Synthesis of pentopyranosyl-containing thiodisaccharides. Inhibitory activity against β-glycosidases

Article abstract of DOI:10.1016/j.bmc.2009.07.055

β-(1→4)-Thiodisaccharides formed by a pentopyranose unit as reducing or non reducing end have been synthesized using a sugar enone derived from a hexose or pentose as Michael acceptor of a 1-thiopentopyranose or 1-thiohexopyranose derivatives. Thus, 2-pro

Full text of DOI:10.1016/j.bmc.2009.07.055

Bioorganic & Medicinal Chemistry 17 (2009) 6203–6212
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of pentopyranosyl-containing thiodisaccharides. Inhibitory
activity against b-glycosidases
*
Alejandro J. Cagnoni, María Laura Uhrig, Oscar Varela
CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2,
Ciudad Universitaria, 1428-Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
b-(1?4)-Thiodisaccharides formed by a pentopyranose unit as reducing or non reducing end have been
Received 19 May 2009
Revised 21 July 2009
Accepted 23 July 2009
Available online 26 July 2009
synthesized using a sugar enone derived from a hexose or pentose as Michael acceptor of a 1-thiopento-
pyranose or 1-thiohexopyranose derivatives. Thus, 2-propyl per-O-acetyl-3-deoxy-4-S-(b-
D-Xylp)-4-thi-
ohexopyranosid-2-ulose (3) and benzyl per-O-acetyl-3-deoxy-4-S-(b- -Galp)-4-thiopentopyranosid-2-
D
ulose (11) were obtained in almost quantitative yields. The carbonyl function of these uloses was reduced
with NaBH4 or K-Selectride, and the stereochemical course of the reduction was highly dependent on the
reaction temperature, reducing agent and solvent. Unexpectedly, reduction of 3 with NaBH4–THF at 0 °C
Keywords:
Thiodisaccharides
Thiopentopyranose
Glycosidase inhibitors
Sugar enones
gave a 3-deoxy-4-S-(b-
yield), with isomerization of the sulfur-substituted C-4 stereocenter of the pyranone. Reduction of 11
gave always as major product the benzyl 3-deoxy-4-S-(Galp)-4-thio-b- -threo-pentopyranoside deriva-
D-Xylp)-4-thio-a-D-ribo-hexopyranoside derivative (6) as major product (74%
D
tive 14, which was the only product isolated (80% yield) in the reduction with K-Selectride in THF at
ꢀ78 °C. Deprotection of 14 and its epimer at C-2 (13) afforded, respectively the free thiodisaccharides
19 and 18. They displayed strong inhibitory activity against the b-galactosidase from Escherichia coli.
Thus, compound 18 proved to be a non-competitive inhibitor of the enzyme (Ki = 0.80 mM), whereas
19 was a mixed-type inhibitor (Ki = 32 lM).
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
In recent years, we have reported diastereoselective procedures
for the construction of the sulfur mediated interglycosidic linkage
of thiodisaccharides. Thus, we have recently described the synthe-
sis of 3,4-epoxides and their use as acceptors of 1-thioaldoses to
give, by ring-opening, (1?3)- or (1?4)-thiodisaccharides.8 Alter-
natively, the S-linkage has been generated by Michael addition of
1-thioaldoses to hexose-derived sugar enones.9 This methodology
led to 3-deoxy-4-S-(1?4)-thiodisaccharides, which proved to be
inhibitors of b-glycosidases. Also thiodisaccharides with a thiofura-
nose as non-reducing end have been prepared10,11 and evaluated as
Glycosidases are vital for living systems as they are involved in
varied biological processes, including metabolic disorders and dis-
eases.1 Inhibition of glycosidases may prevent the proliferation of
pathogenic microorganisms and hence glycosidase inhibitors are
seen as potential therapeutic agents. In fact, they are already used
or tested in the treatment of diabetes and HIV infection and as
antifungal compounds.2 Also, enzyme inhibition constitutes an
innovative approach for drug development in cancer therapies.3
Sugar mimetics in which oxygen atoms have been replaced by
other heteroatoms present altered binding properties or increased
stability toward enzyme degradation, compared with the natural
analogues.4 Among the sugar mimetics, thiooligosaccharides are
interesting compounds as, in many cases, they act as enzyme
inhibitors,5 and they are usually tolerated by biological systems
and resistant to metabolic processes.6 Because of the potential of
thiooligosaccharides as enzyme inhibitors and therapeutics, the
investigation about their synthesis and the study of their biological
activities are topics of current research.6,7
inhibitors of a b-D
-galactofuranosidase.11 The procedure based on
the conjugated addition has been employed by Witczak and
coworkers for the thioglycosylation of levoglucosenone5b,d,12 or
isolevoglucosenone.13 In our case, hexose-derived sugar enones
have also been used as Michael acceptors of 1-thiohexoses to gen-
erate the S-linkage between two hexopyranose units.9 However,
the procedure has not been applied for the synthesis of thiodisac-
charides constituted by a pentopyranose moiety. Furthermore, we
considered that the presence of a pentopyranose as reducing end of
the thiodisaccharide was a relevant issue, as an increased flexibil-
ity at the reducing end of the thiodisaccharide could facilitate its
accommodation in the active site of the enzyme that demands dis-
tortion of both substrates and inhibitors from their more stable
ground-state conformation.14 Therefore, we report herein the syn-
* Corresponding author. Fax: +54 11 4576 3346.
E-mail address: varela@qo.fcen.uba.ar (O. Varela).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.055

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A. J. Cagnoni et al. / Bioorg. Med. Chem. 17 (2009) 6203–6212
thesis of thiodisaccharides with a pentopyranose as reducing or
non-reducing unit. The new products have been evaluated as
inhibitors of glycosidases.
ucts which were separated by column chromatography. The result-
ing 3-deoxy-4-S-glycosyl-4-thiohexopyranosides, having the 4-
thio-a-D-lyxo (4) and a-D-xylo (5) configuration for the reducing
end, were isolated in 23% and 70% yield, respectively. A similar ste-
reoselectivity was observed in previous studies,9,18 suggesting that
the axial thioglycosyl group, b to the carbonyl, has a minor contri-
bution in the steric hindrance than the vicinal axial anomeric iso-
propyl group.
2. Results and discussion
2.1. Synthesis and chemical characterization
The structures of 4 and 5 were confirmed by NMR spectroscopy.
Compound 4 showed the signal of H-1 as a broad singlet, indicating
a diequatorial disposition for H-1 and H-2. In contrast, the H-1 sig-
nal in 5 appeared as a doublet with J1,2 = 3.6 Hz, which is in agree-
ment with an equatorial–axial disposition for H-1 and H-2. The H-4
signal in 4, as well as in 5, appeared protected by the proximity of
sulfur, as multiplets at ꢁ3.27 ppm, with small coupling constants
with their vicinal H-3ax, H-3eq and H-5, confirming that H-4 is
equatorially disposed. The resonances of the b-1-thioxylosyl pro-
tons in 3, 4 and 5 were in accordance with those reported for other
thioxylopyranosides.19
The synthesis of the S-linked disaccharide with xylopyranose as
non-reducing end was conducted by Michael addition of 2,3,4-tri-
O-acetyl-1-thio-b-
D
-xylopyranose15 (2) to the
a,b-unsaturated
system of 2-propyl 6-O-acetyl-3,4-dideoxy-a-D-glycero-hex-3-eno-
pyranosid-2-ulose16 (1). Under optimized conditions (CH2Cl2 at
ꢀ18 °C for 30 min in the presence of triethylamine (TEA) as a cat-
alyst) this reaction led to the thiodisaccharide 3 in 96% yield
(Scheme 1). The addition was highly diastereoselective as 3 was
the only isomer isolated. The R configuration for the new stereo-
center at C-4 in 3 was established on the basis of the 1H NMR spec-
trum. Thus, in accordance with the equatorial orientation of H-4,
the 1H NMR spectrum of 3 showed small coupling constant values
of H-4 with the methylene protons at C-3 (J3a,4 = 4.9 Hz,
J3b,4 = 1.6 Hz) and with H-5 (J4,5 = 1.8 Hz). Similar to previous
results in our laboratory, the stereocontrol was provided by the
Reduction of 3 with an excess of NaBH4 in anhydrous THF led to
a mixture of three reduction products, upon careful separation by
column chromatography. The first fractions afforded 4 and 5 in
5% and 15% yield, respectively. The more polar product (74%) was
identified as the thiodisaccharide 6. Its structure was confirmed
by 1H and 13C NMR spectroscopy, assisted by 2D 1H COSY and
1H–13C HSQC experiments. The large coupling constant values of
H-4 with H-5 (11.0 Hz) and H-3ax (13.0 Hz), and the small one
with H-3eq (4.2 Hz) indicated that 6 had reverse configuration at
C-4 with respect to 4 and 5. This result was rather unexpected
and suggested the isomerization of the C-4 stereocenter in the
axially oriented anomeric substituent of
a
configuration.9 When
the reaction was carried out at room temperature the stereoselec-
tivity was partially lost, which was attributed to the higher reactiv-
ity of the 1-thiopentopyranose, in comparison with the analogous
1-thiohexopyranoses previously studied. The increased nucleophi-
licity of 2 may be due to the lack of the electron-withdrawing acet-
oxymethyl substituent on C-5 of the 1-thiopyranose, which
diminishes the electron density on the ring oxygen atom vicinal
to the sulfur-containing anomeric center.17
ꢀ
starting 3 mediated by the species BH4 and BH3. To explain this
isomerization–reduction we propose the hypothetical mechanism
depicted in Scheme 2. Initial removal of the hydrogen
bonyl (H-3) in 3 by the basic BH4 leads to enolate formation and
a to the car-
The reduction of 3 with NaBH4 was performed in MeOH/THF or
THF, as the ulose was partially soluble in MeOH at 0 °C. In THF–
MeOH the reduction proceeded rapidly at 0 °C to afford two prod-
ꢀ
releasing of borane. The BH3 would promote the elimination of the
OAc
O
O
AcO
AcO
+
SH
OAc
OiPr
O
1
2
O
O
AcO
AcO
AcO
AcO
OAc
OH
S
S
OAc
O
Et3N, CH2Cl2
OAc
OAc
+
O
-18 ºC
NaBH4
MeOH/THF
0°C
HO
OiPr
OiPr
4 (23%)
5 (70%)
O
AcO
AcO
OAc
S
OAc
O
NaBH4/THF
0°C
OAc
O
O
AcO
AcO
4
(5%)
5 (15%)
+
+
S
OAc
HO
OiPr
3
(96%)
O
OiPr
1) K-Selectride/THF
-78 °C
6
(74%)
O
AcO
OAc
2) Ac2O/C5H5N
S
AcO
OAc
O
O
OAc
AcO
AcO
O
S
+
OAc
AcO
OiPr
AcO
OiPr
7 : 8
(
= 3.5 :1, 73%)
8
7
Scheme 1. Synthesis of thiodisaccharides 3–8.

