The synthesis of a novel thio-linked disaccharide of chondroitin as a potential inhibitor of polysaccharide lyases

Article abstract of DOI:10.1016/j.carres.2003.12.011

A thio-linked disaccharide based on the structure of the glycosaminoglycan chondroitin was synthesized as a potential inhibitor of chondroitin AC lyase from Flavobacterium heparinum for structural analysis of the active site. Instead it was found to be a slow substrate, thereby demonstrating that lyases, in contrast to glycosidases, can cleave thioglycoside links between sugars.

Full text of DOI:10.1016/j.carres.2003.12.011

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 699–703
The synthesis of a novel thio-linked disaccharide of chondroitin as a
potential inhibitor of polysaccharide lyases
Carl S. Rye and Stephen G. Withers*
Department of Chemistry, 2036 Main Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z1
Received 5 September 2003; received in revised form 26 November 2003; accepted 11 December 2003
Abstract—A thio-linked disaccharide based on the structure of the glycosaminoglycan chondroitin was synthesized as a potential
inhibitor of chondroitin AC lyase from Flavobacterium heparinum for structural analysis of the active site. Instead it was found to be
a slow substrate, thereby demonstrating that lyases, in contrast to glycosidases, can cleave thioglycoside links between sugars.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Glycosaminoglycan; Polysaccharide lyase; Flavobacterium heparinum; Chondroitin; Chondroitin AC
1. Introduction
RO
R'O
OR
O
CO2H
O
O
OR''
+
The highly heterogeneous and acidic polysaccharide
chains of glycosaminoglycans (GAGs) contribute to a
variety of important physiological roles such as simple
mechanical support, the lubrication and cushioning of
joints, the modulation of cell signals, as well as the
control of cellular adhesion and motility.1;2 GAGs are
composed of a repeating sequence of disaccharide units
consisting of hexosamine and uronic acid residues. The
chondroitin sulfates are the most common type of GAG
HO
NHAc
OH
CO2H
RO
R'O
OR
O
O
HO
OR''
OH
NHAc
OH
-
R = H, SO3
R' = uronic acid
R'' = hexosamine
chain and consist of an N-acetyl-
due (usually O-sulfated at C-4 or C-6) attached through
a b-(1 fi 4) linkage to -glucuronic acid, or sometimes
-iduronic acid (dermatan sulfate), that is in turn
D-galactosamine resi-
Figure 1. Elimination reaction of polysaccharide lyases.
D
L
attached via a b-(1 fi 3) linkage to the next hexosamine
residue. Polysaccharide lyases are one class of enzymes
responsible for the degradation of GAG chains. They do
so via an elimination mechanism, resulting in disaccha-
rides or oligosaccharides with D4,5-unsaturated uronic
acid residues at their nonreducing ends (Fig. 1).3;4
Compared with other carbohydrate-degrading
enzymes, such as the well studied and characterized
glycosidases, there is a paucity of information regarding
the polysaccharide lyases. This is especially true
regarding their mechanism of action and the enzymatic
residues responsible for their function. The structures of
several lyases have been determined,5–11 but few struc-
tures of complexes of native enzymes with substrates, or
analogues thereof bound across the active site are
available.ꢀ Part of the problem here is that no known,
specific, tight-binding inhibitors of polysaccharide lyases
ꢀ
For a more complete listing of the available 3D structures of the
polysaccharide lyases, please go to the Web at http://afmb.
cnrs-mrs.fr/CAZY.
* Corresponding author. Tel.: +1-604-822-3402; fax: +1-604-822-2847;
e-mail: withers@chem.ubc.ca
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2003.12.011

700
C. S. Rye, S. G. Withers / Carbohydrate Research 339 (2004) 699–703
that might be useful in such complexes have been
developed. Not only might such inhibitors prove useful
in the identification of the key residues involved in
binding and catalysis, but they also may provide
important therapeutic agents, since lyases have not been
found in mammals but are common in some bacteria. In
addition, inhibitors may be used to gain detailed infor-
mation regarding the nature of the transition state of the
enzyme-catalyzed reaction.
