Sulfatase-catalyzed assembly of regioselectively O-sulfonated p-nitrophenyl α-D-gluco- and α-D-mannopyranosides

Article abstract of DOI:10.1016/j.carres.2004.03.026

A chemoenzymic methodology is extended to the library synthesis of regioselectively O-sulfonated pNP D-gluco and D-mannopyranosides. The method involves the sequential reactions of chemical O-sulfonation and sulfatase-catalyzed O-desulfonation. pNP 2,6-di-O-sulfo-α-D- glucopyranoside and pNP 3,6-di-O-sulfo-α-D-mannopyranoside were obtained as sodium salts using chemical methods by way of dibutylstannylene acetals or tributylstannyl ethers. They were then applied to enzyme reactions using three molluscan enzymes (snail, limpet, and abalone). The sulfatase reactions cleaved a sulfate group at the secondary O-2 or O-3 position to yield the corresponding pNP 6-O-sulfo sugars. Neither pNP 6-O-sulfo-α-D-glucopyranoside nor 6-O-sulfo-α-D-mannopyranoside became the enzyme substrate. Evidently, the molluscan sulfatases have a tendency to cleave the secondary O-sulfo group with assistance from the 6-O-sulfo group.

Full text of DOI:10.1016/j.carres.2004.03.026

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 1597–1602
Sulfatase-catalyzed assembly of regioselectively O-sulfonated
-gluco- and a-
p-nitrophenyl a-
D
D
-mannopyranosides
a,
b
b
Hirotaka Uzawa, Yoshihiro Nishida, Kenji Sasaki, Takehiro Nagatsuka,
c
*
c
Hideo Hiramatsu and Kazukiyo Kobayashi
b
a
Research Center of Advanced Bionics, National Institute of Advanced Industrial Science and Technology (AIST),
Central 5, 1-1-1 Higashi, Tsukuba 305-8565, Japan
b
Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University,
Chikusa, Nagoya 464-8603, Japan
College of Industrial Technology, Nihon University, 1-2-2 Izumichou, Narashino, Chiba 275-8575, Japan
c
Received 17 December 2003; accepted 21 March 2004
Available online 10 May 2004
Abstract—A chemoenzymic methodology is extended to the library synthesis of regioselectively O-sulfonated pNP
mannopyranosides. The method involves the sequential reactions of chemical O-sulfonation and sulfatase-catalyzed O-desulfona-
tion. pNP 2,6-di-O-sulfo-a- -glucopyranoside and pNP 3,6-di-O-sulfo-a- -mannopyranoside were obtained as sodium salts using
D-gluco and D-
D
D
chemical methods by way of dibutylstannylene acetals or tributylstannyl ethers. They were then applied to enzyme reactions using
three molluscan enzymes (snail, limpet, and abalone). The sulfatase reactions cleaved a sulfate group at the secondary O-2 or O-3
position to yield the corresponding pNP 6-O-sulfo sugars. Neither pNP 6-O-sulfo-a-
D
-glucopyranoside nor 6-O-sulfo-a-D-
mannopyranoside became the enzyme substrate. Evidently, the molluscan sulfatases have a tendency to cleave the secondary O-sulfo
group with assistance from the 6-O-sulfo group.
2004 Elsevier Ltd. All rights reserved.
ꢀ
Keywords: O-sulfonation; Sulfatase; Regioselectivity; Library synthesis
1
. Introduction
reported for mammalian sources possess either a D-
2
galacto- or an N-acetyl-
able sugar species are known to occur in nature like
-fucoses in algae and -glucoses in microbial systems.
