Convenient synthesis of 4-thiolactose, 3,4-dithiolactose and related thiooligosaccharides and disulfides. Inhibitory activity of the glycomimetics against a β-galactosidase

Article abstract of DOI:10.1039/c2ob26388b

The ring-opening reaction of sugar 3,4-epoxides by 2,3,4,6-tetra-O-acetyl- 1-thio-β-d-galactopyranose (7) as a nucleophile led to (1 → 3)- and (1 → 4)-thiodisaccharides. High regio- and diastereoselectivities were achieved in the synthesis of the per-O-acetyl derivative of the β-d-Galp-S-(1 → 4)-4-thio-α-d-Glcp-O-iPr (10). Analogues of the 4-thiolactoside 10 have been prepared, with the β-d-Galp non-reducing end S-linked to d-Glcp, d-Gulp and d-Idop. A similar regioselective attack of 7 on C-4 of 2-propyl 3,6-di-O-acetyl-3,4-epithio-α-d-galactopyranoside (6) led to 2-propyl 3,4-dithiolactoside derivative 15. During this reaction the free 3-SH group of 15 underwent oxidative dimerization or oxidative coupling with the SH function of 7 to give the respective disulfides. Glycosylation of the thiol group of 15 using trichloroacetimidate derivatives of β-d-Galp or β-d-Galf afforded the corresponding branched dithiotrisaccharides. The free compounds were evaluated as inhibitors of the E. coli β-galactoside. The bis(2-propyl 3,4-dithiolactosid-3-yl)-disulfide, obtained from 15, displayed the strongest inhibitory activity in these series of glycomimetics and proved to be a non-competitive inhibitor (K_i = 95 μM).

Full text of DOI:10.1039/c2ob26388b

View Online / Journal Homepage
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This is an Accepted Manuscript, which has been through the RSC Publishing peer
Organic &
review process and has been accepted for publication.
Biomolecular
Accepted Manuscripts are published online shortly after acceptance, which is prior
to technical editing, formatting and proof reading. This free service from RSC
Publishing allows authors to make their results available to the community, in
citable form, before publication of the edited article. This Accepted Manuscript will
be replaced by the edited and formatted Advance Article as soon as this is available.
Chemistry
www.rsc.org/obc
Volume 8 | Number 3 | 7 February 2010 | Pages 481–716
To cite this manuscript please use its permanent Digital Object Identifier (DOI®),
which is identical for all formats of publication.
More information about Accepted Manuscripts can be found in the
Information for Authors.
Please note that technical editing may introduce minor changes to the text and/or
graphics contained in the manuscript submitted by the author(s) which may alter
content, and that the standard Terms & Conditions and the ethical guidelines
that apply to the journal are still applicable. In no event shall the RSC be held
responsible for any errors or omissions in these Accepted Manuscript manuscripts or
any consequences arising from the use of any information contained in them.
ISSN 1477-0520
PERSPECTIVE
FULL PAPER
Laurel K. Mydock and
AlexeiV. Demchenko
Shuji Ikeda et al.
Hybridization-sensitive fluorescent
DNA probe with self-avoidance ability
Mechanism of chemical O-glycosylation:
from early studies to recent discoveries
1477-0520(2010)8:3;1-H
www.rsc.org/obc
Registered Charity Number 207890

Organic & Biomolecular Chemistry
Page 1 ofJ11ournal Name
Dynamic Article Links ►
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
View Online
ARTICLE TYPE
Convenient synthesis of 4-thiolactose, 3,4-dithiolactose and related
thiooligosaccharides and disulfides. Inhibitory activity of the
glycomimetics against a -galactosidase
Verónica E. Manzano, María Laura Uhrig and Oscar Varela*
5
Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X
DOI: 10.1039/b000000x
The ring-opening reaction of sugar 3,4-epoxides by 2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranose (7)
as nucleophile, led to (13)- and (14)-thiodisaccharides. High regio and diastereoselectivities were
achieved in the synthesis of the per-O-acetyl derivative of the β-D-Galp-S-(14)-4-thio-α-D-Glcp-O-iPr
10 (10). Analogues of the 4-thiolactoside 10 have been prepared, with the β-D-Galp non-reducing end S-
linked to D-Glcp, D-Gulp and D-Idop. Similar regioselective attack of 7 to C-4 of 2-propyl 3,6-di-O-
acetyl-3,4-epithio--D-galactopyranoside (6) led to 2-propyl 3,4-dithiolactoside derivative 15. During this
reaction the free 3-SH group of 15 underwent oxidative dimerization or oxidative coupling with the SH
function of 7 to give the respective disulfides. Glycosylation of the thiol group of 15 using
15 trichloroacetimidate derivatives of β-D-Galp or β-D-Galf afforded the corresponding branched
dithiotrisaccharides. The free compounds were evaluated as inhibitors of the E. coli β-galactoside. The
bis(2-propyl 3,4-dithiolactosid-3-yl)-disulfide, obtained from 15, displayed the strongest inhibitory
activity in these series of glycomimetics and proved to be a non-competitive inhibitor (Ki = 95 M).
20
45 and convenient approaches have been reported.3,13,14
Thioglycosidic linkages have also been constructed using
enzymes to promote the coupling of appropriate carbohydrate
moieties.14,15 In our laboratory, we have developed regio and
diastereoselective strategies for the construction of the
50 thioglycosidic linkage of thiodisaccharides. Thus, the Michael
addition of 1-thioaldoses to hexose-16 or pentose-derived sugar
enones17 was the key step in the synthesis of 3-deoxy-4-thio
analogues of natural or non-natural (14)-disaccharides; and
thiodisaccharides with a thiofuranose as non-reducing end have
55 also been prepared.18,19 Similarly, 4,6′-thioether-linked
disaccharides have been obtained as nonglycosidic, hydrolytically
Introduction
In recent years, the development of glycomimetics has received
considerable attention as these compounds display interesting
biological activities and may be involved in many metabolic
25 pathways. The search for new glycomimetics has led to analogues
of carbohydrates in which one or more oxygen atoms have been
replaced by sulfur1 or other heteroatoms.2 In particular,
thiooligosaccharides are sugar mimetics with at least one
interglycosidic linkage mediated by sulfur. They are usually
30 resistant to metabolic processes and are frequently employed as
useful tools for glycobiology.3 The understanding of enzyme-
inhibitor interactions is essential to provide insight into binding
and recognition events, and also for the rational design of
inhibitors of glycosidases.4 Inhibition of these enzymes may
35 disrupt the biosynthesis and catabolism of glycoconjugates, thus
interfering vital processes. Therefore, glycosidase inhibitors have
shown promise as therapeutics in the treatment of diabetes,5
lysosomal storage diseases6 and viral infections,7 including
influenza8 and HIV.9 Indeed several glycosidase inhibitors are
40 already used clinically for treating some of these diseases.10,11
Enzyme inhibition also constitutes an innovative approach for
drug development in cancer therapies.12
stable glycomimetics.20 Moreover, we have described
a
straightforward procedure for the synthesis of (13)- or (14)-
thiodisaccharides based on the ring-opening of sugar epoxides
60 with 1-thioaldoses.21 In addition, the controlled ring-opening
reaction of sugar thiiranes by 1-thiopyranoses was successfully
accomplished to afford, regio and stereoselectively, β-S-(14)-
3,4-dithiodisaccharides that were employed as precursors of
branched dithiotrisaccharides.22
65
The thiodisaccharides obtained by some of these
methodologies have been evaluated as inhibitors of glycosidases,
and many of them were active against the β-galactosidase from
Escherichia coli.16,17 This enzyme has been extensively studied,23
and it has shown to be highly specific for β-galactopyranosyl
Because of all these biologically relevant issues, the synthesis
of thiooligosaccharides is a subject of intensive current research,
70 units linked to
a
wide variety of aglycons. Among the
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

Organic & Biomolecular Chemistry
Page 2 of 11
thioglycomimetics, simple thiogalactopyranosides24 (such as
isopropyl or phenylethyl) are good inhibitors and
thiodisaccharides like 4-thiolactose25 and analogues16,17 display
also inhibitory activity against the enzyme.
