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A. Malleron et al. / Carbohydrate Research 353 (2012) 96–99
Ph
O
O
OBn
O
O
Ph
Ph
OAc
O
O
O
O
O
O
O
O
ii
i
OPMB
BnO
BnO
O
OPMB
AcO
AcO
OPMB
HO
NHAc
NHAc
NHAc
OBn
OAc
21
14
20
OBn
O
OH
OBn
O
OBn
O
OBn
O
HO
O
BnO
BnO
iii
iv
vi
OPMB
BnO
BnO
OPMB
O
NHAc
OBn
NHAc
OBn
22
23
NaSO3O
OH
O
NaSO3O
OBn
O
OBn
O
OH
v
O
HO
HO
BnO
BnO
OPMB
OH
O
O
NHAc
NHAc
OBn
OH
24
25
Scheme 5. Reagents and conditions: (i) Bromo peracetate glucose (1.33 equiv), Hg(CN)2 (1.33 equiv), toluenenitromethane (2:1), 16 h, 55 °C, 76%; (ii) MeOH–NEt3–H20
0
(8:1:1), 4 h, 40 °C then PhCH2Br (8 equiv), NaH (4 equiv), DMF, 3 h, 0 °C, 69%; (iii) Et3SiH (3 equiv), TfOH (2 equiv), MS 4 ÅA, CH2Cl2, 30 min, ꢀ78 °C, 55%; (iv) (a) (COCl2)
(5 equiv), DMSO (25 equiv), NEt3 (25 equiv), CH2Cl2, ꢀ60 to ꢀ30 °C, (b) K Selectride (1.2 equiv), ꢀ78 to ꢀ30 °C, 70%; (v) SO3ꢁpyr (3 equiv), pyridine, 65 °C, 1 h 30 min, 84%; (vi)
H2, Pd/C, MeOH–phosphate buffer (2:1), 25%.
DMF, gave 15 in 64% yield. Hydrolysis of the 4,6-benzylidene ace-
tal, with 60% acetic acid at 60 °C, followed by regioselective stann-
ylene-promoted alkylation, allowed obtaining compound 16 in 82%
yield. Inversion of the configuration at C-4 of 16 was achieved by
SN2 displacement of C-4 triflate, as described above for compound
10, giving the D-galacto derivative 17 in 51% yield. Further treat-
ment with the sulfurtrioxide–pyridine complex in DMF gave the
4-O-sulfonato derivative 18 in 85% yield.
the substrate structural requirements of this enzyme, compounds
1, 7, 12, 13, 19, and 25 were tested as potential substrate for this
4-O-sulfatase using capillary electrophoresis to follow the desulf-
ation reaction. Interestingly, none of the benzylglycoside monosac-
charides were found to be a substrate: benzyl galactopyranoside 1,
benzyl glucopyranoside 7, and benzyl N-acetylgalactosaminopyr-
anoside 12 remained fully intact upon incubation with the sulfa-
tase. The benzyl aglycon moiety was not at the origin of the
absence of activity, since compound 13, obtained by hydrogenoly-
sis of 12 was not a substrate either. Not surprisingly, a methyl at
the position 3 of N-acetylgalactosamine was not sufficient to mi-
mic a pyranosyl ring and no reaction was observed when com-
pound 19 was incubated with the sulfatase. In contrast, the
Alternatively, monosaccharide 14 was glycosylated with acet-
obromoglucose to give 20 in 76% yield, following standard proce-
dure.13 Zemplèn deacetylation, followed by benzylation using
NaH (1 equiv/OH) and benzyl bromide (2 equiv/OH) in DMF at
0 °C gave 21 in 69% yield, without noticeable side reaction on the
N-acetamido moiety. Further treatment with triethylsilane and tri-
fluoromethanesulfonic acid allowed regioselective opening of the
benzylidene acetal to provide alcohol 22 in 55% yield. Swern oxida-
tion followed by stereoselective K Selectride reduction, gave the
disaccharide 25, that contains a b-D-glucopyranosyl moiety in posi-
tion 3 of the galactosaminyl ring, was accepted as substrate. As a
result of the sulfatase action, compound 25 was converted into
the non-sulfated corresponding disaccharide as demonstrated by
the capillary electropherograms (Fig. 1). Although, in this case,
the reaction was slower than with original CS disaccharides (factor
15 at 2 mM), these results clearly indicated that the presence of a
glucuronic acid residue at the non-reducing end is not necessary
for the enzyme activity.
In conclusion, we have prepared four 4-O-sulfated monosaccha-
rides and one 4-O-sulfated disaccharide that allowed us to better
characterize the substrate specificity of a newly cloned chondroitin
4-O-sulfatase. We have shown that the enzyme needed a minimum
structure, which should be a disaccharide. In contrast to the natu-
ral substrate, which is supposed to bear a 4,5-insaturation, this
disaccharide can contain a saturated glucuronic acid at the non-
reducing end or even a glucopyranosyl moiety. The presence of car-
boxylic acid is thus not essential to the recognition by the sulfa-
tase. Interestingly, these results show that the enzyme from
Bacteroides thetaiotaomicron had a substrate profile similar to the
one found for the 4-O-sulfatase purified from Proteus vulgaris.1
D
-galacto derivative 23 in 70% yield.5 As shown previously on other
disaccharides,5 this oxidation/reduction method gave a better yield
than SN2 displacement of a C-4 triflate. Then, sulfation of the alco-
hol with the sulfurtrioxide–pyridine complex in pyridine gave the
4-O-sulfonato derivative 24 in 85% yield. The 1H NMR spectrum of
12, 18, and 24 showed signals at relatively high field (4.6–5.1) and
display small coupling constant (J3,4 = 3.0 Hz), as awaited for 4-O-
sulfonato-D-galacto derivatives.
Hydrogenolysis of benzyl group from compounds 12, 18, and 24
using Pd/C as catalyst, afforded monosaccharide 13, its 3-O-meth-
ylated counterpart 19, and disaccharide 25, all isolated as their
sodium salts.
Recently, we used capillary electrophoresis and a synthetic
library of the eight sulfo forms of the CS basic disaccharide5 to
screen a library of potential new sulfatases, cloned notably from
Bacteroides thetaiotaomicron. Interestingly, only in the case of one
enzyme, we were able to detect sulfatase activity.6 We further
demonstrated that this enzyme hydrolyzes regioselectively sulfate
at the 4 position of the different libraries members, establishing it
as an authentic and specific chondroitin 4-O-sulfatase. In addition,
we observed that the 4,6-disulfated CS disaccharides were desulf-
ated five times slower than their non-sulfated counterpart in posi-
tion 6. Considering that 4,6-disulfated moieties are rare in CS
polymers, such result was not unexpected. Nevertheless, the sensi-
tivity of this new sulfatase to the overall pattern of sulfation of CS
disaccharides appeared to be a decisive advantage for its use in
analytical or synthetic applications. In order to better characterize
1. Experimental
1.1. Enzymatic tests for chondroitin 4-O-sulfatase
For the libraries, 1 mL of enzyme was added to 68 mL Tris–HCl
buffer 34 mM, pH 7.5 containing 6 mL of libraries solution (1 mg/
mL). The hydrolysis was followed by capillary electrophoresis.
For other compounds, sulfatase activity was assayed at 25 °C using
Malleron, Annie