For example, 2-O-sulfotrehalose was found in myco-
D-glucosamino backbone, vari-
The O- and N-sulfonated oligosaccharides occurring
widely in mammalian glycoproteins and lipids are
thought to have many biological roles. Among them,
O-sulfonated -galacto sugars involved in GlyCAM-1
and glycosaminoglycans have received particular atten-
L
D
1;2
5
;6
D
bacteria and archebacteria, and 6-O-sulfo-D-mannose
was identified in Dictyostelium discoideum as a compo-
3
7;8
tion because they are considered to serve as informa-
nent of secreted glycoproteins. These situations indi-
tional molecules associated with cell–cell recognition
and differentiation events. Some of the synthetic sulfo
sugars, after being converted to cluster models, work as
selectin antagonists that inhibit the binding of selectins
cate that the assembly of various types of sulfo sugars
provides a key step to elucidate the biological roles of
these natural and synthetic sulfo sugars. On the other
hand, synthesis and handling of the sulfo sugars are
believed to require special techniques and care different
from those for neutral sugars. This may be linked also to
the fact that little is known about the biological poten-
tial of sulfo sugars in applications to carbohydrate-
based drugs and materials.
x
a
to cell-surface sialyl Lewis (or Lewis ) that may lead to
4
malignant transformations. Although the sulfo sugars
*
Corresponding author. Tel.: +81-298-61-4431; fax: +81-298-61-4680;
e-mail: h.uzawa@aist.go.jp
0
008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.03.026

1598
H. Uzawa et al. / Carbohydrate Research 339 (2004) 1597–1602
Regarding chemical syntheses, regioselective O-
sulfonation methods assisted by Bu SnO and (Bu Sn)
have been proposed and applied to the synthesis, for
example, of 3-O-sulfo Lewis , 3-O-sulfo Lewis , and
their cluster models like glycopolymers and dendri-
OR2
OR2
HO
2
3
2
O
HO
HO
O
O
HO
1
R O
OR1
OpNP
a
x
OpNP
R2
R1
H
R2
R1
9
–12
mers.
received increasing attention because of their efficiency
Recently, chemoenzymatic approaches have
1
2
3
4
H
5
6
7
8
H
H
SO Na
3
SO Na
3
i
SO3Na
SO3Na
SO Na
3
iii
0
ii
SO3Na
H
ii
iv
and green processes. A 6-sulfotransferase using 3 -
0
H
H
SO3Na
i
phosphoadenosine-5 -phosphosulfate (PAPS) as the
donor substrate was applied to the construction of the 6-
SO3Na
H
Scheme 1. Reagents and conditions: (i) Bu
2
SnO, THF–benzene
O-sulfo-
and chitotetraose.
ferase was used for the synthesis of 6-O-sulfo-N-acetyl-
D-GlcNAc residue in chitobiose, chitotriose,
(
50:50 fi 2:98, v/v), 4 h, reflux, then SO NMe , DMF, 50 °C, 16 h; (ii)
3
3
13;14
A bovine UDP-galactosyltrans-
Sulfatases from snail (Helix pomatia), abalone or limpet (Patella
vulgata), 0.25 M HOAc–NaOAc buffer (pH 6.8), 37 °C, 75 h.; (iii)
(Bu Sn) O, THF–benzene (1/1(v/v)), 4 h, then SO NMe , DMF, 40 °C,
15
lactosamines. Recently, a novel methodology based on
glycosidase-catalyzed transglycosylation was proposed
3
2
3
3
1
3 3
1 h; (iv) SO NMe , DMF, 40 °C, 48 h.
0
for the assembly of 6 -O-sulfo-disaccharides carrying the
D
16
-GlcNAc residue at the nonreducing moiety. Though
Table 1. Conditions and products in the chemical O-sulfonation of 1
and 5
these chemical and enzymatic methods may provide
practical ways for the synthesis of natural sulfo sugars,
there is no established way for the library assembly of
sulfo sugars and their cluster models.
Starting
materials
Reagents
Solvents
Main products
b
(yields )
1
Bu
2
2
SnO
SnO
THF–benzene
(50:50 fi 2:98, v/v)
MeOH
2 (35%) and 3
(21%)
In our preceding studies, we have searched for a
chemoenzymic pathway leading to the library assembly
c
1
5
5
5
Bu
––
ꢀ
(Bu Sn) O
3
THF–benzene (1:1) 6 (48%)
a
2
of pNP O-sulfo-glycosides, and we have disclosed the
high potential of molluscan sulfatases (E.C. 3.1.6.1,
a
––
Bu
–– 7 (37%)
THF–benzene (1:1) 8 (54%)
2
SnO
17;18
abalone, snail, and limpet).