As continuation of this previous work we report here
convenient procedures for the synthesis of thiooligosaccharides
and disulfides with a basic structure of 4-thiolactose. The ring-
opening of the epoxide or thiirane derivatives of sugars was
employed as key reaction to control the regio and
View Online
5
55
Scheme 1 Synthesis of epoxides 3-5 and thiirane 6
10 stereoselectivity in the construction of the thioglycosidic linkage.
The resulting products have been evaluated as inhibitors against
the -galactosidase from E. coli.
Furthermore, the large value for the coupling constant between
H-1’ and H-2’ ( 10 Hz) in both 8 and 9 is indicative that the β-
configuration of the 1-thioaldose 7 is maintained during the
reaction and remains in the final product.
60
Thiodisaccharide 9 is the expected product of trans-diaxial
opening of the oxirane ring of 3, according to the Fürst-Plattner
rule. However, no regioselectivity was found as 9 and the trans-
diequatorial product 8 were produced in similar amounts. The
poor regioselectivity observed in the formation of 8 and 9 could
Results and discussion
15
Synthesis of thioglycomimetics
65 be explained, as reported for analogous compounds,26 according
to stabilizing or repulsive effects that operate during the ring-
opening reaction. Thus, formation of the trans-diaxial product 9
by attack of thiolate 7 to the C-4 of 3 is expected to produce a
slight distortion of the conformation of the pyranose ring, but a
70 1,3-diaxial interaction between the substituent at C-3 and the
anomeric isopropyl group is generated during the opening of the
epoxide. In contrast, the transition state that leads to 8 is free of
such an a respulsive interaction and, in addition, 8 is an stable
product as all the substituents of the chair, from C-2 to C-5, are
75 equatorially oriented.
To obtain a 4-thiolactose derivative, we conducted the reaction
of 1-thioaldose 7 with the epoxide 4, which has opposite
stereochemistry at C-3 and C-4 compared to that of 3. This
reaction afforded the expected thiodisaccharides 10 and 11,
80 together with a product which was isolated and identified as the
thiodisaccharide 12. Formation of 12 could be the result of
The oxirane and episulfide derivatives 3-6 were prepared as
thioglycosyl acceptors. These compounds possess the convenient
configuration at C-3 and C-4 to provide, via a ring-opening
20 reaction, a glucopyranose unit as reducing end of the resulting
thiodisaccharides. Thus, the starting 2,3,4,6-tetra-O-acetyl-D-
galactal (1) was converted into the allylic alchohol 2, which upon
oxidation, afforded the epoxides derivatives 3-521 (Scheme 1).
The thiirane derivative 6 was also synthesized using the sugar
25 epoxide 3 as precursor.22
As we wanted to have a galactopyranose unit as non-reducing
end of the ring-opening products, 2,3,4,6-tetra-O-acetyl-1-thio-β-
D-galactopyranose (7) was employed as nucleophilic thioglycosyl
donor (Scheme 2). The ring-opening reaction was performed in
30 methanol containing LiOMe to convert the thiol group of 7 into
the more nucleophilic thiolate. As the basic medium promotes O-
deacetylation, when the reaction was finished, the base was
neutralized and the mixture was reacetylated. This procedure
applied for the ring-opening reaction of the epoxide group of 3 by
35 the 1-thioaldose 7, gave the expected thiodisaccharide derivatives
8 and 9 in an excellent overall yield (95%). The stereochemistry
of 8 and 9 was deduced from their NMR-spectra (1D/2D). The
site of attack of the thiol was readily established because of the
shielding effect of the sulfur atom, which induces an upfield
40 shifting of the signal of the S-linked carbon and that of the
corresponding proton. As the latter appears in a clean region of
the spectrum (2.803.50 ppm) the coupling constants (J) could be
accurately measured. For example, H-3 ( = 3.27) in 8 gave large
values for the J2,3 (11.7 Hz) and J3,4 (11.3 Hz) indicating a trans-
45 diaxial disposition for the coupled protons, and hence a gluco
configuration for the reducing end of 8. In contrast, the H-4
signal ( = 3.28) in 9 showed small values for J3,4 and J4,5 (both
2.7 Hz) suggesting a gulo configuration for the sulfur-containing
moiety of 9. In the 13C NMR spectra, the signals of the carbons
50 bonded to sulfur, C-3 (in 8) and C-4 (in 9) appear, respectively, at
46.7 and 44.8 ppm, whereas the anomeric carbons linked to sulfur
(C-1’) resonate at higher field (83.8 and 83.0 ppm) than the O-
glycosidic anomeric carbons (C-1, 93.7 and 94.4 ppm).
3,42,3 isomerization in O-deacetylated
4
by epoxide
migration,21,26 followed by diaxial ring-opening by attack of the
thiol group of 7 to C-2. The stereochemistry of the reducing-end
85 of 10-12 was established from the NMR spectra, as already
described for the related thiodisaccharides 8 and 9. It is worth to
mention that the spectral data of 10 were coincident with those
reported for the analogue methyl 4-thiolactoside.27 Unfortunately,
the reaction between 4 and 7 afforded a low yield (21%) of the
90 desired per-O-acetyl derivative of 4-thiolactoside 10. However,
we have previously reported that the regioselectivity in the ring-
opening of 3,4-epoxides like 4 could be improved by installing a
bulky substituent on OH-2.21 For example, a bulky TBS group at
HO-2 is expected to hinder the attack of the nucleophile to the
95 vicinal C-3. Furthermore, if the HO-2 remains protected during
the reaction, the epoxide migration should be prevented. For
these reasons, the epoxide 5 was treated with 1-thiogalactose 7 as
described for the previous ring-opening reactions. After the usual
work-up, two products were isolated by column chromatography,
100 the expected thiodisaccharide 13 (51%) and the second
component that was identified as 10. The latter seems to be
formed by O-desilylation once that 13 has been obtained, since
12 has the same 4-thio-D-gluco configuration for the reducing-
end as 13.
2
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Organic & Biomolecular Chemistry
Page 3 ofJ11ournal Name
Dynamic Article Links ►
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
View Online
ARTICLE TYPE
AcO
AcO
OAc
O
AcO OAc
O
OAc
O
SH
OAc
O
AcO
S
AcO OAc
OAc
O
OAc
O
OAc
O
AcO
S
OAc
7
AcO
AcO
AcO
OAc
O
O
+
OAc
OAc
O
S
1) LiOMe/MeOH
AcO
AcO
AcO
AcO
S
OiPr
AcO
O
HO
OAc
OAc
O
S
2) AcOH, pH = 7
3) Ac2O, C5H5N
OAc
AcO
AcO
OiPr
OiPr
OiPr
OiPr
HS
OiPr
OAc
AcO
+
S
+
3
8 (45%)
S
9
(50%)
OAc
O
OAc
O
AcO
AcO
OiPr
AcO
15 (5%)
16 (32%)
18-crown-6
THF
SR
OAc
AcO
OAc
OAc
+
OAc
OAc
O
AcO
OAc
OAc
O
AcO
AcO
AcO
OAc
OAc
O
O
OiPr
AcO
AcO
O
7 R = H
S
AcO
6
S
NaH
+
+
AcO
AcO
S
O
14 R = Na
S
OiPr
OAc
OAc
AcO
OAc
O
AcO
OiPr
OAc
O
OiPr
O
i) 18-crown-6, THF
ii) LiAlH4, THF
iii) Ac2O, C5H5N
OAc
OAc
1) 7, LiOMe/MeOH
10 (21%)
O
11 (20%)
AcO
AcO
OAc
S
2) AcOH, pH = 7
3) Ac2O, C5H5N
AcO
AcO
S
O
OAc
S
AcO
OAc
OiP
AcO
OAc
O
OAc
O
17
(3%)
OAc
r
4
O
S
AcO
OAc
RS
AcO
iPrO
OAc
AcO
OiPr
12 (20%)
SH
MeCN
18 R = Ac (70%)
15 R = H (71%)
NH2
AcO
AcO
OAc
O
AcO
AcO
OAc
O
OAc
O
O
35
OAc
OAc
7
1) , LiOMe/MeOH
O
O
S
AcO
S
AcO
+
Scheme 3 Products obtained by ring-opening of thiirane 6
2) AcOH, pH = 7
3) Ac2O, C5H5N
AcO
AcO
TBSO
AcO
TBSO
OiPr
OiPr
13 (51%)
10 (33%)
5
The free thiol group of the dithiodisaccharide 15, obtained
under the optimized conditions described above, was subjected to
glycosylation (Scheme 4). This reaction was conducted, in first
1) TBAF, THF
2) Ac2O, C5H5N
10 (85%)
Scheme 2 Synthesis of thiodisaccharides from epoxides 3-5
40 instance, using an excess of the trichloroacetimidate 19 as
glycosyl donor. Reaction of 15 and 19 under catalysis with
trimethylsilyl triflate (TMSOTf), and in the presence of 4 Å
molecular sieves, led to the dithiotrisaccharide 20 in a moderate
yield (35%). The yield could not be improved even though a
45 number of different conditions were employed. The resistance of
the 3-thiol group of 15 to glycosylation may be attributed to the
sterical hindrance of the vicinal gauche-disposed thiopyranose
moiety at C-4 and the acetoxy group at C-2. Probably due to the
low reactivity of the thiol group, part of the trichloroacetimidate
50 19 is converted into the Galp-(11)-Galp derivative 21.28 The
sterical hindrance on the SH group of 15 justifies why disulfides
16 and 17 are formed during the ring-opening reaction of thiirane
6. The longer and more flexible S-S bond, compared to the 3-
thioglycosidic linkage in 20, would alleviate the crowd in the C-3
55 region of the 3,4-dithioglucopyanose moiety. In agreement with
this behaviour, glycosylation of 15 with the galactofuranose
trichloroacetimidate 22, as less bulkier analogue of 19, led to the
dithiotrisaccharide 23 in a very good yield (81%).