A. J. Cagnoni et al. / Bioorg. Med. Chem. 17 (2009) 6203–6212
6205
RS
H
OAc
O
OAc
O
RS
RS
OAc
H2
OAc
RS
O
O
H
OiPr
OiPr
H3B
OiPr
BH4
H3B
O
H
OiPr
O
O
O
I
H
II
3
BH4
H
B
H
H
H
OAc
O
RS
OH
OiPr
6
Scheme 2. Proposed mechanism for the isomerization of C-4 during the NaBH4/THF reduction of 3.
allylic hydride in the enolate, giving rise to I, stabilized by reso-
nance of the non shared electron pair of sulfur that is conjugated
niques. Consistent with the equatorial disposition of the substi-
tuent at C-4, the H-4 signal appeared as multiplet that
a
with the
a
,b-unsaturated carbonyl system. Reduction of the sulfo-
exhibited relatively small coupling constants with the vicinal
H-3a, H-3b, H-5a, H-5b (5.5, 4.3, 3.0, 3.8 Hz, respectively). Fur-
thermore, the long-range coupling constant J3b,5b (1.8 Hz), also
observed in pyranoses,21 was in accordance with the respective
quasy-equatorial/equatorial disposition of H-3b and H-5b in 11.
The configuration of C-4 in this compound indicates the ap-
proach of the 1-thioaldose to the enone system of 9 from the
b face, opposite to the anomeric substituent. Lowering the tem-
perature of the Michael addition resulted in an increase in the
diastereoselectivity. Thus, the reaction of 9 with 10 performed
at ꢀ78 °C afforded only 11 in almost quantitative yield.
nium ion and the carbonyl group of II, gives rise to 6. This reduc-
tion follows the previous tendency observed, with approach of
hydride from the less hindered b face, resulting in an S configura-
tion for the C-4 stereocenter. This approach allows the bulky 1-
thioxylopyranose residue at C-4 to adopt an equatorial disposition
in the final product 6. The isomerization seems to be inhibited in
the presence of a protic solvent such as MeOH.
The carbonyl group of 3 was also reduced with K-Selectride in
THF at ꢀ78 °C. After the usual work-up, the reaction mixture
showed by TLC some spots of lower mobility than those corre-
sponding to thiodisaccharides 4–6, suggesting partial O-deacetyla-
tion. Therefore, the mixture was acetylated under standard
conditions to afford, after purification, a chromatographically
homogenous product that revealed to be a 3.5:1 mixture of 7:8.
The identity of 7 was confirmed by acetylation of its precursor 5.
The 1H NMR spectrum of 8 in the mixture showed, similar to that
of 6, large coupling constant values for H-4 with H-3ax and H-5,
indicating isomerization of C-4 during the reduction. The fact that
the extent of isomerization is diminished respect to the analogous
NaBH4/THF reduction of 3 may be attributed to the axially oriented
thioglycosyl and isopropoxy groups, which hinder both faces of the
molecule for the approach of the bulky K-Selectride to remove H-3
(Scheme 2).
To go further into the study of structure–activity relationship in
thiodisaccharides, we explored the use of a pyranone derived form
a pentose as Michael acceptor or 1-thiogalactopyranose (Scheme
3). The target thiodisaccharides are expected to act as inhibitors
of the b-galactosidase from Escherichia coli, since analogous
S-(1?4) linked disaccharides of Galp bonded to a 3-deoxy-4-thioh-
exopyranose were inhibitors of this enzyme.9 Also, the thiodisac-
charides were designed taking into account some stereoelectronic
and conformational requirements of carbohydrate derived inhibi-
tors to fit in the binding site of the enzyme, as explained later
(see Section 2.2). Thus, 2-(S)-benzyloxy-2H-pyran-3(6H)-one (9)
Reduction of 11 with NaBH4 in MeOH at 0 °C gave the thiodisac-
charides 13 and 14 in 39% and 45% yield, respectively. They possess
the b-D-erythro (13) and b-D-threo (14) configuration for the reduc-
ing end, according to their 1H-NMR data. Furthermore, the large
coupling constants observed for the trans-diaxial protons of 13
(J1,2 = 7.0 Hz, J2,3ax = 11.5 Hz and J4,5ax = 10.7 Hz) indicated that
the 4-thio-3-deoxy unit adopts mainly the 4C1 conformation, with
all substituents (at C-1, C-2 and C-4) in an equatorial disposition.
On the other hand, the coupling constant values for the major
product 14 (J2,3a = 7.2 Hz, J2,3b = 3.8 Hz, J3a,4 = 3.7 Hz and
J3b,4 = 7.5 Hz) suggests contribution of the 1C4 conformer to the
conformational equilibrium. These results are in accordance with
the behavior of similar compounds having small S-linked residues
at C-4.18
Reduction of 11 with K-Selectride in THF at ꢀ78 °C produced 14
as a single product in 80% yield. It is interesting to note that
according to this and previous studies,18 K-Selectride is useful to
reduce 4-thio-2-uloses derived from pentoses with high stereose-
lectivity, no matter the size of the sulfur substituent. Furthermore,
in contrast with the analogous reduction of 3 with NaBH4/THF no
isomerization was observed for the reduction of 11. This fact
may be explained on the basis of the mechanism proposed for
the isomerization (Scheme 2). The electron-withdrawing effect of
the acetoxymethyl group in the non-reducing end of 11 would
diminish the electron donation of sulfur, affecting the stabilization
of the intermediate species. In contrast, the 1-thioxylopyranoside
in 3 lacks of the acetoxymethyl substituent. Furthermore, the con-
formational flexibility of the pentopyranoside ring may contribute
to relieve the instability caused by the axial 4-thioglycopyranosyl
substituent in 14.
was prepared in enantiomerically pure form from benzyl b-D-
arabinopyranoside, following the procedure previously described
by us.20
Michael addition of per-O-acetyl-b-D
-1-thiogalactopyranose9
(10) to the enone 9 in CH2Cl2 was carried out at ꢀ18 °C, in
the presence of TEA as catalyst. Under these conditions, a 4:1
mixture of 11:12 was formed. Separation of these stereoisomers
by column chromatography was difficult, although in some cases
partial separation was possible by adding TEA to the eluting sol-
vent. The 1H NMR spectrum of 11 admitted a first order analysis
and the assignments were fully confirmed by 2D NMR tech-
The reduction of the original 4:1 mixture of 11 and 12 (obtained
as described above by coupling of 9 and 10 at ꢀ18 °C) was con-
ducted with NaBH4 in MeOH at ꢀ18 °C. In this case, three isomeric
thiodisaccharides were obtained, which were purified by column
chromatography. The previously described 13 and 14 were isolated

6206
A. J. Cagnoni et al. / Bioorg. Med. Chem. 17 (2009) 6203–6212
Ph
O
OAc
AcO
AcO
OAc
Ph
Ph
O
O
Ph
+
O
O
OAc
O
O
AcO
AcO
O
Et3N/CH2Cl2
+
S
SH
O
O
AcO
AcO
O
S
OAc
OAc
O
O
OAc
10
9
-18 °C
-78° C
12 (20%)
11
11
(80%)
(~100%)
Ph
O
O
O
OAc
AcO
AcO
O
OAc
AcO
AcO
OAc
O
AcO
AcO
O
O
OH
OH
O
S
O
Ph
+
S
S
OAc
13
OAc
OAc
14
11
Isolated Yields (%)
Reducing agent/Temp.
13
14
39
18
33
45
NaBH4/MeOH, 0 ºC
NaBH4/MeOH, -18 ºC
NaBH4/THF, 0 ºC
71
42
80
K-Selectride/THF, -78 ºC
OAc
AcO
AcO
Ph
O
NaBH4/MeOH
-18 °C
O
O
+
+
+
12
S
11
13 (14%)
14 (54%)
OH
OAc
15 (21%)
(~80 : 20)
Scheme 3. Synthesis of thiodisaccharides 11–15.
first in 14% and 54% yield, respectively. The more polar thiodisac-
charide 15 was then obtained in 21% yield. It was clear that this
last compound was the only reduction product of 12, as the config-
uration of the C-2 and C-4 stereocenters was established by 1H
NMR as S and R, respectively. The J1,2 value (3.6 Hz) was consistent
with an equatorial-axial disposition for H-1 and H-2, and the large J
values (J2,3ax = J3ax,4 = 12.2 Hz, J4,5ax = 11.5 Hz) indicated that the
coupled protons were axially disposed. A long-range coupling con-
stant J3eq,5eq (1.8 Hz) was measured and the value is characteristic
of protons equatorially oriented and connected through four
bonds.21
Thiodisaccharide 17 is an analogue of the structure found in aga-
rans from the red seaweed Georgiella confluens.22 The agarans iso-
lated from red seaweeds are usually galactans constituted by a
regular backbone of alternating (1?3) b-
units. Avarietyof substituents(sulfate, methyl, pyruvateoreven b-
xylopyranose residues) appeared linked to this regular chain. How-
ever, polysaccharides having the motif b- -xylopyranosyl(1?4)-
galactopyranose are rather unusual in nature. The thiodisaccharide
18 is structurally related to the fragment b- -Gal(1?4)- -Xyl pres-
D-Galp and (1?4) a-L-Galp
D
-
D
D-
D
D
ent in the linkage region between the protein and the polysaccharide
chain of heparin and other animal proteoglycans.23
Thiodisaccharides 4 and 5 were O-deacetylated by treatment
with MeOH/Et3N/H2O (Scheme 4). The free isopropyl 3-deoxy-4-S-
2.2. Evaluation of the inhibitory activity
(b-D-xylopyranosyl)-4-thio-a-D-lyxo- and a-D-xylo-hexopyranosides
(16 and 17, respectively) obtained were purified by ion-exchange
chromatography with Dowex MR-3C resin followed by elution
through a reverse-phase column. Similar O-deacetylation of 13
and 14 afforded, respectively the free thiodisaccharides 18 and 19.