Though the sulfatases
a
b
c
Without the tin activation using SO
Isolated yields.
3 3
NMe (3.0 mol equiv) in DMF.
are still thought to hold rigid specificity for 3-O-sulfo-b-
1
9
D
-galactopyranoside in natural sulfatides, we recently
found that they can also accept multiply sulfated pNP
-galactopyranosides. For example, a snail sulfatase
cleaves selectively the 3-O-sulfo group in pNP 3,6-di-O-
Unidentified complicated products.
D
Table 1. Compound 2 was obtained in 35% yield by
applying conventional dibutylstannylene acetal
method to pNP a- -glucopyranoside (1). The product 2
was obtained together with 2-O-sulfo-a- -Glc-OpNP
17;18
sulfo-galactosides to afford 6-O-sulfo-galctosides.
a
The results have allowed us to propose a synthetic
methodology involving sequential reactions of chemical
O-sulfonation and sulfatase-catalyzed O-desulfonation.
The combinatorial method has allowed us to assemble
the molecular library of regioselectively O-sulfonated
D
D
(3), which were, however, separable with an ODS
1
column. The products 2 and 3 were characterized by H
NMR and FAB mass spectroscopy. The signals of H-2 (d
0
pNP
lactosides.
aminophenyl group, which is also extendable to multi-
D
-galactosides, N-acetyl
D
-galactosaminides, and
4.42) and H-6 and H-6 (d 4.28–4.19) in 2 were largely
deshielded in comparison with the corresponding signals
of 1. In the H NMR spectrum of 3, a similar downfield
shift was observed selectively for H-2 (d 4.39), supporting
17;18
The pNP group can be reduced to p-
1
20–22
valent models.
the potential utility of our approach for the assembly of
regioselectively O-sulfated pNP -gluco- and -manno-
pyranosides as non -galacto-types of hexopyranosides.
In the present study, we examined
the 2-O-sulfo-a-D-Glc structure. The tin-activation
D
D
method, when carried out in MeOH for the activation
followed in DMF for the sulfation, gave a complicated
mixture of nonselectively O-sulfonated products (Table
D
1
). The observed solvent effect was in good accordance
17;18
2
. Results and discussion
with the results for
D
-galacto sugars.
-a- -Man-OpNP (6) was
-Man-OpNP (5) in 48% yield using
(Bu Sn) O (ca. 1.5 mol equiv) in THF–benzene, fol-
In the same way, 3,6-O-SO
derived from a-
3
D
2
.1. Chemical syntheses of pNP O-sulfo-a-
D
-gluco- and a-
-mannopyranosides as substrates of molluscan sulfatases
D
D
3
2
lowed by O-sulfonation with SO Me N in DMF. For
3
3
Five enzyme substrates of O-sulfo-a-
3) and O-sulfo-a-D-Man-OpNP (6, 7, and 8) (Scheme 1)
were prepared by chemical methods as summarized in
D
-Glc-OpNP (2 and
the transformation of 5 to 3-O-sulfo-a-
D-Man-OpNP
(8), Bu SnO was applied instead of (Bu Sn) O for the tin
2
3
2
1
activation. These products were assigned by H NMR
and FAB mass data in the same way as mentioned
above. Moreover, as a reference compound to assign
ꢀ
pNP ¼ p-nitrophenyl.

H. Uzawa et al. / Carbohydrate Research 339 (2004) 1597–1602
1599
OSO -
products in following sulfatase reactions, 6-O-sulfo-a-
Man-OpNP (7) was prepared from 5 in a random sul-
fonation with SO Me N.