The per-O-acetyl derivatives of thiodisaccharides 8-10 and
60 dithiotrisaccharides 20 and 23 were fully deprotected by a smooth
hydrolysis of the acetyl esters with an aqueous methanol solution
of triethylamine at room temperature (Scheme 5). The resulting
free products were purified by elution of their respective
solutions in water through a column filled with a mixed-bed ion-
65 exchange resin and then through a reverse phase minicolumn.
The NMR spectra of thiodisaccharides 24 -26 exhibited the
expected values for the signals of the anomeric protons (H-1 at
~5.0 ppm, J1,2 = 3.54.0 Hz and H-1’ at ~4.5 ppm, J1’,2’ = 9.69.8
Hz); whereas the anomeric carbon of the thioglycoside appeared
70 shifted upfield (~12 ppm) with respect to that of the O-glycoside.
The dithiotrisaccharides 29 and 30 showed the additional signals
due to the respective S-linked Galp or Galf units.
To avoid the separation of 10 and 13 by chromatography, in
further preparations the crude mixture of reaction was treated
with TBAF for the O-desilylation prior to reacetylation. This way
compound 13 was converted into 10, which was isolated in 85%
overall yield.
Our next target was the 3,4-dithiolactoside derivative 15
(Scheme 3), an analogue of 10 having the HO-3 group replaced
5
10 by thiol. Compound 15 was prepared by ring-opening of the 3,4-
thiirane 6 with the thiolate 14. In these reactions the expected
ring-opening products are accompanied by (13)-disulfides,
formed by oxidative coupling of the thiol groups present in the
starting 1-thioaldose or in the resulting dithiodisaccharides.21
15 However, the reduction of disulfides in the reaction mixture with
LiAlH4, led to the major expected product. Thus, the 3,4-
dithiodisaccharide 18 (70%) was obtained after LiAlH4 reduction
of the crude mixture of reaction of 6 with 14, followed by
acetylation. The thiol group was released from its acetyl
20 derivative by treatment of 18 with 2-aminoethanethiol
(cysteamine) to give 15. We have now identified all the products
formed by reaction of 6 with 14. These products were isolated by
careful column chromatography and repurification of the fraction
that contained disulfides 16 and 17.
Once isolated, the structure of 16 and 17 was readily
established by NMR (1D/2D). The signal of C-3, linked to the
disulfide group, was shifted downfield (48.9 in 16 and 50.9 ppm
in 17) with respect to the C-3 signal in 15 (40.2 ppm). As
previously reported,22 these relative shiftings may be used as
30 diagnostic of disulfide formation. Similarly, the signal of the
anomeric carbon (C-1’) linked to the disulfide in 16 appeared at a
 value (88.5 ppm) intermediate between that of O- and S-
glycosides (94.2 and 81.5 ppm, respectively).
25
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 3

Organic & Biomolecular Chemistry
Page 4 of 11
The conformation of the pyranose rings of dithiodisaccharides
24, 25 and 26 was established by 1H-NMR. As expected, the Galp
and Glcp rings of 24 and 26 adopt the 4C1 conformation
AcO
OAc
AcO
AcO
OAc
O
OAc
O
AcO
AcO
OAc
O
AcO
O
O
S
AcO
OAc
3
OAc
+
S
O
AcO
according to the J coupling constants. Also, the Galp ring of 25
AcO
AcO
AcO
O
OAc
NH
CCl3
OiPr
View Online
AcO
O
AcO
OAc
19
TMSOTf, CH2Cl2
35 adopts the same conformation as indicated by the small value for
AcO
OAc
OAc
OAc
O
20 (35%)
21
J3,4 (3.6 Hz). Similarly, the large values for J2,3, J3,4 and J4,5 in 28
O
S
HS
AcO
4
AcO
OAc
AcO
OAc
AcO
are indicative of the C1 chair for the 3,4-dithioGlcp ring. In this
OiPr
O
O
S
S
15
NH
CCl3
AcO
case, the J values are even larger (0.61.4 Hz) than those found
for the respective coupled protons in 26, due to the replacement
40 of OH by SH and being the sulfur atom less electronegative than
oxygen. In the field of thiooligosaccharides, is always interesting
to evaluate how the substitution of the interglycosidic oxygen by
sulfur modifies the conformational behavior of the analogues
with respect to their natural counterparts. Differences should be
45 expected as the C-S bond length (1.78 Å) and the C-S-C bond
angle (99°) differ from the values for C-O (1.41 Å) and C-O-C
(116°) and the size of stereoelectronic effects, in particular the
anomeric effects, should be different as well. In fact, the
conformation of methyl 4-thiolactoside was studied using a
50 combination of NMR spectroscopy and molecular mechanics and
dynamics calculations.25a The theoretical results were
experimentally confirmed by the interresidue NOEs that
unequivocally characterize the minimum energy regions of the
conformational maps. Thus, according with the torsional angles 
55 (H1’-C1’-S-C4) and  (C1’-S-C4-H4), three main conformations
were found for methyl 4-thiolactoside: syn /anti , syn /syn 
and anti /syn , which were confirmed by the respective
interresidue observed NOE contacts H1’-H3, H1’-H4, and H2’-
H4. To determine the NOE correlations that suggest the presence
60 of given conformers, the NOESY spectrum of the
thiodisaccharides were recorded and analyzed. Thus, in the
NOESY spectrum of compound 26, the isopropyl glycoside
analogue of methyl 4-thiolactoside, the same NOEs were detected
AcO
OAc
BzO
OiPr
BzO
O
O
O
BzO
BzO
BzO
BzO
OBz
22
OBz
TMSOTf, CH2Cl2
(81%)
23
Scheme 4 Synthesis of dithiotrisaccharides 20 and 23
5
Interestingly, the deprotection of 15 afforded a single product
with spectral properties that were in agreement with those
expected for the resulting dithiodisaccharides. However, the Rf
value was lower than those measured for the analogue
10 thiodisaccharide 24-26. Finally, the MS of the compound
revealed that we were dealing with a dimeric structure, assigned
as the disulfide 27. The structure of 27 was confirmed by
treatment of the compound with tris(2-carboxyethyl)phosphine
hydrochloride (TCEP). This reagent is employed for the reductive
15 cleavage of the disulfide linkages in water and polar solvents.29
As expected, TLC examination of the reaction showed the
conversion of the disulfide into the faster moving
dithiodisaccharide 28. The NMR spectra of 27 and 28 were quite
similar, but a careful analysis showed some differences in the
20 chemical shifts of the signals of the carbon atoms (C-3 and C-4)
linked to sulfur, and also for those of H-3 and H-4. For example,
H-3 and H-4 signals appeared in 27 overlapped as a multiplet at
3.13-3.02 ppm; whereas in 28 they exhibited well resolved
triplets at 3.29 and 2.85 ppm, respectively. We have also
25 observed that 28 in solution is rapidly oxidized to 27, even under
an inert Ar atmosphere.
suggesting
a
similar conformational behavior for the
65 thioglycosidic linkage of both compounds. In contrast, for methyl
-lactoside was predicted and experimentally proven,30 the
almost exclusive contribution of the syn /syn  conformation.