Thioglycosides, in which the glycosidic oxygen atom
has been replaced by a sulfur atom, have been shown to
be stable glycoside analogues, acting as competitive
inhibitors of many glycosidases.12–15 We were therefore
interested in exploring whether or not the polysaccha-
ride lyase enzymes could cleave a thiol linkage. If they
are stable towards elimination, then thioglycosides
might prove to be useful inhibitors of these enzymes,
and help expand our understanding of these enzymes,
which are still in their infancy of mechanistic investi-
gation. The thio-linked disaccharide 5 was therefore
synthesized with the hope that it would be an inhibitor
of chondroitin AC lyase from Flavobacterium hepari-
num8;16 that might be useful for structural analyses.
AcO
AcO
AcO
AcO
OAc
O
OAc
O
OR
O
i, ii
S
SH
OBn
AcO
NHAc
NHAc
OAc
1
2 R = 4-methoxybenzyl
3 R = H
iii
RO
OR
O
CO2H
iv
O
RO
S
OBn
RO
NHAc
OR
4 R = Ac
5 R = H
v
Scheme 1. Reagents and conditions: (i) NaH, THF, 0 °C fi rt; (ii)
benzyl 2,3-di-O-acetyl-6-O-(4-methoxybenzyl)-4-O-trifluoromethane-
sulfonyl-b-D
-galactopyranoside,4 DMF; (iii) 10% TFA in CH2Cl2; (iv)
CrO3, H2SO4, acetone, water, sonication, 35 °C; (v) NaOMe, MeOH.
previously been oxidized to the methyl ester, resulted in
only the elimination product being isolated, with no
substitution product evident. Thus the oxidation of the
C-6 hydroxyl was left until the end of the synthesis.
Following the coupling reaction, the 4-methoxybenzyl
ether protecting group was selectively removed by a
brief exposure to a solution of 10% TFA in CH2Cl2,
giving compound 3 as a white solid (86%). Formation of
a b-glycosidic linkage was confirmed by the 10.4 and
10.7 Hz coupling constants of the anomeric proton of
the GlcNAc moiety with H-2 in 2 and 3. The selective
oxidation of the newly exposed primary hydroxyl group
over that of the sulfur moiety was accomplished using a
sonicated Jones oxidation19 with CrO3 and H2SO4, in
acetone/water at 35 °C, to give 4 as a white solid (73%).
The global deprotection of the remaining O-linked
acetates was accomplished using sodium methoxide in
methanol, to give the desired target 5 as a white solid
(46% isolated). High resolution mass spectrometric
analysis of 5 confirmed its structure, and particularly
that oxidation had not taken place at the sulfur atom.
1H NMR analysis was used to determine whether or
not compound 5 was a substrate for chondroitin AC
lyase. Upon addition of enzyme to compound 5 in D2O
buffer, the 1H NMR spectrum revealed the gradual
appearance of peaks characteristic of the unsaturated
uronic acid product of the enzymatic reaction.4 Clearly
visible were doublets at 5.95 ppm (J 4.0 Hz) and
5.26 ppm (J 5.4 Hz) corresponding to the C-4 alkene and
anomeric protons, respectively. The H-3proton of the
product was also evident at 4.26 ppm (dd, J 4.0, 0.8 Hz),
however the signal of the H-2 proton was obscured
under peaks arising from the starting compound (5).
The enzymatic cleavage of the disaccharide 5 was very
slow (ꢀ9% product after 24 h) and was not observed to
go to completion, even after a few days under the con-
ditions used (12.5 mg/mL 5, 0.4 mg/mL enzyme, pH 7).