Specific glycosidases are involved in the hydrolysis of xylose
residues in polysaccharides. Thus, xylanase and b-xylosidase are
key enzymes for the degradation of the plant polysaccharide xy-
lan.24 The b-xylosidase (or xylobiases, or exo-b-1,4-xylanase, EC
O
O
HO
HO
HO
OH
OH
S
S
HO
OH
OH
OH
TEA/MeOH/H2O
TEA/MeOH/H2O
O
O
5
4
HO
OiPr
OiPr
17 (77%)
16 (76%)
Ph
O
OH
O
HO
HO
O
OH
O
TEA/MeOH/H2O
O
HO
HO
TEA/MeOH/H2O
OH
13
S
14
O
Ph
S
OH
18 (75%)
Scheme 4. Synthesis of thiodisaccharides 16–19.
OH
OH
19 (88%)

A. J. Cagnoni et al. / Bioorg. Med. Chem. 17 (2009) 6203–6212
6207
3.2.1.37) is responsible for the complete degradation of xylan, since
it cleaves short chain xylooligosaccharides from the reducing end,
liberating
-xylose as the only product of hydrolysis.24 The best
D
substrate of xylosidase is xylobiose and some thioxylobiosides
have been synthesized and tested as inducers and inhibitors of this
enzyme.25 Also, imidazolo[1,2-a]piperidinoses derived from
D
- and
L-treose and D- and L-erythrose have been synthesized and their
inhibitory activity against b-xylosidase from Aspergillus niger has
been determined.26 Recently, simple amino alcohols have been
described as inhibitors of b-xylosidase from Selenomonas
ruminantium.27
The inhibitory activity of thiodisaccharides 16 and 17 against
the b-xylosidase from A. niger was studied following the methodol-
ogy described in detail in Section 4.3.1. No inhibition of this
enzyme was detected even at large concentrations of thiodisaccha-
rides. They neither display any inhibitory activity towards the b-
glucosidase from almonds nor to the b-galactosidase from E. coli.
The free thiodisaccharides 18 and 19 were also evaluated as
inhibitors of the b-galactosidase from E. coli. This enzyme has been
extensively studied,28 and it has shown to be highly specific for the
b-galactopyranosyl non-reducing end of the substrate, which may
be linked to a wide variety of aglycons. Enzyme–substrate interac-
tions have been studied by labeling of specific amino acids located
at the active site,29 and also by X-ray30 and NMR14,31a experiments.
Among the thiosugars, simple thiogalactopyranosides, such as iso-
Figure 1. Lineweaver–Burk plot for inhibition of E. coli b-galactosidase by thiodi-
saccharide 18 at concentration: e: 0.00, h: 0.40, 4: 0.80 and s: 1.20 mM of
inhibitor.
propyl b-
D
-thiogalactoside are good inhibitors.31 We have re-
-1-thiogalactose S-
ported9 that thiodisaccharides having a b-
D
bonded (1?4) to a 3-deoxy-4-thiohexopyranose moiety display
inhibitory activity against the b-galactosidase from E. coli. Jimé-
nez-Barbero and coworkers14 demonstrated that 4-thiolactose
competes with lactose for the same binding site of the enzyme
and they showed, by NMR and ab initio quantum mechanical stud-
ies, that the 3D-shapes of the ligand-inhibitor within the enzyme
binding site depend on the relative size of the stereoelectronic bar-
riers for the chair conformation and for the rotation of the intergly-
cosidic linkage. Therefore, as mentioned above, in the design of the
inhibitor we took into account the higher flexibility of a pentopyr-
anose ring compared to that of a hexopyranose ring. Also, as the
presence of a hydrophobic group distal to the thiogalactose residue
Figure 2. Lineweaver–Burk plot for inhibition of E. coli b-galactosidase by thiodi-
saccharide 19 at concentration: e: 0.00, h: 0.01, 4: 0.02 and s: 0.05 mM of
inhibitor.
enhances the inhibitory activity32 (for example, phenylethyl b-
D-
most quantitative yields. The stereochemical course of the car-
bonyl reduction in the ulose thiodisaccharides was highly
sensitive to the reducing agent and the reduction conditions em-
ployed. In general, a higher diastereoselectivity was observed at
lower temperatures. In some particular cases (with NaBH4 or K-
Selectride in anhydrous THF), the reduction took place with inver-
sion of the C-4 stereocenter. A distinct structural feature of this
unexpected isomerization is that it seems to take place only when
the substituent at C-4 is a 1-thiopentopyranosyl derivative, as it
was not observed for similar reductions of 4-thiohexopyranosyl
analogues studied in the present and previous reports.9
thiogalactopyranoside behaves as a strong inhibitor31b) we judged
convenient to increase the hydrophobicity of the reducing end of
the thiodisaccharide by introducing a benzyl 3-deoxy pentopyr-
anoside unit. For all these reasons we selected benzyl pent-2-
ulo-3-enopyranoside 9 as Michael acceptor.
In accordance with our expectations, thiodisaccharides 18 and
19 displayed inhibitory activity against the b-galactosidase from
E. coli. The inhibition was evaluated using o-nitrophenyl b-D-galac-
topyranoside as substrate, which was incubated with increasing
concentrations of the thiodisaccharides. The released o-nitrophe-
nol was quantified spectrophotometrically and the Lineweaver–
Burk plots (Figs. 1 and 2) were constructed. Thiodisaccharide 18
proved to be a non-competitive inhibitor of the enzyme, with
Ki = 0.80 mM, whereas 19 was a strong mixed-type inhibitor with
Among the new thiodisaccharides synthesized, benzyl 3-deoxy-
4-S-(b-
stronger inhibitor of the b-galactosidase from E. coli compared to
benzyl 3-deoxy-4-S-(b- -Galp)-4-thio-b- -erythro-pentopyrano-
D-Galp)-4-thio-b-D-threo-pentopyranoside (Ki = 32 lM) was
D
D
Ki = 32 lM.
side (Ki = 0.80 mM), which indicates that the configuration of the
C-2 stereocenter of the pentose is relevant for the activity. The for-
mer thiodisaccharide was also a better inhibitor than the ana-
logues of Galp-S-(1?4) linked to 3-deoxy-4-thiohexopyranose
3. Conclusion
unit having the D-lyxo or D-xylo configurations (Ki = 0.12 mM and
0.16 mM, respectively). The increment of the inhibitory activity
seems to correlate with the higher conformational flexibility of
The Michael addition showed to be a very useful reaction for the
construction of the sulfur mediated interglycosidic bond between a
pentopyranose S-(1?4)-linked to a hexopyranose and viceversa. In
these reactions sugar enones (dihydropyranones) derived from
pentoses or hexoses were useful Michael acceptors of 1-thioaldos-
es to give, with very high diastereoselectivity, the corresponding 3-
the 3-deoxy-4-thio-b-D-threo-pentopyranose ring with respect to
that of the hexopyranoses, which could facilitate the accommoda-
tion of the inhibitor in the binding site of the enzyme. Moreover,
the inhibition may be additionally increased by the higher hydro-
deoxy-4-S-(b-D-glycopyranosyl)-4-thiopyranosyd-2-ulose in al-

6208
A. J. Cagnoni et al. / Bioorg. Med. Chem. 17 (2009) 6203–6212
phobicity of the benzyl 3-deoxypentopyranoside regarding the 3-
deoxyhexopyranoside.
mp 113 °C (MeOH), ½a 20
ꢂ
ꢀ3.7 ((c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3)
d = 5.20 (t, 1H, J2 ,3Dꢃ J3 ,4 = 8.2 Hz, H-30), 4.97 (t, 1H, J1 ,2
ꢃ
0
0
0
0
0
0
J2 ,3 = 8.3 Hz, H-20), 4.96 (ddd, 1H, J4 ,5 eq = 4.9, J3 ,4 = 8.2, J4 ,5 ax = 8.7 Hz,
0
0
0
0
0
0
0
0
H-40), 4.89 (br s, 1H, H-1), 4.76 (d, 1H, J1 ,2 = 8.3 Hz, H-10), 4.43 (ddd, 1H,
J4,5 = 2.7, J5,6b = 4.3, J5,6a = 7.3 Hz, H-5), 4.25 (dd, 1H, J5,6a = 7.3,
0
0
4. Experimental
0
0
0
0
J6a,6b = 11.7 Hz, H-6a), 4.24 (dd, 1H, J4 ,5 eq = 4.9, J5 ax,5 eq = 11.8 Hz, H-
4.1. General methods
50eq), 4.21 (dd, 1H, J5,6b = 4.3, J6a,6b = 11.7 Hz, H-6b), 3.96 (m, 1H,
0
0
J = 6.2 Hz, CH(CH3)2), 3.61 (m, 2H, H-2 + HO), 3.41 (dd, 1H, J4 ,5 ax = 8.7,
Analytical thin layer chromatography (TLC) was performed on
Silica Gel 60 F254 (Merck) aluminum supported plates (layer thick-
ness 0.2 mm) with solvent systems given in the text. Visualization
of the spots was effected by exposure to UV light and charring with
a solution of 5% (v/v) sulfuric acid in EtOH, containing 0.5% p-anis-
aldehyde. Column chromatography was carried out with Silica Gel
60 (230–400 mesh, Merck). Optical rotations were measured with
a Perkin–Elmer 343 digital polarimeter. Nuclear magnetic reso-
nance (NMR) spectra were recorded with a Bruker AC 200 or with
a Bruker AMX 500 instruments. Assignements of 1H and 13C were
assisted by 2D 1H-COSY and HSQC experiments.