The variation in bond length and angles may explain the higher
flexibility of 26 versus its natural O-analogue.
Preliminary conformational information was also obtained
from the NOESY spectra of thiodisaccharides 24 and 25. The
interresidue NOE contacts between H1’-H3 and H1’-H4 observed
for 24 suggested the presence of the respective syn /syn  and
syn /anti  conformations around the thioglycosidic bond
HO
OH
OH
O
S
O
HO
MeOH/H2O/Et3N
OH
HO
S
HO
HO
OH
HO
MeOH/H2O/Et3N
O
O
8
9
HO
OiPr
70
HO
HO
25 (95%)
OH
OiPr
24 (92%)
OH
HO
OH
O
O
MeOH/H2O/Et3N
S
HO
10
HO
HO
OH
OiPr
26 (93%)
75 (Figure 1). On the other hand, the NOESY spectrum of 25
exhibited characteristic interresidue cross peaks between H1’-H3,
H2’-H4 and H1’-H4 that justify, respectively, the presence of the
syn /anti , anti /syn  and syn /syn  conformations. It is
worth to mention that all the conformations found for the
80 dithiodisaccharides are stabilized by the exo-anomeric effect.
Therefore, similar to the O-glycosides, the thiodisaccharides
adopt exo-anomeric conformations around , but the orientations
around the aglyconic bond  are rather different for S- and O-
glycosides. The major conformer is always centered at the syn 
HO
OH
OH
O
S
HO
HO
OH
O
OH
HO
HO
O
TCEP/H2O
MeOH/H2O/Et3N
O
S
S
HO
OH
HO
OH
15
OiPr
HS
HO
HO
O
O
OiPr
S
HO
S
28 (92%)
HO
27 (89%)
HO
OiPr
OH
OH
HO
HO
OH
HO
HO
OH
O
O
O
S
O
S
S
S
HO
OH
HO
HO
MeOH/H2O/Et3N
MeOH/H2O/Et3N
OiPr
OiPr
HO
OH
20
23
HO
HO
O
O
HO
HO
OH
HO
(83%)
30
29 (95%)
85 region,25 but anti
 conformers are also found for the
thioglycosides. For the binding of a flexible compound to a
protein, one of the existing conformations could be selected and
bound to the binding site without major energy conflicts.25b
Scheme 5 O-Deacetylation of thioglycomimetics 8-10, 15, 20 and 23
| Journal Name, [year], [vol], 00–00
30
4
This journal is © The Royal Society of Chemistry [year]

Page 5 of 11
Organic & Biomolecular Chemistry
1,0
24
View Online
26
0,5
29
25
30
27
syn Ф/ syn Ψ
syn Ф/ anti Ψ
1
2
3
4
5
[I] (mM)
50
Figure 1. Conformations proposed for 24 according to NOE contacts.
5
Figure 2. Effect of concentration of thioglycomimetics on the enzymatic
activity of the-galactosidase from E. coli. Each point is the mean
obtained from three replicate experiments.
The free compounds 25-30 were evaluated as inhibitors of the
β-galactosidase from E. coli. Thiodisaccharides 24 and 25 are
useful to establish the influence of structural changes on the
10 reducing end, in comparison with the 4-thiolactoside 26; whereas
27, 29 and 30 will show the effect of the glycosylation or
disulfide formation involving the thiol group at C-3 of 28.
Unfortunately, this compound could not be evaluated as
underwent rapidly oxidative dimerization during the isolation and
15 incubation.
140
120
100
control
I=0,05
80
I=0,10
60
I=0,25
I=0,4
40
20
0
Evaluation of the inhibitory activity
For the evaluation of the inhibitory activity of thiodisaccharides
24-26, disulfide 27 and dithiotrisaccharides 29 and 30 against the
20 E. coli β-galactosidase a standard protocol was followed.16 The
substrate employed was o-nitrophenyl β-D-galactopyranoside, and
-0,7
-0,2
0,3
0,8
1,3
1,8
2,3
2,8
1/ [S](mM-1
)
-20
55
Figure 3. Lineweaver–Burk plot for inhibition of E. coli
β-galactosidase by disulfide 27
the
release
of
o-nitrophenol
was
measured
spectrophotometrically. The enzymatic reaction was performed in
the presence of the potential inhibitor at concentrations ranging
25 from 0.05 to 5.0 mM, and the release of o-nitrophenol is referred
to the control (Figure 2).
These results suggest that small molecules, like the S-
60 disaccharides, structurally similar to the O-glycoside substrate,
compete with this compound for binding to the active site of the
β-galactosidase. Contrarily, a bulky molecule, such as 27, is
probably not able to interact with the catalytic site, but decreases
the efficiency of the hydrolysis of the O-glycoside by interacting
65 with some other sites of the enzyme.
The isopropyl 4-thiolactoside 26 showed only
a weak
inhibitory activity. Similarly, methyl 4-thiolactoside was
determined to be a weak competitive inhibitor of the enzyme (Ki
30 = 7.7 mM).25 The thiodisaccharide 24 was not active in the range
of concentrations employed. Therefore, the change of position of
the thiogalactopyranosyl unit from C-4 to C-3 of the
glucopyranose residue has a negative effect on the inhibition. In
Conclusions
contrast, compound 25, with
a D-gulo-hexopyranoside as
35 reducing end, showed an increased activity with respect to 26.
Interestingly, the dithiotrisaccharides that contain a 3-S-Galp (29)
or 3-S-Galf (30) unit were stronger inhibitors of E. coli β-
galactosidase than 26. The IC50 values determined for 25 (3.53 ±
0.02 mM), 29 (3.79 ± 0.02 mM) and 30 (1.16 ± 0.06 mM) allows
40 the comparison of their relative activity. The best inhibitor of
The ring–opening reaction of 3,4-epoxide or 3,4-thiirane
derivatives of sugars by the nucleophilic 1-thiogalactose was
70 successfully applied for the respective synthesis of 4-
thiolactosides and 3,4-dithiolactosides. Under optimized
conditions, the formation of the thioglycosidic linkage took place
with high regio and diastereoselectivities to give S-(14)-linked
disaccharides having the 4-thio- or 3,4-dithio-D-Glcp as reducing
75 end.
The 2-propyl per-O-acetyl-3,4-dithiolactoside possesses a thiol
group free that can be glycosylated or converted into disulfides,
molecules with potential biological activity.31,32 Thiols can also
act as antioxidants33 and have an essential role in many
80 biochemical redox reactions.34
these series of compounds was the symmetric disulfide 27 (IC50
=
0.111 ± 0.005 mM). The kinetics of inhibition of 27 was
determined on the basis of Lineweaver-Burk plot (Figure 3),
which indicated that 27 in a non-competitive inhibitor with Ki =
45 95 M (Km
= 1.15 mM). On the other hand, the
dithiotrisaccharide 30 is a mixted-type inhibitor, while the
analogue 25, as methyl 4-thiolactoside,25 is a competitive
inhibitor.
One of the synthetic mimetics, the symmetric bis(2-propyl 3,4-
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5

Organic & Biomolecular Chemistry
Page 6 of 11
dithiolactosid-3-yl)-disulfide (27), showed to be a good non-
galactopyranosyl)-3-thio-α-D-glucopyranoside (8) and 2-
propyl 2,3,6-tri-O-acetyl-4-S-(2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyl)-4-thio-α-D-gulopyranoside (9)
competitive inhibitor of the E. coli β-galactosidase. From the
inhibition studies we can conclude that the activity of the
thiodisaccharides depends on the site of S-bonding (13 or
14) of GalpSH to the hexose at the reducing-end, and the
configuration of this unit. Thus, the S-(13) linked disaccharide
24, is the less active compound. The inhibitory activity increases
for the 4-thiolactoside 26, the analogue of a natural substrate of
60
View Online
2-Propyl 6-O-acetyl-3,4-anhydro--D-allopyranoside21 (3, 0.25 g,
0.87 mmol) and 2,3,4,6-tetra-O-acetyl-1-thio-β-D-
galactopyranose (7, 0.45 g, 1.2 mmol) were dissolved in 2 M
LiOMe/MeOH (3.1 mL) and the mixture was stirred in a screw-
5
the enzyme. The S-disaccharide 25, which has the C-3 and C-4 65 cap tube under Ar at 65 oC for 24 h. The solution was neutralized
10 stereocenters with opposite configuration than those in 26, is
more active than this compound. Interestingly, the 3-deoxy
analogue of 25,16 is an even more potent inhibitor (Ki = 0.16 mM)
than 25 (Ki = 1.24 mM). Therefore, the change of the
with acetic acid, concentrated and the residue was acetylated with
pyridine (1 mL) and Ac2O (1 mL) at room temperature for 15 h
and concentrated. Monitoring of the mixture by TLC (1:1
toluene/EtOAc) showed two main products (Rf 0.37 and 0.46),
configuration at C-4 and the deoxygenation at C-3 of the reducing 70 which were separated by column chromatography (3:1→2:1
15 end of these series of S-(14) linked analogues of 4-thiolactoside
provide more potent inhibitors.