2. Results and discussion
The protected and activated monosaccharide moieties
used to create the target disaccharide compound were
prepared as described in the literature.4;17;18 Briefly,
compound 1 was synthesized starting from 2-acetamido-
2-deoxy-
neat acetyl chloride to give the fully acetylated a-
D-galactopyranose, which was reacted with
-
D
galactopyranosyl chloride. This crude material was then
treated with thiourea in acetone at 80 °C, yielding the
thiopseudourea hydrochloride salt, which was subse-
quently treated with K2S2O5 to yield the free thiol 1.18
To produce the other coupling partner, benzyl 2,3-di-O-
acetyl-6-O-(4-methoxybenzyl)-4-O-trifluoromethanesulfo-
nyl-b-
D
-galactopyranoside,4 benzyl b-
D-galactopyrano-
side was treated with p-anisaldehyde dimethyl acetal and
p-TsOH in DMF to install a benzylidene functionality
across hydroxyls 4 and 6. The remaining hydroxyls were
acetylated, and the benzylidene ring was selectively
opened with trifluoroacetic acid (TFA) and sodium
cyanoborohydride, leaving the 4-hydroxyl free, onto
which the triflate leaving group was installed via reac-
tion with trifluoromethanesulfonic anhydride and pyri-
dine.
The coupling of the two monosaccharides was
accomplished by deprotonating the thiol 1 with an
equivalent of sodium hydride, followed by the addition
of benzyl 2,3-di-O-acetyl-6-O-(4-methoxybenzyl)-4-O-
trifluoromethanesulfonyl-b-D
-galactopyranoside4 (Scheme
1). Prior experiments attempting to carry out this SN2
displacement of a triflate in a compound where C-6 had
This corresponds to a kcat value of less than 0.3min ꢁ1
.

C. S. Rye, S. G. Withers / Carbohydrate Research 339 (2004) 699–703
701
25
20
15
10
5
3. Experimental
3.1. Inhibition kinetics
Assays were carried out in quartz cuvettes (1 cm path
length), total solution volume 125 lL. Mixtures con-
taining buffer (50 mM sodium phosphate, 100 mM
NaCl, pH 6.8) and the desired amount of substrate
(phenyl 4-O-(20,40-dinitrophenyl)-b-
D-glucopyranosid-
uronic acid)4 and inhibitor 5 were incubated at 30 °C for
at least 15 min to thermally equilibrate them. Enzyme
(9 lL of 9.0 · 10ꢁ5 M chondroitin AC lyase in buffer)
was added, the solution mixed, and the change in
absorption at 400 nm was monitored over 3min. Rates
were calculated using an extinction coefficient of
e ¼ 10,910 Mꢁ1 cmꢁ1 for 2,4-dinitrophenolate.
1/Vmax
0
-50
-30
-10
[5] (mM)
10
Figure 2. Dixon plot showing the inhibition by compound 5. Kinetic
analysis was carried out as described in Ref. 4 using substrate con-
centrations of 0.25Km and 0.75Km. A Km value of 45 mM was deter-
mined.
3.2. General methods
All reagents were obtained from commercial suppliers
and were used without further purification. Column
chromatography was performed with Silica Gel (230–
400 mesh). TLC was performed on E. Merck pre-coated
60 F-254 silica plates and visualized using UV light and/
or by applying a solution of 10% ammonium molybdate
in 2 M H2SO4 followed by heating. CH2Cl2 was distilled
over CaH2. MeOH was distilled over magnesium and
A control experiment consisting of the same concen-
tration of compound 5 in D2O buffer in the absence of
enzyme showed no degradation whatsoever as observed
by H NMR after a few days.
The thio-disaccharide 5 displayed surprisingly weak
1
binding to the enzyme, acting as a competitive inhibitor
with a Ki value of 45 mM, measured using phenyl 4-O-
ꢁ
(20,40-dinitrophenyl)-b- -glucopyranosiduronic acid4;20;
D
ꢀ
iodine. DMF was dried successively over 4 A molecular
sieves (3Â). THF was distilled over sodium benzophe-
as substrate (Fig. 2). This low binding was quite unex-
pected, given that the monosaccharide substrate has a
Km value of 7 mM. The presence of the second sugar
moiety in the disaccharide should provide additional
binding interactions that improve affinity. It is possible
that differences in bond length and bond angle associ-
ated with the thioglycosidic bond versus the O-linked
parent might be responsible. However it has been
1
none. All H NMR assignments were based on COSY
experiments.