D-
3
OH
O
O
HO
HO
HO
HO
3
3
-O3SO
-
3
O SO
O
O
2
.2. Substrate- and regioselectivity in sulfatase reactions
2
3
snail
abalone
for O-sulfo-a- -Glc-OpNP and O-sulfo-a- -Man-OpNP
D
D
NO2
abalone
NO2
Each of the synthetic substrates was applied to enzyme
reactions with three sulfatases from snail (Helix poma-
tia), abalone (not specified), and limpet (Patella vulgata)
OSO3-
OSO3-
O
HO
HO
O
(
Table 2). When 2 was treated with the snail sulfatase
-O3SO
O
HO
under the conditions established previously [see (ii) in
Scheme 1], a less polar compound appeared on a silica
gel TLC plate (2:1 CHCl –MeOH, v/v, twice developed).
The product was isolated in 32% yield after chromato-
graphic purification with Sephadex LH-20, ODS C-18,
OH
-O3SO
O
NO2
917,18
snail
018
1
abalone
limpet
snail
NO2
3
abalone
limpet
þ
1
OSO3
-
and ion-exchange columns (Dowex Na ). In the
H
NMR spectroscopic study, the doublet of doublet (dd)
OH
HO
HO
O
HO
-O3SO
O
HO
signals of H-2 appeared at d 3.80. The chemical shift
indicates that the product is 6-O-sulfo-a-D-Glc-OpNP 4
-O3SO
O
6
8
O
carrying no 2-O-sulfo group. No other product was
isolated from the enzyme reaction, indicating that the
snail enzyme regiospecifically cleaved the 2-O-sulfo
group. The abalone sulfatase also gave 4 as a single
product (40% yield) in the reaction with 2. In contrast,
the limpet sulfatase gave the same product but in poor
yields (<5%, determined by TLC) showing a consider-
able decrease in the reactivity. The results summarized
snail
limpet
abalone
limpet
NO2
NO2
Scheme 2.
(10) as the substrates (Scheme 2). This means that the
stereochemistry at position C-4 is not of crucial impor-
tance for the enzyme reactions as long as both the O-2
in Scheme 2 and Table 2 show that no a-
(
D
-Glc-OpNP
1) was produced in the reactions of 2 examined here.
and O-6 positions are sulfated. For 2-O-sulfo-a-
OpNP (3), only the abalone enzyme showed reactivity to
produce a- -Glc-OpNP (1). Comparing the enzyme
D-Glc-
Thus, 6-O-sulfo-a-
D
1
-Glc-OpNP (4) becomes the final
D
7;18
product. Previously,
Gal-OpNP and its b-isomer become the final products in
the sulfatase reactions of 2,6-di-O-sulfo-a- -Gal-OpNP
-Gal (9), respectively
Scheme 2). Thus, the enzyme reactions are selective for
we showed that 6-O-sulfo-a-
D
-
reactions between 2 and 3, it is obvious that the snail
enzyme cleaves the O-2 sulfo group in 2 with assistance
from the O-6 sulfo group. In our preceding studies for
D
17;18
(
(
10) and 3,6-di-O-sulfo-b-
D
D
-galacto-type substrates,
the three enzymes showed
a common tendency. The presence of a 6-O-sulfo group
promotes the hydrolysis of an O-sulfo group at sec-
ondary positions O-2 and O-3. In the present case, the
snail enzyme showed a similar tendency. On the other
hand, the abalone enzyme required no O-6 sulfo group
essentially to cleave the O-2 sulfo group in 3.
the secondary sulfates in both the
lacto-type of hexopyranosides.
D-gluco- and D-ga-
Here, it is of interest to note that the snail and abalone
enzymes accepted both 2,6-di-O-sulfo-a- -glucopyr-
anoside (2) and 2,6-di-O-sulfo-a- -galactopyranoside
D
D
a
Table 2. The results of sulfatase reactions with mono- and di-O-sulfonated pNP a-
D
-glycopyranosides
b
c
Substrates
Location of de-O-sulfation
Products (yields )
Snail
Abalone
Limpet
Snail
Abalone
Limpet
d
2
3
6
8
O-2
––
O-2
O-2
O-3
––
O-2
––
4 (32%)
e
4 (40%)
1 (28%)
7 (60%)
––
––
e
––
7 (21%)
O-3
––
O-3
O-3
7 (10%)
5 (21%)
e
e
f
––
––
a
b
c
d
e
f
Sulfatases from snail (Helix pomatia), abalone, and limpet (Patella vulgata) are commercially available.