J5 ax,5 eq = 11.8 Hz, H-50ax), 3.28 (br q, 1H, J3b,4 ꢃ J4,5 = 2.7, J3a,4 = 3.6 Hz,
H-4), 2.29 (ddd, 1H, J2,3a ꢃ J3a,4 = 3.6, J3a,3b = 15.0 Hz, H-3a), 2.24 (ddd,
1H, J2,3a ꢃ J3a,4 = 2.6, J3a,3b = 15.0 Hz, H-3b), 2.08, 2.06 (ꢄ2), 2.05 (s, 3H
each, CH3CO), 1.22, 1.16 (2d, 6H, CH(CH3)2). 13C NMR (125.7 MHz,
CDCl3) d = 170.6, 169.8, 169.7, 169.5 (COCH3), 99.0 (C-1), 82.1 (C-10),
71.7 (C-30), 69.7 (C-20), 69.5 [OCH(CH3)2], 68.4 (C-40), 68.0 (C-5), 67.7
(C-2), 65.3 (C-50), 65.1 (C-6), 39.2 (C-4), 30.4 (C-3), 23.2, 21.5
[(CH3)2CH], 20.8 (ꢄ2), 20.7 (ꢄ2) (CH3CO). Anal. Calcd for C22H34O12S:
C, 50.57; H, 6.56; S, 6.14. Found: C, 50.45; H, 6.63; S, 6.04.
0
0
Further elution of the column afforded crystalline 5 (105 mg,
70%), mp 135 °C (MeOH), ½a D20
ꢂ
ꢀ7.8 (c 0.5, CHCl3). 1H NMR
(500 MHz, CDCl3) d = 5.15 (t, 1H, J2 ,3 ꢃ J3 ,4 = 7.8 Hz, H-30), 4.93
0
0
0
0
4.2. Synthesis
(dd, 1H, J2 ,3 = 7.8, J1 ,2 = 8.0 Hz, H-20), 4.92 (ddd, 1H, J4 ,5 eq = 5.1,
0
0
0
0
0
0
J3 ,4 = 7.8, J4 ,5 ax = 8.6 Hz, H-40), 4.92 (d, 1H, J1,2 = 3.6 Hz, H-1),
0
0
0
0
4.2.1. 2-Propyl per-O-acetyl-3-deoxy-4-S-(b-D-Xylp)-4-thiohexo-
pyranosid-2-ulose (3)
4.65 (d, 1H, J1 ,2 = 8.0 Hz, H-10), 4.28–4.26 (m, 1H, H-5), 4.23 (dd,
0
0
1H, J4 ,5 eq = 5.1, J5 eq,5 ax = 11.5 Hz, H-50eq), 4.19 (dd, 1H, J5,6a = 4.6,
J6a,6b = 11.9 Hz, H-6a), 4.14 (dd, 1H, J5,6b = 8.0, J6a,6b = 11.9 Hz, H-
6b), 4.02–3.99 (m, 1H, H-2), 3.94 (m, 1H, J = 6.0 Hz, CH(CH3)2),
0
0
0
0
2-Propyl 6-O-acetyl-3,4-dideoxy-
sid-2-ulose16 (1, 323 mg, 1.42 mmol) and 2,3,4-tri-O-acetyl-1-thio-
b-
-xylopyranose15 (2, 410 mg, 1.42 mmol) were dissolved in
anhydrous CH2Cl2 (6 mL). The reaction mixture was cooled to
L) was added. After 30 min of stirring at
a-D-glycero-hex-3-enopyrano-
D
3.38 (dd, 1H, J4 ,5 ax = 8.6, J5 ax,5 eq = 11.5 Hz, H-50ax), 3.26 (m, 1H,
H-4), 2.20 (m, 1H, H-3a), 2.08, 2.06 (ꢄ2), 2.05 (4 s, 3H each, CH3CO),
1.22, 1.16 (2 d, 6H, CH(CH3)2), 2.00 (m, 1H, H-3b). 13C NMR
(125.7 MHz, CDCl3) d = 169.8, 169.7 (ꢄ2), 169.4 (COCH3), 96.8 (C-
1), 83.1 (C-10), 71.6 (C-30), 70.9 [OCH(CH3)2], 69.9, 68.4 (C-20, C-
40), 67.9 (C-5), 65.4 (C-6), 64.9 (C-50), 64.1 (C-2), 43.4 (C-4), 34.6
(C-3), 23.2, 22.0 [(CH3)2CH], 20.8 (ꢄ2), 20.7 (ꢄ2) (CH3CO). Anal.
Calcd for C22H34O12S: C, 50.57; H, 6.56; S, 6.14. Found: C, 50.74;
H, 6.60; S, 5.84.
0
0
0
0
ꢀ18 °C and Et3N (10
l
ꢀ18 °C, TLC showed complete consumption of the starting materi-
als, and a single spot of Rf = 0.49 (hexane/EtOAc 1:1). The mixture
was evaporated to dryness and the residue was purified by column
chromatography using hexane/EtOAc 2.5:1 to afford 3 (710 mg,
96%) as a white solid, which was recrystallized from EtOH. Mp
152 °C (EtOH); ½a 20
ꢂ
ꢀ18.9 (c 0.6, CHCl3). 1H NMR (500 MHz, CDCl3)
d = 5.16 (t, 1H, J2 ,3D ꢃ J3 ,4 = 8.3 Hz, H-30), 4.93 (ddd, 1H, J4 ,5 eq = 5.0,
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
J4 ,3 = 8.3, J4 ,5 ax = 8.9 Hz, H-4 ), 4.91 (t, 1 H, J1 ,2 = 8.5, J2 ,3 = 8.3 Hz,
4.2.2.2. Reduction with NaBH4/THF. Synthesis of 2-propyl 6-O-
H-20), 4.78 (ddd, 1H, J4,5 = 1.8, J5,6a = 4.6, J5,6b = 7.4 Hz, H-5), 4.73 (br
acetyl-3-deoxy-4-S-(2,3,4-tri-O-acetyl-b-
D-xylopyranosyl)-4-thio-
-ribo-hexopyranoside (6). To a solution of 3 (430 mg, 0.83
s, 1H, H-1), 4.63 (d, 1H, J1 ,2 = 8.5 Hz, H-10), 4.27 (dd, 1H, J5,6a = 4.6,
J6a,6b = 11.7 Hz, H-6a), 4.23 (dd, 1H, J5,6b = 7.4, J6a,6b = 11.7 Hz, H-
0
0
a-D
mmol) in anhydrous THF (5 mL) was added NaBH4 (78.2 mg,
2.08 mmol) and stirred at 0 °C. After 30 min, TLC showed no start-
ing material remaining and one major product of Rf = 0.38 (hexane/
EtOAc 1:1.5). Fainter spots of Rf = 0.50 and 0.43, comigrating
respectively with 4 and 5, were also observed. The mixture was di-
luted with MeOH and treated with Dowex 50 H+, then concentrated
and subjected to column chromatography (hexane/EtOAc 1.5:1).
Thiodisaccharides 4 (20 mg, 5%) and 5 (66 mg, 15%) were isolated
from the first fraction of the column and they exhibited the same
properties reported above. The less polar, major product was ob-
tained crystalline and identified as 6 (320 mg, 74%), mp 132 °C
6b), 4.20 (dd, 1H, J4 ,5 eq = 5.0, J5 ax,5 eq = 11.7 Hz, H-50eq), 4.00 (m,
1H, J = 6.2 Hz, CH(CH3)2), 3.63 (ddd, 1H, J3ax,4 = 1.6, J4,5 = 1.8,
0
0
0
0
0
0
0
0
J3eq,4 = 4.9 Hz, H-4), 3.33 (dd, 1H, J4 ,5 ax = 8.9, J5 eq,5 ax = 11.7 Hz, H-
50ax), 3.16 (dd, 1H, J3a,4 = 4.9, J3a,3b = 15.2 Hz, H-3a), 2.77 (dd, 1H,
J3b,4 = 1.6, J3a,3b = 15.2 Hz, H-3b), 2.08, 2.07, 2.05, 2.04 (4s, 12H,
4CH3CO), 1.27, 1.18 (2 d, 6H, CH(CH3)2). 13C NMR (50.28 MHz,
CDCl3) d = 198.8 (C-2), 170.6, 169.8, 169.7, 169.6 (COCH3), 97.9
(C-1), 82.3 (C-10), 71.9, 71.8, 69.7, 68.6, 68.4, 65.3, 64.7 (C-5, C-6,
C-20, C-30, C-40, C-50, CH(CH3)2), 44.7, 43.6 (C-3, C-4), 23.3, 21.8
[(CH3)2CH], 20.8 (ꢄ2), 20.7 (ꢄ2) (CH3CO). Anal. Calcd for
C22H32O12S: C, 50.76; H, 6.20; S, 6.16. Found: C, 50.60; H, 6.12; S,
6.10.