The thiolactosides obtained are relevant not only as analogues
of the naturally occurring lactose, but also because the motif
hexane/EtOAc). The first compound eluted from the column was
identified as the thiodisaccharide 8 (0.31 g, 45%); [α] +62.7 (c
1.1 in CHCl3); 1H RMN (CDCl3, 500 MHz): δ 5.42 (d, 1H, J3’,4’
=
3.1 Hz, H-4’), 5.14 (t, 1H, J1’,2’ = J2’,3’ = 9.9 Hz, H-2’), 5.10 (d,
Galp-β-(14)-Glcp is found in complex molecules. For example, 75 1H, J1,2 = 3.4 Hz, H-1), 5.03 (dd, 1H, J2’,3’ = 9.9, J3’,4’ = 3.1 Hz,
20 such a moiety is a component of gangliosides of marine
organisms,35 and also of the GM3 ganglioside, a characteristic
antigen of murine B16 melanoma.36 A trimeric β-lactosyl cluster
has been synthesized as a GM3 analogue that was an effective
H-3’), 4.92 (t, 1H, J3,4 = J4,5 = 11.3 Hz, H-4), 4.82 (dd, 1H, J1,2
3.4, J2,3 =11.3 Hz, H-2), 4.77 (d, 1H, J1’,2’ = 9.9 Hz, H-1’), 4,20
=
(dd, 1H, J5,6a = 4.9, J6a,6b = 12.2 Hz, H-6a), 4.17 (dd, 1H, J5’,6’a
6.7, J6’a,6’b = 11.1 Hz, H-6’a), 4,08 (dd, 1H, J5,6b = 2.3, J6a,6b
=
=
acceptor of a sialyltransferase.37 The exopolysaccharide from 80 12.2 Hz, H-6b), 4.05 (m, 1H, H-5), 4.03 (dd, 1H, J5’,6’b = 6.7,
25 Lactobacillus delbrueckii contains a lactosyl residue as a lateral
chain.38 With regard to the branched dithiotrisaccharides, a
pattern of a Glcp core with glycosyl residues at HO-3 and HO-4
is found in the sialyl X Lewis epitope39 and related compounds; 40
J6’a,6’b = 11.1 Hz, H-6’b), 3,98 (t, 1H, J5’,6’a = J5’,6’b = 6.7 Hz, H-
5’), 3.89 (m, 1H, J = 6.2 Hz, Me2CHO), 3.27 (dd, 1H, J2,3 = 11.7,
J3,4 = 11.3 Hz, H-3), 2.15, 2.13, 2.10, 2.09, 2.06, 2.02, 1.97 (7 s,
21H, CH3CO), 1.25, 1.14 (2 d, 3H each, J = 6.2 Hz, (CH3)2CHO);
whereas the motif β-D-Galf(13)Glcp was found in 85 13C RMN (CDCl3, 125.7 MHz): δ 170.7, 170.4, 170.2, 170.0,
30 oligosaccharides structures from Streptococcus thermophilus. 35
169.8, 169.4, 169.3 (CH3CO), 93.7 (C-1), 83.8 (C-1’), 74.0 (C-
5’), 72.5 (C-2), 71.8 (C-3’), 71.1 (Me2CHO), 68.4 (C-5), 67.5 (C-
2’), 67.0 (C-4’), 66.8 (C-4), 62.5 (C-6), 61.1 (C-6’), 46.7 (C-3),
23.0, 21.6 [(CH3)2CHO], 20.8 – 20.6 (CH3CO). Found: C, 50.52;
90 H, 6.19; S, 4.40. Calc. for C29H42O17S: C, 50.14; H, 6.09; S,
4.62%.
Experimental
From further fractions from the column compound was isolated
syrupy 9 (0.35 g, 50%); [α] +37.5 (c 1.0 in CHCl3); H RMN
General Methods
1
35
(CDCl3, 500 MHz): δ 5.43 (dd, 1H, J3’,4’ = 3.2, J4’,5’ < 1.0 Hz, H-
95 4’), 5.35 (m, 1H, H-3), 5.34 (d, 1H, J1,2 = 4.2 Hz, H-1), 5.24 (t,
1H, J1’,2’ = J2’,3’ = 10.0 Hz, H-2’), 5.06 (dd, 1H, J2’,3’ = 10.0, J3’,4’
= 3.2 Hz, H-3’), 5.03 (d, 1H, J1,2 = 4.2 Hz, H-2), 4.68 (d, 1H, J1’,2’
= 10.0 Hz, H-1’), 4.65 (m, 1H, H-5), 4.22 – 4.08 (m, 4H, H-6a,
6b, 6’a, 6’b), 3.95 (dd, 1H, J5’,6’a = 6.7, J = 6.5 Hz, H-5’), 3.87
100 (m, 1H, J = 6.2 Hz, Me2CHO), 3.28 (t, 1H, J3,4 = J4,5 = 2.7 Hz, H-
4), 2.16, 2.14, 2.10, 2.07, 2.06, 2.04, 1.98 (7 s, 3H each, CH3CO),
1.25, 1.13 (2 d, 3H each, J = 6.2 Hz, (CH3)2CHO); 13C-RMN
(CDCl3, 50.3 MHz) δ 170.6, 170.5, 170.3 (×2), 170.0, 169.9,
169.8 (CH3CO), 94.4 (C-1), 83.0 (C-1’), 74.7, 71.8, 70.6 (C-3, 3’,
105 5’), 70.4 (Me2CHO), 67.1, 66.9, 65.4, 65.1 (C-2, 5, 2’, 4’), 63.8
(C-6), 61.2 (C-6’), 44.8 (C-4), 23.0, 21.4 [(CH3)2CHO], 21.1 –
20.5 (CH3CO). Found: C, 50.22; H, 6.21; S, 4.57. Calc. for
C29H42O17S: C, 50.14; H, 6.09; S, 4.62%.
Column chromatography was carried out with Silica Gel 60 (230-
400 mesh). Analytical thin layer chromatography (TLC) was
performed on Silica Gel 60 F254 aluminium supported plates
(layer thickness 0.2 mm). Visualization of the spots was effected
40 by exposure to UV light and by charring with a solution of 5 %
(v/v) sulfuric acid in EtOH, containing 0.5% p-anisaldehyde.
Optical rotations were measured at 25 °C in a 1 dm cell in the
solvent indicated. The nuclear magnetic resonance (NMR)
spectra were recorded at 500 MHz (1H) or 125.7 MHz (13C);
45 chemical shifts are relative to tetramethylsilane or a residual
solvent peak (CHCl3: 1H: δ = 7.26 ppm, 13C: δ = 77.2 ppm).
1
1
Assignments of H and 13C were assisted by 2D H–COSY and
1
2D H–13C HSQC experiments. In the description of the NMR
spectra of dithiotrisaccharides or the asymmetric disulfide, the H
50 and C atoms of the S-glycosyl substituent at C-3 of the pyranose
core have been labelled as H’ and C’; whereas those of C-4 of the
pyranose, as H’’ and C’’. High resolution mass spectra (HRMS)
were obtained by Electrospray Ionization (ESI) and Q-TOF
detection.