1
Determined with H NMR analyses.
Isolated yields.
The yield was less than 5%.
The starting material was recovered.
À1
Determined with HPLC analyses. Chromatographic conditions: temperature, 40 °C; flow rate, 1.0 mL min ; detection, 300 nm; mobile phase, 10%
MeOH–H O containing 0.1% TFA. Retention time (RT): 22.8 min (compound 8) and 45.9 min (compound 5).
2

1600
H. Uzawa et al. / Carbohydrate Research 339 (2004) 1597–1602
For
every sulfatase cleaved the O-3 sulfo group of 3,6-di-O-
sulfo-a- -Man-OpNP (6) to afford 6-O-sulfo-a- -Man-
OpNP (7) exclusively. On the other hand, only a limpet
sulfatase recognized 3-O-sulfo-a- -Man-OpNP (8) as
the substrate to give a nonsulfated a- -Man-OpNP (5).
D
-manno-type substrates (Scheme 2 and Table 2),
purchased from Sigma Chemical Co. and used without
further purification. All other reagents were obtained
from Aldrich or Sigma Chemical Co. and used as
received. Reactions were monitored by thin-layer chro-
matography (TLC) on Silica Gel 60 F254 (E. Merck),
which were visualized by UV light and by spraying with
D
D
D
D
These results are in accordance with the tendency as
mentioned before as well as the assisting role of the 6-O-
sulfo group.
20% H
2
SO
4
in EtOH followed by charring at 180 °C.
Flash column chromatography was performed on Silica
Gel 60 RP-18 (ODS-C18, E. Merck, 40–63 lm). Gel-fil-
tration chromatography was performed on Sephadex
LH-20 (Amersham Pharmacia Biotech). Optical rota-
tions were measured with JASCO DIP-1000 digital
polarimeter at ambient temperature, using a 10-cm mi-
As can be seen in product yields, however, the enzyme
reaction for the 3,6-di-O-sulfo-a-D-Man-OpNP (6) was
obviously slower than that for 2, 9, and 10. Although
the reactivity varies also among the three enzymes, the
1
13
overall relative reactivity among the three
D
-hexopyr-
anoside substrates can be summarized in the order of
cro cell. H and C NMR spectra were recorded on a
Varian G300, a Varian INOVA 400 MHz or a Bruker
3
,6-di-O-sulfo-b-
Gal-OpNP (10) > 2,6-di-O-sulfo-a-
-di-O-sulfo-a- -Man-OpNP (6). Apparently, the
manno- and
OH-2 and an equatorial OH-4 group, respectively,
provide poorer substrates than the -galacto-configu-
D
-Gal-OpNP (9) ¼ 2,6-di-O-sulfo-a-
D
-
AVANCE-500 spectrometer for solutions in D
Chemical shifts are given in ppm and referenced to
internal tert-butyl alcohol (d 1.23 in D O or d 31.2 in
2
O.
D
-Glc-OpNP (2) > 3,
6
D
D
-
H
2
C
D
-gluco-configurations carrying an axial
D O). All data are assumed to be first order with
2
apparent doublet and triplets reported as ‘d’ and ‘t’,
respectively. Resonances that appear broad are desig-
nated ‘b’. FAB mass spectra (FABMS) were recorded
using a JEOL DX 303 mass spectrometer, and high-
resolution mass spectra (HRMS) were recorded using a
Hitachi M 80 mass spectrometer. Elemental analyses
were performed with a Carlo Elba EA-1108 or Perkin–
Elmer EA-2400 instrument. The HPLC measurements
were carried out on a TOSOH HPLC system with a PD-
8020 photodiode-array detector from TOSOH Corpo-
ration, Tokyo, Japan. The chromatographic system was
equipped with an autosampler AS-8020 (TOSOH Cor-
poration, Tokyo, Japan). The column used was TSKgel
ODS-80Ts QA (ODS-C18 column) with 5-lm particle
size, 250 mm · 4.6 mm i.d. (TOSOH Corporation,
Tokyo, Japan).