(iPrOH), ½a 2D0
ꢂ
+23.8 ((c 1.0, CHCl3). 1H NMR (CDCl3, 500 MHz)
0
0
0
0
0
0
d = 5.17 (t, 1H, J3 ,4 ꢃ J2 ,3 = 8.3 Hz), 4.94 (ddd, 1H, J4 ,5 a = 5.0,
J3 ,4 = 8.3 Hz, J4 ,5 b = 8.8 Hz, H-40), 4.92 (d, 1H, J1,2 = 3.7 Hz, H-1),
0
0
0
0
4.91 (t, 1H, J1 ,2 ꢃ J2 ,3 = 8.3 Hz, H-20), 4.70 (d, 1H, J1 ,2 = 8.3 Hz, H-
0
0
0
0
0
0
4.2.2. Reduction of 3
4.2.2.1. Reduction with NaBH4 in MeOH/THF 1:1. Synthesis of
10), 4.44 (dd, 1H, J5,6a = 2.0, J6a,6b = 11.9 Hz, H-6a), 4.34 (dd, 1H,
0
0
2-propyl 6-O-acetyl-3-deoxy-4-S-(2,3,4-tri-O-acetyl-b-
anosyl)-4-thio- -lyxo-hexopyranoside (4) and 2-propyl
6-O-acetyl-3-deoxy-4-S-(2,3,4-tri-O-acetyl-b- -xylopyranosyl)-
4-thio- -xylo-hexopyranoside (5). Compound 3 (150 mg, 0.29
mmol) was dissolved in a mixture of MeOH/THF 1:1 (5 mL) and NaBH4
(11 mg, 0.29 mmol) was added at 0 °C with stirring. After 30 min two
main products were detected by TLC (Rf = 0.50 and Rf = 0.43, hexane/
EtOAc 1:1.5). The mixture was made neutral by addition of Dowex
50 H+ resin, the solvent was evaporated and purification was per-
formed by column chromatography (hexane/EtOAc 1.5:1). From the
first fractions of the column, was isolated crystalline 4 (35 mg, 23%),
D-xylopyr-
J5,6b = 5.0, J6a,6b = 11.9 Hz, H-6b), 4.23 (dd, 1H, J4 ,5 a = 5.0,
J5 a,5 b = 11.8 Hz, H-50a), 3.95 (m, 1H, J = 6.2 Hz, CH(CH3)2), 3.86
(ddd, 1 H, J5,6a = 2.0, J5,6b = 5.0, J4,5 = 11.0 Hz, H-5), 3.71 (dddd, 1H,
J1,2 = 3.7, J2,3eq = 4.6, J2,OH = 11.4, J2,3ax = 12.2Hz, H-2), 3.38 (dd, 1H,
0
0
a-D
D
a-D
J4 ,5 b = 8.8, J5 a,5 b = 11.8 Hz, H-50b), 2.89 (ddd, 1H, J3eq,4 = 4.2,
J4,5 = 11.0, J3ax,4 = 13.0 Hz, H-4), 2.24 (ddd, 1H, J3eq,4 = 4.2,
J2,3eq = 4.6, J3eq,3ax = 12.2 Hz, H-3 eq), 2.11, 2.08, 2.07, 2.06 (4s,
12H, CH3CO), 1.90 (d, 1H, J2,OH = 11.4 Hz, HO–), 1.83 (ddd, 1H,
J2,3ax = J3eq,3ax = 12.2, J3ax,4 = 13.0 Hz, H-3ax), 1.26, 1.20 (2d, 6H,
CH(CH3)2). 13C NMR (125.7 MHz, CDCl3) d = 170.6, 169.8, 169.7,
169.4 (COCH3), 96.2 (C-1), 83.0 (C-10), 71.8 (C-30), 70.6 (OCH(CH3)2),
0
0
0
0

A. J. Cagnoni et al. / Bioorg. Med. Chem. 17 (2009) 6203–6212
6209
70.0 (C-20), 69.9 (C-50), 68.4 (C-40), 67.4 (C-2), 65.0 (C-50), 64.0 (C-6),
39.8 (C-4), 35.7 (C-3), 23.2, 21.9 (CH(CH3)2), 20.8, 20.7 (ꢄ2)
(CH3CO). Anal. Calcd for C22H34O12S: C, 50.57; H, 6.56; S, 6.14.
Found: C, 50.35; H, 6.68; S, 6.04.
(d, 1H, J1 ,2 = 9.9 Hz, H-10), 4.42 (dd, 1H, J4,5a = 3.0, J5a,5b = 12.0 Hz,
0
0
H-5a), 4.15 (dd, 1H, J5 ,6 a = 7.0, J6 a,6 b = 11.4 Hz, H-60a), 4.10 (dd,
0
0
0
0
1H, J5 ,6 b = 6.3, J6 a,6 b = 11.4 Hz, H-60b), 3.93 (br t, 1H, J4 ,5 = 0.8,
0
0
0
0
0
0
J5 ,6 b = 6.3, J5 ,6 a = 7.0 Hz, H-50), 3.80 (ddd, 1H, J3b,5b = 1.8,
J4,5b = 3.8, J5a,5b = 12.0 Hz, H-5b), 3.74 (m, 1H, H-4), 3.14 (dd, 1H,
J3a,4 = 5.5, J3a,3b = 15.5 Hz, H-3a), 2.61 (br ddd, 1H, J3b,5b = 1.8,
J3b,4 = 4.3, J3a,3b = 15.5 Hz, H-3b), 2.15, 2.06, 2.05, 1.98 (4s, 12H,
CH3CO). 13C NMR (125.7 MHz, CDCl3) d = 199.1 (C-2), 170.5,
170.2, 170.0, 169.6 (COCH3), 136.4, 128.3, 128.2 (C-aromatic),
98.4 (C-1), 83.7 (C-10), 74.7 (C-50), 71.7 (C-30), 70.1 (CH2Ph), 67.2
(C-40), 67.0 (C-20), 63.4 (C-5), 61.5 (C-60), 42.5 (C-3, C-4), 20.8,
20.7 (ꢄ2), 20.6 (CH3CO). Anal. Calcd for C26H32O12S: C, 54.92; H,
5.67; S, 5.64. Found: C, 54.80; H, 5.82; S, 5.79.
0
0
0
0
4.2.2.3. Reduction with K-Selectride/THF. Synthesis of 2-propyl
2,6-di-O-acetyl-3-deoxy-4-S-(2,3,4-tri-O-acetyl-b-
syl)-4-thio- -xylo-hexopyranoside (7) and 2-propyl 2,6-di-O-
acetyl-3-deoxy-4-S-(2,3,4-tri-O-acetyl-b- -xylopyranosyl)-4-thio-
-ribo-hexopyranoside (8). To a solution of compound 3
D-xylopyrano-
a-D
D
a-D
(70 mg, 0.15 mmol) in THF (1.5 mL), cooled at ꢀ78 °C, was added
K-Selectride (0.23 mL, 0.23 mmol) with constant stirring. After 3 h
the mixture was made neutral by addition of Dowex 50 H+ resin
and the solvent was evaporated. The residue showed by TLC some
spots of low mobility, suggesting partially O-deacetylated prod-
ucts. Therefore, the mixture was treated with 1:1 acetic anhy-
dride-pyridine (1 mL). Upon acetylation, TLC (hexane/AcOEt 1:1)
exhibited a major product (Rf = 0.41), which was purified by col-
umn chromatography (hexane/EtOAc 2.5:1). Although the product
(56 mg, 73%) was chromatographically homogeneous, the NMR
analysis revealed the presence of two components (3.5:1 ratio).
The mixture could not be separated with all the solvent systems at-
tempted. The major component of the mixture was identified as 7,
and its structure was confirmed as this product showed identical
properties to that obtained by acetylation of 5. Compound 7: 1H
When the reaction was conducted at ꢀ18 °C, an inseparable
mixture of 11 and its a-L-glycero isomer 12 was obtained (4:1 ratio,
estimated by NMR). Compound 12 partial 13C NMR (125.7 MHz,
CDCl3) d = 97.7 (C-1), 84.4 (C-10), 63.2 (C-5), 42.9, 42.7 (C-3, C-4).
4.2.4. Reduction of 11
4.2.4.1. Reduction with NaBH4/MeOH. Synthesis of benzyl 3-
deoxy-4-S-(2,3,4,6-tetra-O-acetyl-b-
b- -erythro-pentopyranoside (13) and benzyl 3-deoxy-4-S-(2,
3,4,6-tetra-O-acetyl-b-
pyranoside (14).