3.2.1. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-b-
D
-galactopyranose (1).18 Prepared from 2-acetamido-2-
deoxy-D-galactopyranose according to the procedure
for the corresponding glucopyranose compound:17;18;22
1H NMR (CDCl3, 400 MHz): d 5.54 (1H, d, J 9.4 Hz,
NH), 5.35 (1H, dd, J 3.4, 1.2 Hz, H-4), 5.04 (1H, dd, J
11.0, 3.4 Hz, H-3), 4.58 (1H, dd, J 9.8, 9.1 Hz, H-1), 4.26
(1H, ddd, J 9.7, 9.7, 9.4 Hz, H-2), 4.14–4.05 (2H, m, H-
6a, H-6b), 3.90 (1H, ddd, J 6.7, 6.7, 1.2 Hz, H-5), 2.59
(1H, 9.1 Hz, SH), 2.15, 2.03, 1.99, 1.97 (12H, 4s, 4Ac).
Anal. Calcd for C14H21NO8S: C, 46.27; H, 5.82; N, 3.85.
Found: C, 46.53; H, 5.76; N, 3.83.
pointed out13 that the greater C–S bond length (1.8 A vs
ꢀ
ꢀ
1.4 A for C–O) is somewhat countered by the smaller C–
S–C bond angle (105° vs 110° for C–O–C)21 resulting in
almost equivalent relative positioning of the two sugar
moieties. Indeed thioglycosides typically bind to glyco-
sidases with similar affinities to those of their oxygen-
linked counterparts.12;13
In summary, a thio-linked disaccharide, based on the
structure of chondroitin, was synthesized and observed
to be slowly cleaved by chondroitin AC lyase from F.
heparinum. This, combined with the fact that the enzyme
binds this disaccharide with a lower affinity than the
monosaccharide substrate, suggests that the strategy of
replacing the glycosidic oxygen with sulfur is not the
correct avenue to follow in the quest for a useful
inhibitor of the polysaccharide lyase enzymes.
3.2.2. Benzyl S-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
-galactopyranosyl)-(1 ! 4)-2,3-di-O-acetyl-4-deoxy-6-
b-D
(4-methoxybenzyl)-4-thio-b-
D
-glucopyranoside (2).
A
suspension of 1 (345 mg, 0.95 mmol), in dry THF
(10 mL) was added to a suspension of NaH (38 mg of a
60% dispersion in mineral oil, 0.95 mmol, 1 equiv) in dry
THF (9 mL) at 0 °C. The reaction mixture was then
allowed to warm to ambient temperature until the evo-
lution of hydrogen gas ceased (ꢀ15 min), concentrated
under diminished pressure and the residue dissolved in
dry DMF (7 mL). To this was added a solution of benzyl
ꢁ
Synthesized as for the appropriate benzyl glycoside found in Ref. 4.

702
C. S. Rye, S. G. Withers / Carbohydrate Research 339 (2004) 699–703
2,3-di-O-acetyl-6-O-(4-methoxybenzyl)-4-O-trifluoro-
methanesulfonyl-b-
-galactopyranoside4 (750 mg, 1.24
10.7 Hz, H-4), 2.14, 2.05 (6H, 2s, 2Ac), 2.01 (6H, s, 2Ac),
1.96, 1.89 (6H, 2s, 2Ac); 13C NMR (CDCl3, 100 MHz): d
171.35, 170.57, 170.29, 170.26, 169.80, 169.23 (6C@O),
136.85 (C), 128.49, 128.04, 127.45, 99.62, 83.04, 76.03,
74.47, 72.50, 71.96 (9CH), 70.80 (CH2), 70.46, 66.74
(2CH), 62.23, 62.20 (2CH2), 48.29, 46.17 (2CH), 23.10,
20.94, 20.72, 20.66, 20.62 (5Ac).
D
mmol, 1.3equiv) in dry DMF (10 mL), and the reaction
mixture was stirred for 2 h at rt, concentrated under
diminished pressure, redissolved in CH2Cl2, washed
with water (1Â), saturated NaHCO3 (1Â), water (1Â).
