Cl2, −20 oC
O
HO
70%
70% α-anomer only
N3
N3
OAr
OAr
22
23
24
O
=
O
Ar
Ph
(a) Pd/C, 1 atm H2, MeOH
(b) Ac2O, pyr.
(c) Na(s), MeOH/CH2Cl2
OH
O
OH
OAc
O
HO
HO
O
7, cat. TMSOTf,
CH2Cl 2, −20 oC
AcO
AcO
O
oC
O
O
O
(d) 90% AcOH(aq), 60
HO
O
AcHN
OH
1
75% β-anomer
OAc
N
3OAr
40%
OAr
only
25
Scheme 3. Stereospecific synthesis of 4-MU
a
-T-anitgen 1.
3.2. p-Nitrophenyl 2,3,4-tri-O-acetyl-L-fucopyranosides 4
3.3.1. p-Nitrophenyl 2,3,4,6-tetra-O-acetyl-a-D-glucopyranoside
15a
To a solution of 2,3,4-tri-O-acetyl
L
-fucopyranosyl trichloro-
(36 mg, 38%):54 1H NMR (300 MHz, CDCl3) d 8.24–8.20 (m, 2H),
7.22–7.19 (m, 2H), 5.85 (d, J = 3.6 Hz, 1H), 5.72 (t, J = 9.9 Hz, 1H),
5.20 (t, J = 10.2 Hz, 1H), 5.09 (dd, J = 3.6, 10.2 Hz, 1H), 4.26 (dd,
J = 4.5, 12.3 Hz, 1H), 4.05–4.00 (m, 2H), 2.07 (s, 3H), 2.06 (s, 3H),
2.04 (s, 3H), 2.03 (s, 3H); 13C NMR (75 MHz, CDCl3) d 170.3,
170.07, 170.03, 169.4, 143.0, 125.7, 116.4, 94.0, 69.9, 69.5, 68.5,
67.8, 61.2, 20.58, 20.54, 20.50, 20.4. HRMS ES (m/z) (M+H)+ calcd
for C20H23NO12 470.1299, found 470.1308.
acetimidate 344 (100 mg, 0.23 mmol) in CH2Cl2 (2.0 mL), phenol
(1.2 mol equiv) and MS-4 Å (300 mg) were added. Upon further
addition of TMSOTf (40 lL, 0.23 mmol), the mixture was stirred
at ꢀ20 °C under N2. Complete glycosylation was assessed by
TLC and the reaction mixture was diluted with CH2Cl2 (10 mL).
After filtration removal of MS-4 Å, the CH2Cl2 solution was
washed with sat. NaHCO3 soln (20 mL ꢂ 2), water (20 mL ꢂ 1),
and brine (20 mL ꢂ 1), dried over MgSO4, filtered, and concen-
trated for column chromatography over silica gel to furnish p-
3.3.2. p-Nitrophenyl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside
nitrophenyl 2,3,4-tri-O-acetyl-
L
-fucopyranosides 4. For p-nitro-
4a (53.0 mg,
15b
phenyl 2,3,4-tri-O-acetyl- -L-fucopyranoside
a
(12 mg, 13%):55 1H NMR (300 MHz, CDCl3) d 8.24–8.19 (m, 2H),
7.26–7.04 (m, 2H), 5.47–5.07 (m, 3H), 4.31–3.90 (m, 4H), 2.09 (s,
3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.01 (s, 3H); 13C NMR (75 MHz,
CDCl3) d 170.3, 170.1, 169.3, 169.1, 143.2, 125.7, 116.5, 97.9,
72.4, 72.3, 70.8, 67.9, 61.7, 20.6, 20.59, 20.55, 20.52.
56%):39 1H NMR (300 MHz, CDCl3) d 8.22–8.19 (m, 2H), 7.17–
7.13 (m, 2H), 5.86 (d, J = 4.0 Hz, 1H), 5.58 (dd, J = 6.0, 12.0 Hz,
1H), 5.37 (ddd, J = 6.0, 6.0, 12.0 Hz, 2H), 4.29 (q, J = 7.0, 14.0 Hz,
1H), 2.19 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H), 1.21 (d, J = 7.0 Hz,
3H); 13C NMR (75 MHz, CDCl3) d 170.67, 170.65, 170.3, 161.3,
143.0, 126.1, 116.5, 94.9, 70.7, 67.8, 67.6, 66.3, 20.9, 20.9, 20.8,
16.0. HRMS FAB (m/z) (M+H)+ calcd for C18H21NO10 412.1244,
found 412.1245.