D-galactopyranosyl)-4-thio-
D
D-galactopyranosyl)-4-thio-b-D-threo-pento-
To a solution of 11 (751 mg, 1.32 mmol) in
MeOH (7.0 mL), cooled to 0 °C, NaBH4 (55 mg, 1.48 mmol) was
added under stirring. After 30 min, TLC (hexane/EtOAc 1:1.5)
showed the complete conversion of the starting material into
two compounds of Rf = 0.40 and 0.33. The mixture was treated
with Dowex 40 (H+) resin and concentrated, and the residue was
purified by column chromatography (hexane/EtOAc 2.5:1). From
the first fractions of the column was isolated syrupy 13 (290 mg,
NMR (500 MHz, CDCl3) d = 5.16 (t, 1H, J2 ,3 ꢃ J3 ,4 = 8.0 Hz, H-30),
0
0
0
0
5.13 (dt, 1H, J1,2 = 3.7, J2,3eq = 4.6, J2,3ax = 12.0, H-2), 5.07 (d, 1H,
J1,2 = 3.7 Hz, H-1), 4.93 (dd, 1H, J2 ,3 = 7.8, J1 ,2 = 8.0 Hz, H-20), 4.92
0
0
0
0
(ddd, 1H, J4 ,5 eq = 5.1, J3 ,4 = 7.8, J4 ,5 ax = 8.6 Hz, H-40), 4.68 (d, 1H,
0
0
0
0
0
0
J1 ,2 = 8.0 Hz, H-10), 4.37 (ddd, 1H, J4,5 = 2.3, J5,6a = 4.1, J5,6b = H-5),
0
0
4.23 (dd, 1H, J4 ,5 eq = 4.8, J5 eq,5 ax = 11.8 Hz, H-50eq), 4.19 (dd, 1H,
J5,6a = 4.1, J6a,6b = 11.7 Hz, H-6a), 4.14 (dd, 1H, J5,6b = 7.8,
J6a,6b = 11.7 Hz, H-6b), 3.89 (m, 1H, J = 6.2 Hz, CH(CH3)2), 3.38 (dd,
0
0
0
0
39%);
½
a 2D0
ꢂ
ꢀ64.5 (c 0.4, CHCl3). 1H NMR (500 MHz, CDCl3)
0
0
d = 7.38–7.30 (m, 5H, H-aromatic), 5.43 (br d, 1H, J4 ,5 <1,
1H, J4 ,5 ax = 8.4, J5 ax,5 eq = 11.8 Hz, H-50ax), 3.26 (m, 1H, J4,5 = 2.3,
J3eq,4 = 3.0, J3ax,4 = 3.7, H-4), 2.29 (td, 1H, J3ax,4 = 3.7, J2,3ax = 12.0,
J3ax,3eq = 12.6, H-3ax), 2.07 (m, 16H, 3H each + 1H, CH3CO + H-
3 eq), 1.24, 1.13 (2d, 6H, J = 6.2, CH(CH3)2), 2.00 (m, 1H, H-3 eq).
13C NMR (125.7 MHz, CDCl3) d = 170.5, 170.2, 169.8, 169.3, 169.1
(COCH3), 94.3 (C-1), 82.9 (C-10), 71.5 (C-30), 70.5 [OCH(CH3)2],
69.7, 68.3 (C-20, C-40), 67.6 (C-5), 65.4, 64.9, 64.1 (C-2, C-50, C-6),
43.2 (C-4), 30.6 (C-3), 23.1, 21.7 [(CH3)2CH], 20.8 (ꢄ2), 20.7 (ꢄ2),
20.6 (CH3CO).
J3 ,4 = 3.0, H-40), 5.25 (t, 1H, J1 ,2 = J2 ,3 = 10.0 Hz, H-20), 5.05 (dd,
0
0
0
0
0
0
0
0
0
0
1H, J3 ,4 = 3.0, J2 ,3 = 10.0 Hz, H-30), 4.90, 4.58 (2 d, 1H each,
0
0
0
0
J = 11.8 Hz, PhCH2), 4.49 (d, 1H, J1 ,2 = 9.9 Hz, H-10), 4.27 (d, 1H,
0
0
0
0
J1,2 = 7.0 Hz, H-1), 4.15 (m, 1H, H-5 eq), 4.14 (dd, 1 H, J5 ,6 a = 7.0,
J6 a,6 b = 11.5 Hz, H-60a), 4.10 (dd, 1H, J5 ,6 b = 6.3, J6 a,6 b = 11.5 Hz,
0
0
0
0
0
0
H-60b), 3.92 (br t, 1H, J4 ,5 <1, J5 ,6 a = 6.3, J5 ,6 b = 7.0 Hz, H-50), 3.58
(ddd, 1H, J2,3eq = 4.8, J1,2 = 7.0, J2,3ax = 11.5 Hz, H-2), 3.35 (dd, 1H,
J4,5ax = 10.7, J5ax,5eq = 11.4 Hz, H-5ax), 3.21 (dddd, 1H, J3eq,4 = 4.3,
J4,5eq = 4.5, J4,5ax = 10.7, J3ax,4 = 11.6 Hz, H-4), 2.45 (br s, 1H, HO),
2.34 (dddd, 1H, J3eq,5eq = 1.8, J3eq,4 = 4.3, J2,3eq = 4.8, J3a,3b = 13.0 Hz,
H-3eq), 2.17, 2.07 (ꢄ2), 1.99 (4s, 3H each, CH3CO), 1.49 (ddd, 1H,
J2,3ax = 11.5, J3ax,4 = 11.6, J3ax,3eq = 13.0 Hz, H-3ax). 13C NMR
(125.7 MHz, CDCl3) d = 170.2 (ꢄ2), 170.0, 169.5 (COCH3), 137.1,
128.6, 128.1 (ꢄ2) (C-aromatic), 103.7 (C-1), 82.9 (C-10), 74.8 (C-
50), 71.8 (C-30), 70.8 (CH2Ph), 69.9 (C-5), 69.2 (C-2), 67.2 (C-40),
66.9 (C-20), 61.5 (C-60), 37.5 (C-4), 35.7 (C-3), 20.8, 20.7, 20.6,
20.5 (CH3CO). Anal. Calcd for C26H34O12S: C, 54.73; H, 6.01; S,
5.62. Found: C, 54.55; H, 6.12; S, 5.94.
0
0
0
0
0
0
Compound 8 showed the following diagnostic signals: 1H NMR
(500 MHz, CDCl3) d = 5.03 (d, 1H, J1,2 = 3.5 Hz, H-1), 4.79 (ddd, 1H,
0
0
J1,2 = 3.5, J2,3eq = 4.6, J2,3ax = 11.8 Hz, H-2), 4.69 (d, 1H, J1 ,2 = 8.0 Hz,
H-10), 3.94 (ddd, 1H, J4,5 = 11.0, J5,6a = 2.0, J5,6b = 4.7 Hz, H-5), 2.96
(m, 1H, J3eq,4 = 4.6, J4,5 = 11.0, J3ax,4 = 12.6 Hz, H-4); 13C NMR
(125.7 MHz, CDCl3) d = 93.7 (C-1), 83.0 (C-10), 39.8 (C-4), 31.0 (C-3).
4.2.3. Benzyl 3-deoxy-4-S-(2,3,4,6-tetra-O-acetyl-b-
anosyl)-4-thio-b- -glycero-pentopyranosid-2-ulose (11)
solution of (2S)-2-benzyloxy-2H-pyran-3(6H)-one20 (9,
414 mg, 2.03 mmol) and 2,3,4,6-tetra-O-acetyl-1-thio-b- -galacto-
pyranose9 (10, 739 mg, 2.03 mmol) in dry CH2Cl2 (2.5 mL) was
cooled to ꢀ78 °C and Et3N (13 L) was added. After stirring at
ꢀ78 °C for 3 h very major product was detected by TLC
D-galactopyr-
D
A
Further fractions from the column afforded 14 (342 mg, 45%) as
D
a syrup; ½a 2D0
ꢂ
ꢀ70.2 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3)
0
0
0
0
d = 7.35–7.29 (m, 5H, H-aromatic), 5.43 (d, 1H, J4 ,5 <1, J3 ,4 = 3.4,
l
H-40), 5.24 (t, 1H, J1 ,2 = J2 ,3 = 10.0 Hz, H-2), 5.05 (dd, 1H,
´
0
0
0
0
a
J3 ,4 = 3.4, J2 ,3 = 10.0 Hz, H-30), 4.85, 4.60 (2 d, 1H each,
0
0
0
0
(Rf = 0.22, hexane/EtOAc 1:1). The solvent was evaporated to dry-
ness to afford 11 (1.15 g, ꢁ100%) as a white foam. This crude prod-
uct was pure enough to be used as starting material in the
following reactions. Purification of a portion of this material by col-
umn chromatography (hexane/EtOAc 2.5:1) gave an analytical
J = 11.8Hz, PhCH2), 4.65 (d, 1H, J1,2 = 2.2 Hz, H-1), 4.54 (d, 1H,
J1 ,2 = 10.0 Hz, H-10), 4.15 (dd, 1H, J5 ,6 a = 6.9, J6 a,6 b = 11.3 Hz, H-
0
0
0
0
0
0
60a), 4.12–4.07 (m, 2H, H-5a + H-60b), 3.93 (br t, 1H, J4 ,5 <1,
0
0
J5 ,6 a ꢃ J5 ,6 b = 6.9 Hz, H-50), 3.90 (ddd, 1H, J1,2 = 2.1, J2,3b = 3.8,
J2,3a = 7.2 Hz, H-2), 3.48–3.43 (m, 2H, H-4 + H-5b), 2.23 (ddd, 1H,
J3a,4 = 3.7, J2,3a = 7.2, J3a,3b = 12.2 Hz, H-3a), 2.17, 2.07, 2.05, 1.98
0
0
0
0
sample of 11. ½a 2D0
ꢂ
ꢀ69.8 ((c 0.9, CHCl3). 1H NMR (500 MHz, CDCl3)
0
0
d = 7.38–7.30 (m, 5H, H-aromatic), 5.43 (dd, 1H, J4 ,5 = 0.8,
(4s, 12H, CH3CO), 1.83 (ddd,
1
H, J2,3b = 3.8, J3b,4 = 7.5,
J3 ,4 = 3.3, H-40), 5.23 (t, 1H, J1 ,2 = J2 ,3 = 10.0 Hz, H-20), 5.05 (dd,
J3a,3b = 12.2 Hz, H-3b). 13C NMR (125.7 MHz, CDCl3) d = 170.5,
170.3, 170.0, 169.5 (COCH3), 137.1, 129.2, 128.0 (C-aromatic),
98.3 (C-1), 83.8 (C-10), 74.6 (C-50), 71.8 (C-30), 69.9 (CH2Ph), 67.2
0
0
0
0
0
0
1H, J3 ,4 = 3.4, J2 ,3 = 10.0 Hz, H-30), 4.81 (d, 1H, J = 11.7 Hz, PhCH2),
4.75 (br s, 1H, J1,3eq <1 Hz, H-1), 4.63 (d, 1H, J = 11.7, PhCH2), 4.57
0
0
0
0

6210
A. J. Cagnoni et al. / Bioorg. Med. Chem. 17 (2009) 6203–6212
(C-40), 67.2 (C-20), 66.6 (C-5), 65.8 (C-2), 61.5 (C-60), 38.0 (C-4), 34.7
(C-3), 20.8, 20.7 (ꢄ2), 20.6 (CH3CO). Anal. Calcd for C26H34O12S: C,
54.73; H, 6.01; S, 5.62. Found: C, 54.52; H, 6.24; S, 5.61.