D
ration can do. Most of the 2,3- and 3,6-di-O-sulfo-
derivatives, however, can satisfy the minimal substrate
structure required for the binding and reaction with the
three molluscan sulfatases.
In conclusion, we have demonstrated the utility of our
chemoenzymatic method for the library assembly of
regioselectively O-sulfonated pNP
mannopyranosides. The method allowed us to assemble
not only 2,6-di-O-sulfo-a- -Glc-OpNP (2) and 3,6-di-O-
sulfo-a- -Man-OpNP (6) by chemical tin-activation
methods and but also 6-O-sulfo-a- -Glc-OpNP (4) and
-O-sulfo-a- -Man-OpNP (7) in the following enzymic
sulfatase reactions. We assembled also 2-O-sulfo-a-
Glc-OpNP (3) and 3-O-sulfo-a- -Man-OpNP (8) along
D-gluco- and D-
D
D
D
6
D
D
-
D
the chemical way. The pNP group can be converted to
the 4-aminophenyl function by catalytic hydrogenation
3.2. pNP 2,6-di-O-sulfo-a-D-glucopyranoside, disodium
salts (2) and pNP 2-O-sulfo-a-D-glucopyranoside, sodium
salt (3)
20;21
with Pd(OH)
2
irrespectively of the O-sulfo group.
Thus, these pNP-based O-sulfonated glycosides provide
a useful tool for analytical studies to determine carbo-
hydrate–protein interactions with surface plasmon res-
onance, a quartz crystal microbalance, and other
A mixture of pNP a-
D-glucopyranoside (1) (500 mg,
1.7 mmol) and Bu SnO (992 mg, 4.0 mmol) was refluxed
2
23;24
sensing.
carbohydrate modules
In addition, they can be applied as the key
for the assembly of multi-
in THF–benzene (changed from 50:50 gradually to 2:98,
v/v, 500 mL) for 4 h with continuous azeotropic removal
of water. After the evaporation of the solvents, the
20;21
valent glycoconjugates having higher potential in bio-
logical and medicinal applications. These application
studies presently in progress will be reported elsewhere.
3 3
residue was treated with SO NMe (554 mg, 4.0 mmol)
in DMF at 50 °C for 16 h. The reaction mixture was
diluted with MeOH (10 mL) and concentrated in vacuo.
The crude product was subjected to reversed-phase
chromatography on a column of ODS C18, and the
3. Experimental
column was eluted with H O. Each of fractions con-
2
3
.1. General methods
taining the desired di-O-sulfonated 2 and mono-O-sul-
fonated 3 was collected, combined, and concentrated.
Each of the products (2 and 3) was treated with ion
exchange resin (Dowex Na form) to obtain di-O-sul-
fonated 2 (297 mg, 35%) and mono-O-sulfonated 3
Sulfatases (E.C. 3.1.6.1) from snail (Helix pomatia, 16.1
units/mg), abalone entrails (not specified, 23 units/mg)
and limpet (Patella vulgata, 7.6 units/mg) were
þ

H. Uzawa et al. / Carbohydrate Research 339 (2004) 1597–1602
1601
(
141 mg, 21%), respectively. 2: ½aꢀ þ114 (c 0.7, H
2
O).
H NMR (300 MHz): d 8.23 and 7.30 (d, J 9.3 Hz,
Anal. Calcd for C12
H
13NNa
2
O
14
S
2
ÆH
2
O: C, 27.54; H,
2.89; N, 2.68. Found C, 27.11; H, 3.10; N, 2.86. FABMS
D
1
pNP), 6.09 (d, J1;2 3.6 Hz, H-1), 4.42 (dd, J 9.9 Hz,
H-2), 4.28–4.19 (m, H-6 and H-6 ), 4.08 (t, J3 9.9 Hz,
(positive-ion): 528 [M þ Na]þ.