3.3.3. p-Nitrophenyl 2-azido-2-deoxy-a-D-glucopyranoside 17
(29.0 mg, 80%): 1H NMR (300 MHz, CDCl3) d 8.24 (dd, 2H), 7.31
(dd, 2H), 5.76 (d, J = 3.6 Hz, 1H), 4.05 (dd, J = 8.4, 10.2 Hz, 1H),
3.72–3.68 (m, 2H), 3.64–3.61 (m, 1H), 3.56–3.55 (m, 1H), 3.41–
3.35 (m, 2H); 13C NMR (75 MHz, CDCl3) d 161.8, 143.1, 125.7,
116.9, 97.3, 74.4, 71.7, 70.4, 63.2, 61.0; HRMS ES (m/z) (M+Na)+
calcd for C12H14N4O7 349.0760, found 349.0760.
3.3. p-Nitrophenyl per-O-acetyl glycopyranosides 15ꢀ21
To a solution of per-O-acetyl glycopyranosyl trichloroacetimi-
dates 5ꢀ10 (100 mg, 1.0 mol equiv) in CH2Cl2 (2.0 mL), p-nitro-
phenol (1.2 mol equiv) and MS-4 Å (300 mg) were added at
ꢀ20 °C. Upon addition of TMSOTf (1.0 mol equiv), the mixture
was stirred at ꢀ20 °C under N2 for 0.5ꢀ2 h and progress of reac-
tion was monitored by TLC. Upon complete glycosylation, CH2Cl2
(10 mL) was added to the mixture, and the organic phase was
washed with satd NaHCO3 soln (20 mL ꢂ 2), water (20 mL ꢂ 1),
and brine (20 mL ꢂ 1), dried over MgSO4, filtered, and concen-
trated for column chromatography purification over silica gel.
3.3.4. p-Nitrophenyl 2,3,4,6-tetra-O-acetyl-a-D-
galactopyranoside 18a
(49.0 mg, 51%):55 1H NMR (300 MHz, CDCl3) d 8.25–8.19 (m, 2H),
7.20–7.15 (m, 2H), 5.89 (d, J = 3.6 Hz, 1H), 5.58–5.51 (m, 2H), 5.33
(dd, J = 3.6, 10.2 Hz, 1H), 5.27 (t, J = 6.0 Hz, 1H), 4.14–4.02 (m, 2H),
2.17 (s, 3H), 2.08 (s, 3H), 2.02 (s, 3H), 1.93 (s, 3H); 13C NMR
(75 MHz, CDCl3) d 170.4, 170.3, 170.2, 170.1, 160.8, 143.0, 125.8,
116.5, 94.6, 67.7, 67.5, 67.3, 67.1, 61.3, 20.68, 20.63, 20.59, 20.54.
Elution with EtOAc–hexane mixture afforded the respective
a-
and b-anomers of the titled p-nitrophenyl per-O-acetyl glycopyr-
anosides 15, 18ꢀ21. To purify 16, the crude anomeric mixture of
glycoside 16 (50 mg, 0.11 mmol) was dissolved in MeOH
(2.0 mL) at 0 °C, which was followed by the addition of a piece
of freshly cut sodium (ca. 20 mg). The mixture was stirred from
0 °C to rt for 2 h and then neutralized with IR-120 H+. After fil-
tration removal of resin and removal of solvent by rotary evap-
orator, the crude deacetylated product was purified by column
chromatography over silica gel with 1:20 v/v MeOH–CH2Cl2 elu-
3.3.5. p-Nitrophenyl 2,3,4,6-tetra-O-acetyl-b-D-
galactopyranoside 18b
(8.0 mg, 9%):55 1H NMR (300 MHz, CDCl3) d 8.24–8.21 (m, 2H),
7.15–7.08 (m, 2H), 5.50 (d, J = 3.6 Hz, 1H), 5.20 (d, J = 8.1 Hz, 1H),
5.16 (dd, 1H), 4.23–4.11 (m, 4H), 2.20 (s, 3H, AcO), 2.08 (s, 6H,
AcO), 1.94 (s, 3H, AcO).