When the reaction was carried out at ꢀ18 °C, and treated as de-
scribed above, compounds 13 and 14 were obtained in 18% and
71% yield, respectively.
TLC (hexane/EtOAc 1:1 or 1:1.5) showed complete consumption
of the starting material. The solution was concentrated and the res-
idue was dissolved in water (1 mL) and eluted through a column
filled with Dowex MR-3C mixed bed ion-exchange resin. The elu-
ate was concentrated and additionally purified by filtration
through an octadecyl C18 minicolumn (Amprep, Amersham Biosci-
ences). Evaporation of the solvent afforded the free thiodisaccha-
ride, which showed a single spot by TLC (n-BuOH/EtOH/H2O
2.5:1:1). The respective Rf values are given in each individual case.
4.2.4.2. Reduction of 11 with NaBH4/THF or with K-Selectride/
THF. Synthesis of 13 and 14.
To a solution of 11 (100 mg, 0.18
mmol) in anhydrous THF (7.4 mL), cooled at 0 °C, NaBH4 (9 mg,
0.23 mmol) was added under stirring. After 20 min the solution
was neutralized with Dowex 50 W (H+) resin, filtered and concen-
trated. The residue was diluted with MeOH, concentrated and then
purified by column chromatography (hexane/EtOAc 2:1). From the
first fractions of the column was isolated 13 (32 mg, 33%) and further
fractions from the column afforded 14 (42 mg, 42%).
The reduction of 11 was also performed with K-Selectride/THF
as follows. To a solution of 11 (400 mg, 0.70 mmol) in anhydrous
THF (7.4 mL), cooled to ꢀ78 °C was added 1 M solution of K-Select-
ride in THF (1.32 mL, 1.32 mmol) under nitrogen. The mixture was
stirred at ꢀ78 °C for 3 h and then the temperature was raised to
ꢀ20 °C. After the usual work-up and purification by column chro-
matography (hexane/EtOAc 2.5:1) was isolated 14 (322 mg, 80%)
as a single product.
4.2.5.1. 2-Propyl 3-deoxy-4-S-(b-D-xylopyranosyl)-4-thio-a-D-
lyxo-hexopyranoside (16).
O-Deacetylation of 4 gave crystal-
line 16 (76%, Rf = 0.56); mp 187–188 °C; ½a D20
ꢂ
+40.3 (c 0.5, H2O). 1H
NMR (D2O, 500 MHz) d = 4.70 (d, 1H, J1,2 = 2.8 Hz, H-1), 4.34 (d, 1H,
J1 ,2 = 9.8 Hz, H-10), 4.08 (ddd, 1H, J4,5 = 3.7, J5,6b = 4.1, J5,6a = 8.1 Hz,
0
0
0
0
H-5), 3.92 (m, 1H, J = 6.2Hz, CH(CH3)2), 3.84 (dd, 1H, J4 ,5 a = 5.4,
J5 a,5 b = 11.4 Hz, H-50a), 3.68 (dd, 1H, J5,6a = 8.1, J6a,6b = 12.1 Hz, H-
6a), 3.61 (dd, 1H, J5,6b = 4.1, J6a,6b = 12.1 Hz, H-6b), 3.53 (ddd, 1H,
0
0
0
0
J1,2 = 2.8, J2,3a = 4.3, J2,3b = 4.9 Hz, H-2), 3.46 (ddd, 1H, J4 ,5 a = 5.4,
J3 ,4 = 9.0, J4 ,5 b = 10.6 Hz, H-40), 3.27 (t, 1H, J2 ,3 ꢃ J3 ,4 = 9.0 Hz, H-
0
0
0
0
0
0
0
0
30), 3.16 (br t, 1H, J4 ,5 b = 10.6, J5 a,5 b = 11.4 Hz, H-50b), 3.15 (m,
0
0
0
0
1H, H-4), 3.11 (dd, 1H, J2 ,3 = 9.0, J1 ,2 = 9.7 Hz, H-20), 2.21 (ddd,
1H, J3a,3b = 14.8 Hz, H-3a), 1.96 (ddd,
0
0
0
0
J2,3a ꢃ J3a,4 = 4.3,
J2,3b ꢃ J3b,4 = 4.9 Hz, J3a,3b = 14.8 Hz, H-3b), 1.09, 1.03 (2d, 3H each,
J = 6.2, CH(CH3)2). 13C NMR (D2O, 127.5 MHz) d = 97.4 (C-1), 87.1
(C-10), 77.1 (C-30), 72.5 (C-20), 72.0 (C-5), 70.5 [CH(CH3)2], 69.0
(C-40), 68.8 (C-50), 66.6 (C-2), 61.9 (C-6), 40.2 (C-4), 32.0 (C-3),
22.3, 20.4 [CH(CH3)2]. Anal. Calcd for C14H26O8S: C, 47.44; H,
7.39; S, 9.05. Found: C, 47.41; H, 7.64; S, 9.14.
4.2.4.3. Reduction of the mixture 11 + 12. Synthesis of benzyl 3-
deoxy-4-S-(2,3,4,6-tetra-O-acetyl-b-
D
-galactopyranosyl)-4-thio-
a
-
-erythro-pentopyranoside (15).
A mixture of 11 and 12
(ꢁL4:1, 200 mg, 0.35 mmol) obtained by coupling of 9 and 10 at
ꢀ18 °C, was reduced with NaBH4 (13.4 mg, 0.35 mmol) in MeOH
(2 mL). The reaction mixture was stirred at ꢀ18 °C for 30 min,
and then it was treated with Dowex 50 W (H+) resin as described
above. TLC (hexane/EtOAc 1: 1.5) analysis showed a major spot
of Rf = 0.40, together with two minor components of Rf = 0.33 and
0.22. The spots of Rf = 0.40 and 0.33 comigrated with samples of
13 and 14, respectively. Careful purification by column chromatog-
raphy (hexane/EtOAc 2.5:1) led to the previously described 13
(28 mg, 14%) and 14 (108 mg, 54%). The third more polar thiodisac-
4.2.5.2. 2-Propyl
3-deoxy-4-S-(b-D-xylopyranosyl)-4-thio-a-
D
-xylo-hexopyranoside (17).
Standard deprotection of 5 gave
17 white crystals (77%, Rf = 0.66); mp 148–150 °C; ½a D20
ꢂ
+18.3 (c 1.2,
H2O). 1H NMR (D2O, 500 MHz) d = 4.88 (d, 1H, J1,2 = 3.9 Hz, H-1),
4.43 (d, 1H, J1 ,2 = 9.7 Hz, H-10), 4.16 (ddd, 1H, J4,5 = 2.2,
J5,6a = J5,6b = 6.0 Hz, H-5), 4.04 (ddd, 1 H, J1,2 = 3.9, J2,3eq = 4.8,
J2,3ax = 11.8 Hz, H-2), 3.91 (m, 1H, J = 6.3 Hz, CH(CH3)2), 3.89 (dd,
0
0
1H, J4 ,5 a = 5.4, J5 a,5 b = 11.3 Hz, H-50a), 3.59 (d, 2H, J5,6a
ꢃ
0
0
0
0
charide eluted was identified as 15 (42 mg, 21%); mp 131 °C; ½a D20
ꢂ
J5,6b ꢃ 6.0 Hz, H-6a + H-6b), 3.53 (ddd, 1H, J4 ,5 a = 5.4, J3 ,4 = 9.0,
0
0
0
0
ꢀ37.7 (c 0.8, CHCl3). 1H NMR (500 MHz, CDCl3) d = 7.42–7.36 (m,
J4 ,5 b = 10.7 Hz, H-40), 3.36 (ddd, 1H, J4,5 = 2.2, J3eq,4 = 3.3,
0 0
5H, H-aromatic), 5.45 (br d, 1H, J4 ,5 <1, J3 ,4 = 3.4, H-40), 5.19 (t,
J3ax,4 = 3.6 Hz, H-4), 3.34 (t, 1H, J2 ,3 ꢃ J3 ,4 = 9.0 Hz, H-30), 3.22
0
0
0
0
0
0
0
0
1H, J1 ,2 = J2 ,3 = 10.0 Hz, H-20), 5.05 (dd, 1H, J3 ,4 = 3.4,
(dd, 1H, J4 ,5 b = 10.7, J5 a,5 b = 11.3 Hz, H-50b), 3.19 (dd, 1H,
0
0
0
0
0
0
0
0
0
0
J2 ,3 = 10.0 Hz, H-30), 4.87 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.80 (d, 1H,