2;3
0
;4
H-3), 4.01–3.93 (m, H-5), 3.71 (t, J 9.9 Hz, H-4).
3.5. pNP 6-O-sulfo-a-D-mannopyranoside, sodium salt (7)
4;5
13
C NMR (75 MHz): d 163.0, 144.4, 127.8, 118.9,
6.7, 78.3, 72.5, 72.3, 70.5, 68.3. Anal. Calcd for
9
3.5.1. By chemical synthesis. A mixture of pNP a-
D
-
C
Found: C, 27.97; H, 3.00; N, 2.43. FABMS (positive-
12
H
13NNa
2
O
14
S
2
ÆH
2
O: C, 27.54; H, 2.89; N, 2.68.
mannopyranoside (5) (98 mg, 0.33 mmol) and SO NMe
3
3
(138 mg, 1.0 mmol) was dissolved in DMF at 40 °C.
After 48 h, the reaction mixture was diluted with MeOH
(5 mL) and concentrated in vacuo. The residue was then
purified in the same way as described above to afford 7
þ
þ
ion): 505 [M] , 528 [M þ Na] . HRMS: Calcd for
þ
C H NNa O S
14 2
[M þ H] :
505.9651.
Found:
12
14
2
1
5
05.9560. 3: ½aꢀ þ158 (c 0.7, H O). H NMR
D
2
1
(
300 MHz): d 8.16 and 7.25 (d, J 9.3 Hz, pNP), 6.08 (d,
(48 mg, 37%). ½aꢀ þ109 (c 0.3, H O). H NMR
D
2
J
1;2 3.6 Hz, H-1), 4.39 (dd, J2;3 9.9 Hz, H-2), 4.08 (t, J3;4
(500 MHz): d 8.25 and 7.28 (d, J 9.2 Hz, pNP), 5.74 (bd,
1;2 1.5 Hz, H-1), 4.25–4.15 (m, H-2, H-6, H-6 ), 4.06 (dd,
2;3 3.4 and J3;4 8.9 Hz, H-3), 3.82 (t, J4;5 10.1 Hz, H-4).
0
0
9
.9 Hz, H-3), 3.82–3.59 (m, H-4, H-5 H-6, H-6 ).
C NMR (75 MHz): d 163.1, 144.2, 127.7, 118.7, 96.6,
8.5, 74.5, 72.4, 70.7, 61.8. FABMS (positive-ion): 426
J
J
13
7
Anal. Calcd for C12
3.83; N, 3.33. Found C, 34.64; H, 3.82; N, 3.49. FABMS
2
H14NNaO11SÆH O: C, 34.21; H,
[
M þ Na]þ.
(
positive-ion): 426 [M þ Na]þ.
3
.3. pNP 6-O-sulfo-a-D-glucopyranoside, sodium salt (4)
3.5.2. By enzymatic synthesis. A mixture of 6 (3.2 mg,
A mixture of 2 (50 mg, 0.10 mmol) and snail sulfatase
25 mg, 402 U) was dissolved in 0.25 M HOAc–NaOAc
buffer (pH 6.8, 2 mL) at 37 °C for 75 h. The reaction
mixture was purified in the same way as described above
6.3 lmol) and abalone sulfatase (3 mg, 69 U) was dis-
solved in 0.25 M HOAc–NaOAc buffer (pH 6.8, 0.5 mL)
at 37 °C for 14 days. The reaction mixture was purified
in the same way as described above to give 7 (1.5 mg,
60%). The physical data were consistent with the pro-
cedure 3.5.1.
(
to give 4 (13 mg, 32%) with recovered 2 (18 mg, 36%).