The combined aq phases were extracted once with
CH2Cl2. The combined organic phases were then dried
over MgSO4, concentrated under diminished pressure
and the resulting residue purified by column chroma-
tography (8:1 PE–EtOAc) giving 2 as a pale yellow foam
3.2.4. Benzyl S-(2-acetamido-2-deoxy-b-
D-galactopyr-
anosyl)-(1 ! 4)-4-deoxy-4-thio-b- -glucopyranosiduronic
D
acid (5). Jones reagent (0.55 mL of a soln made of 1 g
CrO3, 0.857 mL H2SO4 and 7.14 mL water) was added
dropwise to a soln of 3 (220 mg, 0.31 mmol) in acetone
(4 mL). The reaction mixture was sonicated at 35 °C for
1 h, after which time another aliquot of Jones reagent
(0.2 mL) was added and the reaction mixture sonicated
for a further hour. Isopropanol was added to quench,
and the solution was decanted from the resulting green
solid. The solvent was removed under diminished pres-
sure and the residue was passed through a short column
of silica (40:2:1 EtOAc–MeOH–water+0.4% AcOH) to
yield benzyl S-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
1
(334 mg isolated, 43%): H NMR (CDCl3, 400 MHz):
d 7.32–7.20 (7H, m, Ar), 6.86 (2H, d, J 8.5 Hz, Ar), 5.49
(1H, d, J 10.4 Hz, NH), 5.28 (1H, dd, J 3.4, 1.2 Hz,
H-40), 5.09 (1H, dd, J 11.0, 9.1 Hz, H-3), 5.02 (1H, dd,
J 9.1, 7.9 Hz, H-2), 4.85 (1H, d, J 12.5 Hz, OCH2Ar),
4.83(1H, dd, J 10.4, 3.4 Hz, H-30), 4.64 (1H, d, J
10.4 Hz, H-10), 4.56 (1H, d, J 12.5 Hz, OCH2Ar), 4.54
(1H, d, J 11.6 Hz, OCH2Ar), 4.46 (1H, d, J 11.6 Hz,
OCH2Ar), 4.43(1H, d, J 7.9 Hz, H-1), 4.27 (1H, ddd, J
10.4 Hz, H-20), 4.00 (1H, dd, J 11.3, 5.8 Hz, H-60a), 3.98–
3.92 (2H, m, H-6a, H-60b), 3.83 (1H, dd, J 11.0, 1.5 Hz,
H-6b), 3.77 (3H, s, OMe), 3.76 (1H, m, H-50), 3.67 (1H,
ddd, J 10.7, 4.0, 1.5 Hz, H-5), 3.01 (1H, dd, J 11.0,
10.7 Hz, H-4), 2.12, 2.03, 1.99, 1.94, 1.93, 1.85 (18H, 6s,
6Ac); 13C NMR (CDCl3, 100 MHz): d 171.32, 170.47,
170.24, 170.17, 169.66, 169.17 (6C@O), 159.18, 136.98,
130.14 (3C), 129.42, 128.35, 127.80, 127.37, 113.73,
99.34, 82.85, 75.69, 74.27 (9CH), 73.27 (CH2), 72.49,
71.99, 70.56 (3CH), 70.21, 68.69 (2CH2), 66.65 (CH),
62.07 (CH2), 55.194 (CH3), 48.13, 46.03 (2CH), 23.01,
20.90, 20.64, 20.62, 20.55, 20.48 (6Ac).