J2 ,3 = 9.0, J1 ,2 = 9.7 Hz, H-20), 2.06 (ddd, 1H, J3ax,4 = 3.6, J2,3ax
=
0
0
0
0
0
0
J = 11.6, PhCH2), 4.62 (d, 1H, J1 ,2 = 10.0 Hz,H-10), 4.52 (d, 1H,
11.8, J3ax,3eq = 13.2 Hz, H-3ax), 1.98 (ddd, J3eq,4 = 3.3 J2,3eq = 4.8,
J3ax,3eq = 13.2 Hz, H-3 eq), 1.16, 1.08 (2d, 3H each, J = 6.3,
0
0
J = 11.6, PhCH2), 4.17 (dd, 1H, J5 ,6 a = 7.2, J6 a,6 b = 11.4 Hz, H-60a),
0
0
0
0
13
4.11 (dd, 1H, J5 ,6 b = 6.0, J6 a,6 b = 11.4 Hz, H-60b), 3.94 (br t, 1H,
CH(CH3)2)). C NMR (D2O, 127.5 MHz) d = 96.1 (C-1), 85.5 (C-10),
0
0
0
0
J4 ,5 <1, J5 ,6 b = 6.0, J5 ,6 b = 7.2 Hz, H-50), 3.79 (ddd, 1H, J3eq,5eq ꢃ 1.6,
J4,5eq = 4.8, J5eq,5ax = 11.2 Hz, H-5 eq), 3.71 (dddd, 1H, J1,2 = 3.6,
J2,3eq = 4.2, J2,OH = 11.4 Hz, J2,3ax = 12.2 Hz, H-2), 3.60 (t, 1H,
J4,5ax ꢃ J5ax,5eq = 11.5 Hz, H-5ax), 3.04 (dddd, 1H, J3eq,4 = 4.3,
J4,5eq = 4.8, J5ax,4 = 11.5, J3ax,4 = 12.2 Hz, H-4), 2.21 (br ddd, 1H,
J3eq,5eq ꢃ 1.6, J2,3eq = 4.2, J3eq,4 = 4.3, J3eq,3ax = 12.2 Hz, H-3 eq), 2.15,
2.08 (ꢄ2), 2.00 (4s, 3H each, CH3CO), 2.03 (d, 1H, J2,OH = 11.4,
OH), 1.70 (ddd, 1H, J2,3ax = J3ax,3eq = J3ax,4 = 12.2 Hz, H-3ax). 13C
NMR (50.3 MHz, CDCl3) d = 170.3, 170.2, 170.0, 169.5 (COCH3),
137.5, 128.6, 128.0 (C-aromatic), 96.2 (C-1), 84.9 (C-10), 74.5,
77.1 (C-30), 72.3 (C-20), 70.5, 70.4 (C-5, CH(CH3)2), 69.1 (C-40),
68.8 (C-50), 63.9 (C-2), 62.9 (C-6), 42.7 (C-4), 32.4 (C-3), 22.4,
20.5 [CH(CH3)2]. Anal. Calcd for C14H26O8S: C, 47.44; H, 7.39; S,
9.05. Found: C, 47.16; H, 7.35; S, 9.14.
0
0
0
0
0
0
4.2.5.3. Benzyl
3-deoxy-4-S-(b-
D
-galactopyranosyl)-4-thio-b-
O-Deacetylation of 13 gave
D
-erythro-pentopyranoside (18).
crystalline 18 (75%, Rf = 0.64); mp 90 °C; ½a D20
ꢂ
-43.3 (c 0.7, H2O). 1H
NMR (500 MHz, CDCl3) d = 7.23–7.29 (m, 5H, H-aromatic), 4.71,
0
0
4.54 (2d, 1H each, J = 11.6 Hz, PhCH2), 4.38 (d, 1H, J1 ,2 = 9.8 Hz,
0
0
0
0
71.8, 69.4, 67.9, 67.5, 67.2, 63.4, 61.7 (C-2 , C-3 , C-4 , C-5, C-6 , C-
5, C-2, OCH2Ph), 40.4 (C-4), 34.5 (C-3), 20.8, 20.7, 20.6 (ꢄ2)
(CH3CO). Anal. Calcd for C26H34O12S: C, 54.73; H, 6.01; S, 5.62.
Found: C, 54.99; H, 6.01; S, 5.76.
H-10), 4.28 (d, 1H, J1,2 = 7.4 Hz, H-1), 3.95 (ddd, 1H, J3eq,5eq = 2.2,
´
0
0
0
0
J4,5eq = 4.4, J5ax,5eq = 11.6 Hz, H-5eq), 3.80 (br d, 1H, J4 ,5 <1, J3 ,4
=
3.2, H-40), 3.59 (dd, 1H, J5 ,6 a = 8.9, J6 a,6 b = 12.1 Hz, H-60a), 3.53
0
0
0
0
(m, 2H, H-50 + H-60b), 3.47 (dd, 1H, J30;4 = 3.4, J2 ,3 = 9.5 Hz, H-30),
0
0
0
3.39 (ddd, 1H, J2,3eq = 4.8, J1,2 = 7.4, J2,3ax = 10.7 Hz, H-2), 3.36 (br
4.2.5. General procedure for the O-deacetylation of thiodisac-
charides 4, 5, 13 and 14
A suspension of thiodisaccharides 4, 5, 13 or 14 (0.10 mmol) in a
mixture of MeOH/Et3N/H2O 4:1:5 (10 mL) was stirred at room
temperature. The solid was progressively dissolving and after 2 h
t, 1H, J2 ,3 = 9.5, J1 ,2 = 9.8 Hz, H-20), 3.32 (dd, 1H, J4,5ax = 10.9, J5ax,5-
eq = 11.7 Hz, H-5ax), 3.08 (dddd, 1H, J3eq,4 ꢃ J4,5eq = 4.4, J4,5ax = 10.7,
J3ax,4 = 12.0 Hz, H-4), 2.21 (dddd, 1H, J3eq,5eq = 2.2, J3eq,4 = 4.4,
J2,3eq = 4.8, J3a,3b = 12.5 Hz, H-3 eq), 1.40 (ddd, 1H, J2,3ax = 10.7,
J3ax,4 = 12.0, J3ax,3eq = 12.5 Hz, H-3ax). 13C NMR (125.7 MHz, D2O)
0
0
0
0

A. J. Cagnoni et al. / Bioorg. Med. Chem. 17 (2009) 6203–6212
6211
d = 136.6, 128.7 (ꢄ2), 128.4 (C-aromatic), 103.1 (C-1), 84.4 (C-10),
78.9 (C-50), 73.8 (C-30), 71.1 (CH2Ph), 68.7 (C-20), 69.1 (C-5), 68.7
(C-4), 68.3 (C-2), 61.0 (C-60), 37.1 (C-4), 36.3 (C-3). Anal. Calcd for
C18H26O8S.H2O: C, 51.42; H, 6.71; S, 7.63. Found: C, 51.23; H,
6.63; S, 8.00.
pounds 16 (0.6–4.0 mM), 17 (0.6–4.0 mM), 18 (0.4–1.2 mM) and
19 (0.01–0.05 mM); the final volume was adjusted to 0.50 mL.
After 10 min at 37 °C, the reaction was terminated by adding so-
dium borate buffer 0.2 M (4.0 mL, pH 10.0). The concentration of
the released o-nitrophenol was measured by visible absorption
spectroscopy at 410 nm. The absorbance in the presence or ab-
sence of 16 or 17 was similar, indicating no inhibition of the en-
zyme. However, the absorbance in the presence of 18 or 19 was
significantly lower than the absorbance in control experiments.
The Ki and Km values were determined from Lineweaver–Burk plots
(Fig. 1).
4.2.5.4. Benzyl
3-deoxy-4-S-(b-
D-galactopyranosyl)-4-thio-b-
D
-threo-pentopyranoside (19).
Removal of the O-acetyl
groups of 14 gave 19 as white crystals (88%, Rf = 0.68); mp
120 °C;
½
a 2D0
ꢂ
ꢀ92.7 (c 0.6, H2O). 1H NMR (500 MHz, CDCl3)
d = 7.30–7.22 (m, 5H, H-aromatic), 4.65 (m, 2H, PhCH2 + H-1,
superimposed with HDO signal), 4.44 (d, 1H, J = 11.8 Hz, PhCH2),
4.38 (d, 1H, J1 ,2 = 9.8 Hz, H-10), 3.94 (dd, 1H, J4,5a = 3.2, J5a,5b
=
Acknowledgments
0
0
12.0 Hz, H-5a), 3.84 (ddd, 1H, J1,2 = 2.8, J2,3b = 3.5, J2,3a = 8.4 Hz, H-
2), 3.80 (br d, 1H, J4 ,5 <1, J3 ,4 = 3.3, H-40), 3.58 (dd, 1H, J5 ,6 a
=
Support of this work by the University of Buenos Aires (Project
X227), the National Research Council of Argentina (CONICET, Pro-
ject PIP 5011) and the National Agency for Promotion of Science
and Technology (ANPCyT, Project 13922) is gratefully acknowl-
edged. O.V. and M.L.U. are Research Members from CONICET,
A.J.C. is a fellow from CONICET.
0
0
0
0
0
0
8.8, J6 a,6 b = 12.3 Hz, H-60a), 3.55–3.51 (m, 2H, J5 ,6 b = 4.0,
0
0
0
0
J6 a,6 b = 12.3, H-50 + H-60b), 3.47 (dd, 1H, J3 ,4 = 3.3, J2 ,3 = 9.5 Hz,
H-30), 3.42 (dd, 1H, J4,5b = 5.6, J5a,5b = 12.0 Hz, H-5b), 3.38 (br t,
0
0
0
0
0
0
1H, J2 ,3 = 9.5, J1 ,2 = 9.8 Hz, H-20), 3.33 (dddd, 1H, J4,5eq = 3.2,
J3a,4 = 4.2, J4,5b = 5.6, J3b,4 = 7.2 Hz, H-4), 2.05 (ddd, 1H, J3a,4 = 4.2,
J2,3a = 8.6, J3a,3b = 13.5 Hz, H-3a), 1.77 (ddd, 1H, J2,3b = 3.5,
J3b,4 = 7.2, J3a,3b = 13.5 Hz, H-3b). 13C NMR (125.7 MHz, CDCl3)
d = 137.0, 128.7, 128.5, 128.3 (C-aromatic), 98.2 (C-1), 85.3 (C-10),
78.9 (C-50), 73.9 (C-20), 70.0 (CH2Ph), 69.6 (C-30), 68.7 (C-40), 65.4,
65.0 (C-5, C-2), 61.1 (C-60), 38.2 (C-4), 33.2 (C-3). Anal. Calcd for
C18H26O8S: C, 53.72; H, 6.51; S, 7.97. Found: C, 53.38; H, 6.74; S,
8.06.
0
0
0
0
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.07.055.
References and notes
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