3.3.6. p-Nitrophenyl 3,4,6-tetra-O-acetyl-2-azido-2-deoxy-a-D-
galactopyranoside 19
tion to give the
80%).
a
-anomer 17 as white amorphous solid (29.0 mg,
(55.0 mg, 60%): 1H NMR (300 MHz, CDCl3) d 8.25–8.20 (m, 2H),
7.26–7.18 (m, 2H), 5.73 (d, J = 3.6 Hz, 1H), 5.58–5.51 (m, 2H), 5.26

S.-S. Chang et al. / Carbohydrate Research 344 (2009) 432–438
437
(t, J = 6.6 Hz, 1H), 4.12–4.06 (m, 2H), 3.93 (dd, J = 3.6, 10.8 Hz, 1H),
2.19 (s, 3H), 2.15 (s, 3H), 1.94 (s, 3H); 13C NMR (75 MHz, CDCl3) d
170.5, 170.2, 170.1, 161.0, 143.7, 126.2, 117.2, 97.2, 68.6, 68.4,
67.4, 61.6, 57.5, 21.0, 20.97, 20.93. HRMS ES (m/z) (M)+ calcd for
C18H20N4O10 452.1179, found 452.1195.
ded 24 as white amorphous powder (68.0 mg, 70% over two steps):
1H NMR (300 MHz, CDCl3) d 7.56–7.51 (m, 3H, ArH), 7.41–7.38 (m,
3H, ArH), 7.11–7.05 (m, 2H, ArH), 6.19 (s, 1H, C@CH), 5.76 (d,
J = 3.6 Hz, 1H, H-1), 5.62 (s, 1H), 4.42–4.39 (m, 2H, H-4, H-5),
4.28 (d, J = 12.6 Hz, 1H, H-6), 4.09 (d, J = 12.9 Hz, 1H, H-60), 3.85–
3.81 (m, 2H, H-2, H-3), 2.41 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3)
d 161.3, 159.3, 155.2, 152.7, 137.4, 129.8, 128.7, 126.6, 126.2,
115.7, 113.6, 113.4, 104.7, 101.6, 97.8, 75.5, 69.3, 67.8, 64.3, 60.5,
53.8, 19.1. HRMS ES (m/z) (M+H)+ calcd for C23H21N3O7 452.1458,
found 452.1452.
3.3.7. p-Nitrophenyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranoside
20
(43.0 mg, 45%):56 1H NMR (300 MHz, CDCl3) d 8.21–8.16 (m,
2H), 7.18–7.13 (m, 2H), 5.55 (d, J = 3.6 Hz, 1H), 5.54–5.41 (m,
2H), 5.18 (t, J = 9.9 Hz, 1H), 3.90–3.85 (m, 1H), 2.20 (s, 3H), 2.06
(s, 3H), 1.97 (s, 3H), 1.19 (d, J = 6.3 Hz, 3H); 13C NMR (75 MHz,
CDCl3) d 170.0, 169.9, 169.7, 160.3, 142.8, 125.7, 116.2, 95.4,
70.4, 69.4, 69.1, 68.4, 20.7, 20.64, 20.60, 17.3; HRMS ES (m/z)
(M)+ calcd for C18H21NO10 411.1165, found 411.1168.