b-
D
-galactopyranosyl)-(1 fi 4)-2,3-di-O-acetyl-4-deoxy-
-glucopyranosiduronic acid (4) as a white
4-thio-b-
D
solid (164 mg, 73%). This was then dissolved in dry
MeOH (5 mL) to which NaOMe was added until the
soln remained basic. When the reaction was judged to be
complete by TLC (7:4:1 EtOAc–MeOH–water+0.4%
HOAc), the reaction mixture was briefly neutralized
with Amberlite IR-120(Hþ) resin, filtered and concen-
trated under diminished pressure. Note that prolonged
exposure to the acidic resin results in the formation of
the methyl ester. The residue was purified by column
chromatography (17:2:1 to 7:2:1 to 7:4:1 EtOAc–
MeOH–water all include 0.4% acetic acid) and the
resulting material dissolved in water, passed down an
ion exchange column (Bio-Rad AG 50W-X2, 200–
400 mesh, Hþ form), and freeze-dried to give 5 as a white
3.2.3. Benzyl S-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
b-
D
-galactopyranosyl)-(1 ! 4)-2,3-di-O-acetyl-4-deoxy-
4-thio-b-
D
-glucopyranoside (3). Trifluoroacetic acid
(2.0 mL) was added to a soln of 2 (300 mg, 0.37 mmol) in
dry CH2Cl2 (20 mL) and stirred at rt for 20 min. The
reaction mixture was then poured into saturated
NaHCO3 (300 mL). The aq layer was extracted with
CH2Cl2 (3Â) and the combined organic phases were
dried over MgSO4, and concentrated under diminished
pressure. The residue was purified by column chroma-
tography (1:9 PE–EtOAc to 100% EtOAc) giving 3 as an
off-white solid (220 mg, 86%): 1H NMR (CDCl3,
400 MHz): d 7.36–7.21 (5H, m, Ar), 5.62 (1H, d, J
10.7 Hz, NH), 5.33 (1H, dd, J 3.4, 1.2 Hz, H-40), 5.12
(1H, dd, J 10.7, 9.1 Hz, H-3), 5.01 (1H, dd, J 9.1, 7.9 Hz,
H-2), 4.92 (1H, dd, J 10.7, 3.4 Hz, H-30), 4.85 (1H, d, J
12.5 Hz, OCH2Ar), 4.70 (1H, d, J 10.7 Hz, H-10), 4.61
(1H, d, J 12.5 Hz, OCH2Ar), 4.59 (1H, d, J 7.9 Hz, H-1),
4.30 (1H, ddd, J 10.7 Hz, H-20), 4.10–4.02 (3H, m, H-6a,
H-6b, H-60a), 3.87 (1H, ddd, J 7.9, 6.7, 1.2 Hz, H-50), 3.60
(1H, ddd, J 10.7, 4.3, 2.4 Hz, H-5), 2.93 (1H, dd, J
1
solid (55 mg isolated, 48%): H NMR (D2O, 400 MHz):
d 7.55–7.45 (5H, m, Ar), 4.97 (1H, d, J 11.6 Hz,
OCH2Ar), 4.81 (1H, d, J 11.6 Hz, OCH2Ar), 4.63(1H,
d, J 8.0 Hz, H-1), 4.62 (1H, d, J 10.4 Hz, H-10), 4.14 (1H,
d, J 11.1 Hz, H-5), 4.04 (1H, d, J 3.1 Hz, H-40), 3.97 (1H,
dd, J 10.4, 10.4 Hz, H-20), 3.87 (1H, dd, J 11.9, 8.0 Hz,
H-60a), 3.83–3.72 (3H, m, H-30, H-50, H-60b), 3.57 (1H, dd,
J 10.6, 9.2 Hz, H-3), 3.47 (1H, dd, J 9.2, 8.0 Hz, H-2),
3.08 (1H, dd, J 11.1, 10.6 Hz, H-4), 2.11 (3H, s, Ac); 13
C
NMR (D2O, 100 MHz): d 174.71, 172.02 (2C@O),
136.33 (C), 128.67, 101.40, 84.70, 78.97, 76.63, 74.07,
72.68 (7CH), 72.68 (CH2), 71.69, 67.79 (2CH), 61.20
(CH2), 51.19, 48.61 (2CH), 22.20 (Ac); MS calcd for
C21H29NO11S [M)H]ꢁ 502.15, found m=z 502.14. Anal.
Calcd for C21H29NO11SÆ0.5H2O: C, 49.18; H, 5.90; N,
2.73. Found: C, 48.94; H, 5.75; N, 2.74.

C. S. Rye, S. G. Withers / Carbohydrate Research 339 (2004) 699–703
703
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We thank the Canadian Institutes of Health Research,
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