110 2-Propyl
2,4,6-tri-O-acetyl-4-S-(2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyl)-4-thio-α-D-glucopyranoside (10), 2-propyl
2,4,6-tri-O-acetyl-3-S-(2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyl)-3-thio-α-D-gulopyranoside (11) and 2-
55
propyl
2,4,6-tri-O-acetyl-2-S-(2,3,4,6-tetra-O-acetyl-β-D-
2-Propyl
2,4,6-tri-O-acetyl-3-S-(2,3,4,6-tetra-O-acetyl-β-D-
6
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 7 of 11
Organic & Biomolecular Chemistry
galactopyranosyl)-2-thio-α-D-idopyranoside (12).
2-Propyl
2,6-di-O-acetyl-3,4-anhydro--D-galactopyranoside21
J2’,3’ = 10.0 Hz, H-2’), 5.19 (dd, 1H, J1,2 = 3.3, J2,3 = 5.5 Hz, H-2),
60 5.06 (t, 1H, J2’,3’ = 10.0, J3’,4’ = 3.4 Hz, H-3’), 5.02 (br dd, 1H,
J3,4 = 3.1, J4,5 = 1.3 Hz, H-4), 4.95 (d, 1H, J1,2 = 3.2 Hz, H-1),
(4, 0.25 g, 0.87 mmol) and the 1-thioaldose 7 (0.38 g, 1.04 mmol)
were dissolved in 2M LiOMe in MeOH (2.6 mL) and the mixture
was stirred under Ar at 65 ºC for 24 h, and then neutralized,
4.59 (ddd, 1H, J4,5 = 1.3, J5,6a = 5.5, J5,6b = 7.0 Hz, H-5), 4.53 (d,
1H, J1’,2’ = 10.0 Hz, H-1’), 4.15 (dd, 1H, J5,6a = 5.5, J6a,6b = 11.4
Hz, H-6a), 4.144.05 (m, 3H, H-6b, 6’a, 6’b), 3.91 (dt, 1H, J4’,5’
View Online
5
concentrated and acetylated as described in the previous item. 65 = 1.0, J5’,6’a = J5’,6’b = 6.6 Hz, H-5’), 3.89 (m, 1H, J = 6.2 Hz,
Monitoring by TLC (3:1 CH2Cl2/EtOAc) reavealed two main
spots (Rf 0.64 and 0.47). The residue was subjected to column
Me2CHO), 3.58 (dd, 1H, J2,3 = 5.4, J3,4 = 3.0 Hz, H-3), 2.13, 2.12,
2.10, 2.06, 2.05, 2.03, 1.97 (7 s, each 3H, CH3CO), 1.23, 1.13 (2
d, each 3H, J = 6.2 Hz, (CH3)2CHO); 13C NMR (CDCl3, 125.7
MHz): δ 170.5, 170.4, 170.3, 170,2, 170.1, 170.0, 169.2
10 chromatography (9:1 CH2Cl2/EtOAc) to afford first the less polar
product, which was identified as 10 (125 mg, 21%); [α] +46.9
1
(c 1.0 in CHCl3); H NMR (CDCl3, 500 MHz): δ 5.44 (dd, 1H, 70 (CH3CO), 93.9 (C-1), 84.0 (C-1’), 74.6 (C-5’), 72.7 (C-4), 72.0
J2,3 = 9.9, J3,4 = 11.1 Hz, H-3), 5.42 (dd, 1H, J3’,4’ = 3.3 Hz, H-
4’), 5.15 (d, 1H, J1,2 = 3.7 Hz, H-1), 5.11 (t, 1H, J1’,2’ = J2’,3’ = 9.9
15 Hz, H-2’), 5.02 (dd, 1H, J2’,3’ = 9.9, J3’,4’ = 3.3 Hz, H-3’), 4.77
(dd, 1H, J1,2 = 3.7, J2,3 = 9.9 Hz, H-2), 4.71 (d, 1H, J1’,2’ = 9.9 Hz,
(C-3’), 70.5 (Me2CHO), 67.3 (C-4’), 66.9 (C-2’), 66.3 (C-2), 62.9
(C-5), 62,8 (C-6), 61.2 (C-6’), 42.5 (C-3), 23.0, 21.3
[(CH3)2CHO], 21.0, 20.9, 20.7 (2), 20.6 (3) (CH3CO). HRMS
(ESI+): m/z found 717.2060 ([M+Na]+); calc. for C29H42NaO17S
H-1’), 4.50 (dd, 1H, J5,6a = 4.1, J6a,6b = 12.0 Hz, H-6a), 4.45 (dd, 75 717.2035;.
1H, J5,6b = 2.1, J6a,6b = 12.0 Hz, H-6b), 4.20 (ddd, 1H, J4,5 = 11.2,
J5,6a = 4.1, J5,6b = 2.1 Hz, H-5), 4.10–4.04 (m, 2H, J5’,6’a = J5’,6’b
=
2-Propyl
3,6-di-O-acetyl-2-O-tert-butyldimethylsilyl-4-S-
20 6.7, J6’a,6’b = 12.5 Hz, H-6’a, 6’b), 3.89 (dt, 1H, J4’,5’ = 0.8, J5’,6’a
= J5’,6’b = 6.7 Hz, H-5’), 3.89 (m, 1H, J = 6.2 Hz, Me2CHO), 2.91
(t, 1H, J3,4 = J4,5 = 11.1 Hz, H-4), 2.15, 2.09, 2.04 (× 4), 1.96 (6 s,
21H, CH3CO), 1.23, 1.12 (2 d, 3H each, J = 6.2 Hz, (CH3)2CHO);
13C NMR (CDCl3, 125.7 MHz): δ 170.5, 170.3, 170.2, 170.1,
25 169.8, 169.6, 169.5 (CH3CO), 94.2 (C-1), 82.6 (C-1’), 74.0 (C-
5’), 72.4 (C-2), 71.8 (C-4’), 71.0 (Me2CHO), 68.8 (C-5), 67.6 (C-
(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-4-thio-α-D-
glucopyranoside (13) and its conversion into 10.
80
Epoxide 5 (0.25 g, 0.70 mmol) and the 1-thioaldose 7 (0.30 g,
0.83 mmol) were dissolved in 0.8M LiOMe in MeOH (2.1 mL)
o
and stirred under Ar at 60 C for 24 h. The reaction mixture was
processed and acetylated as described above for the analogous
3), 66.9, 66.8 (C-2’, 3’), 63.4 (C-6), 61.3 (C-6’), 46.5 (C-4), 23.1, 85 reactions starting from 3 or 4. The resulting crude product
21.4 [(CH3)2CHO], 20.8 – 20.5 (CH3CO). HRMS (ESI+): m/z
found 717.2039 ([M+Na]+); calc. for C29H42NaO17S 717,2035.