3.6. 4-Methylumbelliferyl 2-azido-4,6-di-O-benzylidene-2-
deoxy-3-(20,30,40,60-tetra-O-acetyl-b-
D-galactopyranosyl)-a-D-
galactopyranoside 25
To a mixture of 2,3,4,6-tetra-O-acetyl-
chloroacetimidate (108.0 mg, 0.22 mmol), (4-MU) 2-azido-
4,6-O-benzylidene-2-deoxy- -galactopyranoside 24 (50 mg,
0.11 mmol) and MS-4 Å (300 mg) in CH2Cl2 (2.0 mL), TMSOTf
(4.0 L, 0.022 mmol) was added and the mixture was stirred
D-galactopyranosyl tri-
3.3.8. p-Nitrophenyl 2,3,4,6-tetra-O-acetyl-a-D-
mannopyranoside 21
7
a-D
(52.0 mg, 55%):57 1H NMR (300 MHz, CDCl3) d 8.25–8.19 (m, 2H),
7.25–7.17 (m, 2H), 5.62 (d, J = 3.6 Hz, 1H), 5.55 (dd, J = 3.3, 9.9 Hz,
1H), 5.46 (dd, J = 3.6, 9.9 Hz, 1H), 5.38 (t, J = 9.9 Hz, 1H), 4.30 (dd,
J = 5.4, 12.0 Hz, 1H), 4.12–3.97 (m, 2H), 2.24 (s, 3H), 2.04 (s, 3H),
2.03 (s, 3H), 1.99 (s, 3H); 13C NMR (75 MHz, CDCl3) d 170.3, 169.9,
169.6, 160.0, 143.0, 125.8, 116.4, 95.6, 69.7, 68.8, 68.4, 65.5, 61.8,
20.8, 20.7, 20.63, 20.60; HRMS ES (m/z) (M+H)+ calcd for
C20H23NO12 470.1299, found 470.1301.
l
at ꢀ20 °C for 2 h. The reaction mixture was then diluted with
CH2Cl2 (6.0 mL), which was washed with satd NaHCO3 soln
(10 mL ꢂ 2), water (10 mL ꢂ 1), and brine (10 mL ꢂ 1), dried
over MgSO4, filtered, and concentrated for column chromatogra-
phy purification over silica gel. Elution with 1:1 v/v EtOAc–hex-
ane elution afforded the titled compound 25 as white
amorphous powder (65.0 mg, 75%): 1H NMR (300 MHz, CDCl3)
d 7.57–7.54 (m, 3H), 7.40–7.38 (m, 3H), 7.10–7.07 (m, 2H),
6.19 (s, 1H), 5.80 (d, J = 3.6 Hz, 1H), 5.60 (s, 1H), 5.44–5.30
(m, 2H), 5.10 (dd, J = 3.0, 10.2 Hz, 1H), 4.91 (d, J = 7.8 Hz, 1H),
4.50 (br, 1H), 4.35–3.99 (m, 7H), 3.79 (br, 1H), 2.41 (s, 3H),
2.16 (s, 3H), 2.09 (s, 3H), 2.03 (s, 3H), 1.97 (s, 3H); 13C NMR
(75 MHz, CDCl3) d 170.7, 170.6, 170.50, 169.8, 161.2, 159.3,
155.3, 152.5, 137.7, 129.4, 128.6, 126.4, 126.3, 115.8, 113.5,
113.3, 105.0, 102.9, 101.0, 98.0, 76.2, 75.8, 71.4, 71.3, 69.3,
69.0, 67.3, 64.6, 61.7, 58.8, 21.15, 21.10, 20.97, 20.90, 19.0.
HRMS ES (m/z) (M)+ calcd for C37H39N3O16 781.2330, found
781.2299.
3.4. 4-Methylumbelliferyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-a-
D
-galactopyranoside 2323
To a mixture of 2,3,4,6-tetra-O-acetyl-D-galactopyranosyl tri-
chloroacetimidate 8 (600 mg, 1.26 mmol), 4-methylumbelliferone
22 (270 mg, 1.51 mmol), and MS-4 Å (1.20 g) in CH2Cl2 (5.0 mL),
TMSOTf (220
l
L, 1.26 mmol) was added. Upon stirring at ꢀ20 °C
for 8 h under N2, the reaction mixture was diluted with CH2Cl2
(10 mL). The resulting CH2Cl2 solution was washed with satd NaH-
CO3 soln (20 mL ꢂ 2), water (20 mL ꢂ 1), and brine (20 mL ꢂ 1),
dried over MgSO4, filtered, and then concentrated for column chro-
matography purification over silica gel. Elution with a 1:6 v/v
EtOAc–hexane mixture provided 23 as a yellowish oily liquor
(430 mg, 70%): 1H NMR (300 MHz, CDCl3) d 7.56 (d, 1H), 7.11 (s,
1H), 7.04 (d, 1H), 6.19 (s, 1H), 5.70 (d, J = 3.6 Hz, 1H), 5.57–5.41
(m, 2H), 4.30 (t, J = 3.6 Hz, 1H), 4.12–4.05 (m, 2H), 3.90 (dd,
J = 8.7, 10.8 Hz, 1H), 2.47 (s, 3H), 2.19 (s, 3H), 2.11 (s, 3H), 1.94
(s, 3H); 13C NMR (75 MHz, CDCl3) d 170.7, 170.3, 170.2, 161.2,
159.0, 155.2, 152.7, 126.2, 115.9, 114.3, 113.5, 104.7, 97.4, 68.5,
68.2, 67.5, 61.6, 57.5, 21.0, 20.98, 20.9, 19.0.