1
½
aꢀ þ93 (c 0.2, H O). H NMR (300 MHz): d 8.27 and
D
2
7
.30 (d, J 9.3 Hz, pNP), 5.80 (d, J 3.6 Hz, H-1), 4.25–
1
;2
0
3
.95 (m, H-6, H-6 ), 3.95 (t, J2;3 9.6 Hz, H-3), 3.80 (dd,
3.6. pNP 3-O-sulfo-a-D-mannopyranoside, sodium salt (8)
13
H-2), 3.59 (t, J3;4 9.6 and J4;5 9.6 Hz, H-4). C NMR
75 MHz): d 138.7, 127.8, 118.6, 98.4, 74.4, 72.6, 72.5,
(
A mixture of pNP a-
D-mannopyranoside (5) (70 mg,
0.23 mmol) and Bu SnO (69 mg, 0.28 mmol) was ref-
7
3
3
0.5, 68.3. Anal. Calcd for C12
H
14NNaO11SÆH
2
O: C,
4.21; H, 3.83; N, 3.33. Found C, 34.55; H, 3.70; N,
2
luxed in THF–toluene (1/1(v/v), 500 mL) for 3 h with
continuous azeotropic removal of water. After the
evaporation of the solvents, the residue was treated with
þ
.17. FABMS (positive-ion): 404 [M þ H] , 426
þ
þ
[
M þ Na] . HRMS: Calcd for C H NNaO S [M] :
12
14
11
4
03.2955. Found: 402.9916.
3 3
SO NMe (53 mg, 0.38 mmol) in DMF at room tem-
perature for 4 h 40 min. The reaction mixture was di-
luted with MeOH (10 mL) and concentrated in vacuo.
The residue was then purified in the same way as de-
3
salts (6)
.4. pNP 3,6-di-O-sulfo-a-D-mannopyranoside, disodium
scribed above to afford 3-mono-O-sulfonated 8 (51 mg,
1
5
8
4 %). ½aꢀ þ99 (c 2.1, H
2
O). H NMR (400 MHz): d
D
A mixture of pNP a-
D
-mannopyranoside (5) (80 mg,
.20 and 7.26 (d, J 9.2 Hz, pNP), 5.76 (bs, H-1), 4.72
0
.27 mmol) and (Bu
3
Sn) O (203 lL, 0.40 mmol) was
2
(
(
bdd, J2;3 3.2 and J3;4 9.6 Hz, H-3), 4.52 (bdd, H-2), 3.92
refluxed in THF–benzene (1/1(v/v), 500 mL) for 4 h with
continuous azeotropic removal of water. After the
evaporation of the solvents, the residue was treated with
0
13
t, J4;5 9.6 Hz, H-4), 3.83-3.65 (m, H-5, H-6, H-6 ).
C
NMR (100 MHz): d 162.3, 143.8, 127.6, 118.2, 99.1,
8
[
0.2, 75.3, 69.6, 65.9, 62.1. FABMS (negative-ion): 401
SO
3
NMe
3
(222 mg, 1.6 mmol) in DMF at 40 °C for 11 h.
M À 2H]À.
The reaction mixture was diluted with MeOH (10 mL)
and concentrated in vacuo. The residue was then puri-
fied in the same way as described above to afford O-
disulfonated 6 (65 mg, 48%). ½aꢀ þ84 (c 0.36, H O). H
NMR (500 MHz): d 8.27 and 7.31 (d, J 9.3 Hz, pNP),
1
D
2
Acknowledgements
5
8
.79 (bd, J1;2 1.9 Hz, H-1), 4.73 (dd, J2;3 3.0 and J3;4
.9 Hz, H-3), 4.54 (bdd, H-2), 4.23 (m, H-6, H-6 ), 3.97
0
The authors gratefully acknowledge Dr. Yukishige Ito,
RIKEN (The Institute of Physical and Chemical Re-
search) for his suggestions.
13
(
1
t, J4;5 8.9 Hz, H-4). C NMR (125 MHz): d 162.3,
44.0, 127.6, 118.4, 99.2, 79.9, 73.3, 69.6, 68.4, 65.6.

1602
H. Uzawa et al. / Carbohydrate Research 339 (2004) 1597–1602
12. Roy, R.; Park, W. K. C.; Zanini, D.; Foxall, C.;
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