30 From further fractions of the column was isolated the component
of Rf 0.47, which although chromatographically homogenous,
showed by TLC (1:1 hexane/EtOAc) two main spots having Rf
0.45 (major) and Rf 0.29. Purification by column chromatography
(5.6:1→2.5:1 hexane/EtOAc) gave first the major product 13 as a
1
syrup (0.27 g, 51%); [α]
+21.3 (c 1.0 in CHCl3); H NMR
was in fact a 1:1 mixture of two thiodisaccharides 11 and 12. 90 (CDCl3, 500 MHz): δ 5.43 (dd, 1H, J3’,4´ = 3.2, J4’,5’ = 0.8 Hz, H-
These two products were partially separated by column
chromatography using 9:1 CH2Cl2/EtOAc as eluent. The first
35 product isolated from the column was the thiodisaccharide 12
4’), 5.35 (dd, 1H, J2,3 = 9.3, J3,4 = 11.2 Hz, H-3), 5.11 (dd, 1H,
J1’,2’ = J2’,3’ = 9.9 Hz, H-2’), 5.04 (dd, 1H, J2’,3’ = 9.9, J3’,4´ = 3.3
Hz, H-3’), 4.87 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.79 (d, 1H, J1’,2’ = 9.9
Hz, H-1’), 4.54 (dd, 1H, J5,6a = 6.5, J6a,6b = 12.9 Hz, H-6a), 4.42
1
(117 mg, 20%); [α]D +31.5 (c 1.7 in CHCl3); H NMR (CDCl3,
500 MHz): δ 5.40 (dd, 1H, J3’,4’ = 3.3, J4’,5’ = 0.9 Hz, H-4’), 5.25 95 (dd, 1H, J5,6b = 1.7, J6a,6b = 12.0 Hz, H-6b), 4.22 (m, 1H, J4,5
=
(dd, 1H, J1’,2’ = J2’,3’ = 10.0 Hz, H-2’), 5.15 (t, 1H, J2,3 ≈ J3,4 = 4.1
Hz, H-3), 5.05 (d, 1H, J1,2 = 2.0, H-1), 5.02 (dd, 1H, J2’,3’ =10.0,
40 J3’,4’ = 3.3 Hz, H-3’), 4.84 (dd, 1H, J3,4 = 3.8, J4,5 = 2.4 Hz, H-4),
4.65 (d, 1H, J1’,2’ = 10.0 Hz, H-1’), 4.46 (ddd, 1H, J4,5 = 2.5, J5,6a
11.2, J5,6a = 6.5, J5,6b = 1.7 Hz, H-5), 4.094.04 (m, 2H, H-6’a,
6’b), 3.91 (dt, 1H, J4’,5’ = 0.8, J5’,6’a = J5’,6’b = 6.6 Hz, H-5’), 3.85
(m, 1H, J = 6.2 Hz, Me2CHO), 3.68 (dd, 1H, J1,2 = 3.6, J2,3 = 9.3
Hz, H-2), 2.79 (t, 1H, J3,4 = J4,5 = 11.2 Hz, H-4), 2.16, 2.10, 2.06
= 7.5, J5,6b = 5.4 Hz, H-5), 4.18 (dd, 1H, J5,6a = 7.5, J6a,6b = 11.5 100 (2), 2.05, 1.96 (6 s, 18H, CH3CO), 1.25, 1.18 (2 d, 3H each, J =
Hz, H-6a), 4.14 (dd, 1H, J5,6a = 5.4, J6a,6b = 11.5 Hz, H-6a),
6.2 Hz, (CH3)2CHO); 0.87 (s, 9H, (CH3)3SiCMe2), 0.06, 0.05 (2
s, 3H each, (CH3)2SiBut); 13C NMR (CDCl3, 125.7 MHz): δ
170.6, 170.2, 169.8, 169.6, 169,5 (×2) (CH3CO), 97.5 (C-1), 82.4
(C-1’), 73.8 (C-5’), 72.5 (C-2), 71.8 (C-3’), 70.9 (Me2CHO), 70.2
4.114.05 (m, 2H, H-6’a, 6’b), 3.96 (dt, 1H, J4’,5’ = 0.9, J5’,6’a
=
45 J5’,6’b = 6.5 Hz, H-5’), 3.89 (m, 1H, J = 6.2 Hz, Me2CHO), 3.28
(dd, 1H, J1,2 = 2.0, J2,3 = 4.1 Hz, H-2), 2.14, 2.09, 2.06, 2,05,
2,03, 2,02, 1,97 (7 s, 21H, CH3CO), 1.21, 1.15 (2d, each 3H, J = 105 (C-3), 68.8 (C-5), 66.9 (C-4’), 66.7 (C-3’), 63.9 (C-6), 61.2 (C-
6.2 Hz, (CH3)2CHO); 13C NMR (CDCl3, 125.7 MHz): δ 170.5,
170.3, 170.2, 170.1, 169.9, 169.6, 169.2 (CH3CO), 98.8 (C-1),
50 84.3 (C-1’), 74.4 (C-5’), 71.9 (C-3’), 69.8 (Me2CHO), 68.9 (C-3),
67.6 (C-4), 67.4 (C-2’), 67.2 (C-4’), 64.1 (C-5), 62.5 (C-6), 61.2
6’), 46.7 (C-4), 25.4 [(CH3)3CSiMe2], 23.3, 21.6 [(CH3)2CHO],
20.7–20.5 (CH3CO), 18.4 [(CH3)3CSiMe2], –4.5, –5.0
[(CH3)2SiBut]. HRMS (ESI+): m/z found 789.2769 ([M+Na]+);
calc. for C33H54NaO16SSi 789.2794.
(C-6’), 43.2 (C-2), 23.2, 21.5 [(CH3)2CHO], 20.9, 20.8, 20.7 (2), 110 The minor product (Rf 0.29) was also obtained as a syrup and
20.6, 20.5 (CH3CO). Found: C, 48.20; H, 6.08. Calc. for
C29H42O17S+H2O: C, 48.87; H, 6.22%. HRMS (ESI+): m/z found
55 717.2064 ([M+Na]+); calc. for C29H42NaO17S 717.2035.
identified as 10 (160 mg, 33%), which showed the same
properties as the product obtained from the oxirane 4.
Alternatively, reaction of 5 (60 mg, 0.19 mmol) and 7 (83 mg,
0.23 mmol), under the conditions employed above, led to a crude
Further fractions from the column led to 11 (116 mg, 20%);
1
[α]
+37.6 (c 1.0 in CHCl3); H NMR (CDCl3, 500 MHz): δ 115 mixture that was subjected to O-desilylation with 1 M TBAF in
5.40 (dd, 1H, J3’,4’ = 3.4, J4’,5’ = 1.0 Hz, H-4’), 5.22 (t, 1H, J1’,2’
=
THF (0.25 mL), followed by acetylation. Purification by column
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 7

Organic & Biomolecular Chemistry
Page 8 of 11
chromatography
(3:12:1
hexane/EtOAc)
led
to
J2’,3’ = 10.0 Hz, H-2’), 5.02 (d, 1H, J1,2 = 3.6 Hz, H-1), 5.00 (dd,
thiodisaccharides 10 (110 mg, 85%) as the only product isolated.
60 1H, J2’,3’ = 10.0, J3’,4’ = 3.4 Hz, H-3’), 4.83 (dd, 1H, J1,2 = 3.6, J2,3
= 10.8 Hz, H-2), 4.61 (d, 1H, J1’,2’ = 10.0 Hz, H-1’), 4.51 (dd, 1H,
J5,6a = 1.9, J6a,6b = 11.8 Hz, H-6a), 4.34 (dd, 1H, J5,6b = 5.5, J6a,6b
= 11.8 Hz, H-6b), 4.13 (ddd, J4,5 = 10.8, J5,6a = 1.9, J5,6b = 5.5
Hz, H-5), 4.06 (dd, 1H, J5’,6’a = 6.4, J6’a,6’b = 11.2 Hz, H-6’a), 4.01
2-Propyl
2,6-di-O-acetyl-4-S-(2,3,4,6-tetra-O-acetyl-β-D-
View Online
5
galactopyranosyl)-3,4-dithio-α-D-glucopyranoside (15), 2-
propyl
2,6-di-O-acetyl-4-S-(2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyl)-
3-S-(2,3,4,6-tetra-O-acetyl-β-D- 65 (dd, 1H, J5’,6’b = 7.2, J6’a,6’b = 11.2 Hz, H-6’b), 3.67 (ddd, 1H,
galactopyranosyl)-disulfide-3,4-dithio-α-D-glucopyranoside
(16) and bis[2-propyl 2,6-di-O-acetyl-4-S-(2,3,4,6-tetra-O-
10 acetyl-β-D-galactopyranosyl)-3,4-dithio-α-D-glucopyranosid-3-
J4’,5’ = 1.0, J5’,6’a = 6.4, J5’,6’b = 7.2 Hz, H-5’), 3.87 (m, 1H, J =
6.2 Hz, Me2CHO), 3.15 (dd, 1H, J2,3 = 10.8, J3,4 = 11.3 Hz, H-3),
2.90 (dd, 1H, J3,4 = 11.3, J4,5 = 10.8 Hz, H-4), 2.10, 2.09, 2.06,
2.04, 1.98, 1.91 (6 s, 18H, CH3CO), 1.19, 1.07 (2 d, each 3H, J =
70 6.2 Hz, (CH3)2CHO); 13C NMR (CDCl3, 50.3 MHz): δ 170.5–
169.5 (CH3CO), 93.9 (C-1), 82.7 (C-1’), 74.2 (C-5’), 74.8 (C-3’),
74.3 (C-2), 70.8 (Me2CHO), 69.7 (C-5), 67.1 (C-2’), 66.9 (C-4’),
64.3 (C-6), 61.1 (C-6’), 50.9 (C-3), 46.8 (C-4), 23.2, 21.6
[(CH3)2CHO], 20,8 – 20,6 (CH3CO); Found: C, 48.32; H, 5.87.
yl]-disulfide (17)
To a solution of 7 (175 mg, 0.49 mmol) in anhydrous THF (20
mL) was added NaH (24 mg, 0.72 mmol) under N2. The mixture
15 was stirred at room temperature until evolution of gases ceased
(~1 h). The solvent was evaporated and the resulting salt 14 was
dissolved in THF (5 mL). To this solution, cooled at –18 oC 75 Calc. for C54H78O30S4: C, 48.57; H, 5.89%. HRMS (ESI+): m/z
(ice/salt bath) a solution of 2-propyl 3,6-di-O-acetyl-3,4-epithio-
-D-galactopyranoside21 (6, 100 mg, 0.32 mmol) in THF (1 mL)
20 was added. The mixture was stirred at –18 oC for 10 minutes and,
upon addition of 18-crown-6 (10 mg, 0.038 mmol), Ar was
found 1357.3355 ([M+Na]+); calc. for C54H78NaO30S4 1357.3353.