3.7. 4-Methylumbelliferyl (4-MU) 2-acetamido-2-deoxy-3-(b-
D-
galactopyranosyl)-
a-D
-galactopyranoside 131
A
mixture of 25 (36.0 mg, 0.046 mmol) and 5% Pd–C
(36.0 mg) in MeOH (2.0 mL) was stirred at rt under 1 atm H2
for 2 h. The reaction mixture was then filtered through Celite
and the filtrate was concentrated by rotary evaporator. The
crude residue was re-suspended in
a mixture of pyridine
(1.0 mL) and Ac2O (1.0 mL), and then stirred at rt for 1 h. Excess
solvent and reagent were removed by rotary evaporator, and
the residue was dissolved in MeOH (2.0 mL), to which a piece
of freshly cut sodium (ca. 20 mg) was added. The resulting mix-
ture was stirred for 2 h from 0 °C to rt and followed by neutral-
ization with IR-120 H+ resin. After filtration removal of resin,
MeOH was reduced by rotary evaporator. The crude residue
was then re-suspended in 90% aqueous acetic acid (1.0 mL)
and stirred at 60 °C for 1 h. Upon complete deacetylation as as-
sessed by TLC, the solution was concentrated by rotary evapora-
tor for column chromatography purification over reversed phase
RP-C18 silica gel with MeOH–H2O mixture elution (gradient
3.5. 4-Methylumbelliferyl 2-azido-4,6-O-benzylidene-2-deoxy-
a-
D-galactopyranoside 24
To a MeOH solution (2.0 mL) of 23 (100 mg, 0.20 mmol), a piece
of freshly cut sodium was added and the mixture was stirred ini-
tially at 0 °C before being gradually brought up to room tempera-
ture. Upon complete deacetylation as assessed by TLC (ca. 2 h),
the reaction mixture was neutralized with IR-120 H+, which was
subsequently removed by filtration. After removal of MeOH by ro-
tary evaporator and drying in vacuo for couple of hours, the crude
product was re-suspended in CH3CN (2.0 mL), to which benzalde-
hyde dimethyl acetal (45 lL, 0.3 mmol) and p-toluenesulfonic acid
from 25% to 30% MeOH) to afford the 4-MU
a-T-antigen 1
monohydrate (TsOH, 4.0 mg, 0.02 mmol) were added. After stirring
at rt for 4 h, the mixture was diluted with CH2Cl2 (6.0 mL), and the
resulting solution was washed with satd NaHCO3 soln (10 mL ꢂ 2),
water (10 mL ꢂ 1), and brine (10 mL ꢂ 1), dried over MgSO4, fil-
tered and concentrated for column chromatography purification
over silica gel. Elution with a 1:1 v/v EtOAc–hexane mixture affor-
(10 mg, 40% over four steps) as white glassy solid: 1H NMR
(300 MHz, CD3OD) d 7.79 (d, 1H), 7.26–7.20 (m, 2H), 6.26 (d,
1H), 5.73 (d, J = 3.6 Hz, 1H), 4.71 (dd, J = 3.6, 11.1 Hz, 1H),
4.61 (d, J = 6.9 Hz, 1H), 4.33 (d, J = 2.4 Hz, 1H), 4.25 (dd,
J = 3.0, 11.1 Hz, 1H), 3.99 (t, J = 5.7, 11.1 Hz, 1H), 3.93 (d,

438
S.-S. Chang et al. / Carbohydrate Research 344 (2009) 432–438
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Acknowledgments
We thank Professor T.-L. Chan for his advice on preparation of
the manuscript, and the National Science Council, Taiwan (ROC)
for financial support (NSC 96-2113-M-009-016).
Supplementary data
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