2-Propyl 2,6-di-O-acetyl-3,4-di-S-(2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyl)-3,4-dithio-α-D-glucopyranoside (20) and
bubbled into the solution and the stirring was continued for 30 80 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl
2,3,4,6-tetra-O-
min, when an additional portion of 18-crown-6 (10 mg) was
added. The solution was allowed to reach room temperature until
25 monitoring by TLC (1:1 toluene/EtOAc) revealed the complete
consumption of the starting 6 (Rf 0.72). The mixture was
acetyl-β-D-galactopyranoside (21)
Compound 15 (36 mg, 0.054 mmol) and the trichloroacetimidate
1941 (53 mg, 0.107 mmol) were mixed with recently activated
concentrated and the residue was subjected to column 85 molecular sieves (4 Å) in CH2Cl2 (1.3 mL). The mixture was
chromatography (hexane/EtOAc 4:11.5:1), to afford the
stirred at room temperature for 90 min. Then, the suspension was
cooled to –18 C and TMSOTf (2.5 L, 13.5 mol) was added
o
thiodisaccharide 15 (11 mg, 5%).22
30 Further fractions of the column afforded a mixture of compounds
16 and 17 (0.12 g). The less polar product was the major
and stirring was continued until TLC (CH2Cl2/EtOAc 3:1)
showed the conversion of 15 (Rf 0.71) and 19 (Rf 0.63) into two
component of the mixture, which was identified as 16 (106 mg, 90 lower moving products (Rf 0.37 y 0.26). After addition of Et3N,
32%); [α]
MHz): δ 5.46 (dd, 1H, J3’’,4’’ = 3.4, J3’’,5’’ = 1.1 Hz, H-4’’), 5.45
35 (dd, 1H, J3’,4’ = 3.5, J4’,5’ = 1.8 Hz, 4’), 5.17 (t, 1H, J1’, 2’ = J 2’, 3’
10.0 Hz, H-2’), 5.16 (t, 1H, J1’’,2’’ = J2’’,3’’ = 10.1 Hz, H-2’’), 5.10
(d, 1H, J1, 2 = 3.3 Hz, H-1), 5.09 (dd, 1H, J2’’,3’’ = 10.0, J4’’,3’’
3.3 Hz, H-3’’), 5.07 (dd, 1H, J2’,3’ = 10.0, J3’,4’ = 3.4 Hz, H-3’),
4.90 (dd, 1H, J1,2 = 3.6, J2,3 = 10.5 Hz, H-2), 4.62 (dd, 1H, J5,6a
+11.3 (c 1.0 in CHCl3); 1H NMR (CDCl3, 500
the solution was concentrated and the residue was purified by
column chromatography (CH2Cl2/EtOAc 9:1 → 4:1). The first
product which eluted from the column was identified as 2,3,4,6-
tetra-O-acetyl-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-1-β-
95 D-galactopyranoside (21, 20 mg), which showed identical
spectroscopic and physical data as those described in the
literature.27
=
=
=
40 2.1, J6a,6b = 12.0 Hz, H-6a), 4.60 (d, 1H, J1’,2’ = 10.0 Hz, H-1’),
4.57 (d, 1H, J1’’,2’’ = 10.2 Hz, H-1’’), 4.53 (dd, 1H, J5,6b = 4.2,
J6a,6b = 12.0 Hz, H-6b), 4.24 (m, 2H, H-5,6’a), 4.12 (m, 4H, H- 100 CHCl3); H NMR (CDCl3, 500 MHz): δ 5.43 (dd, 1H, J3’’,4’’
The more polar compound was characterized as the
dithiotrisaccharide 20 (20 mg, 35%); [α]
+9.5 (c 1.0 in
1
=
6’a, 6’b, 6’’a, 6’’b), 3.98 (ddd, 1H, J4’,5’ = 2.1, J5’,6’a = 5.6 , J5’,6’b
3.4, J4’’,5’’ = 1.0 Hz, H-4’’), 5.42 (dd, 1H, J3’,4’ = 3.4, J4’,5’ = 1.0
Hz, H-4’), 5.16 (t, 1H, J1’, 2’ = J2’, 3’ = 10.0 Hz, H-2’), 5.12 (t, 1H,
1’’, 2’’ = J2’’, 3’’ = 10.1 Hz, H-2’’), 5.11 (d, 1H, J1,2 = 3.6 Hz, H-1),
5.07 (dd, 1H, J2’, 3’ = 10.0, J3’, 4’ = 3.4 Hz, H-3’), 5.04 (dd, 1H,
2.20, 2.16, 2.15, 2.12, 2.10, 2.08, 2.07, 2.02, 2.01, 2.00 (11 s, 105 J2’’, 3’’ = 10.1, J3’’, 4’’ = 3.4 Hz, H-3’’), 4.81 (dd, 1H, J1,2 =11.1, J2,3
= 8.4 Hz, H-5’), 3.92 (m, 1H, J = 6.2 Hz, Me2CHO), 3.88 (dt, 1H,
45 J4’’,5’’ = 1.0, J5’’,6’’a = J5’’,6’’b = 6.1 Hz, H-5’’), 3.32 (t, 1H, J3,4
=
J
J4,5 = 11.1 Hz, H-4), 3.24 (t, 1H, J2,3 = J3,4 = 11.6 Hz, H-3), 2.21,
33H, CH3CO), 1.28, 1.18 (2 d, 3H each, J = 6.2 Hz, (CH3)2CHO);
13C NMR (CDCl3, 125.7 MHz): δ 170.2 – 169.3 (CH3CO), 94.2
50 (C-1), 88.5 (C-1’’), 81.5 (C-1’), 74.5, 74.3 (C-5’, 5’’), 71.8, 71.6
(C-3’, 3’’), 70.7 (Me2CHO), 69.9 (C-2), 69.1 (C-5), 67.6, 67.0,
= 3.6 Hz, H-2), 4.73 (d, 1H, J1’’,2’’ = 10.1 Hz, H-1’’), 4.69 (d, 1H,
J1’,2’ = 10.0 Hz, H-1’), 4.60 (dd, 1H, J5,6a = 4.4, J6a,6b = 11.9 Hz,
H-6a), 4.47 (dd, 1H, J5,6b = 2.0, J6a,6b = 11.9 Hz, H-6b), 4.22 (ddd,
1H, J4,5 = 10.7, J5,6a = 4.4, J5,6b = 2.0 Hz, H-5), 4.16 (dd, 1H,
66.9, 66.7 (C-2’, 2’’, 4’, 4’’), 63.8 (C-6), 61.5, 60.1 (C-6’, 6’’), 110 J5’,6’a = 7.2, J6’a,6’b = 11.2 Hz, H-6’a), 4.15-4.05 (m, 3H, H-6’b, H-
48.9 (C-3), 44.6 (C-4), 23.2, 21.6 [(CH3)2CHO], 21.0 – 20.6
(CH3CO). HRMS (ESI+): m/z found 1053.2381 ([M+Na]+); calc.
55 for C41H58NaO24S3 1053.2378.
6’’a, H-6’’b), 3.97 (ddd, J4’,5’ = 1.0, J5’,6’a = J5’,6’b = 7.2 Hz, H-5’),
3.89 (m, 2H, H-5’’, Me2CHO), 3.39 (dd, 1H, J2,3 =11.0, J3,4
12.0Hz, H-3), 2.81 (dd, 1H, J3,4 =12.0, J4,5 = 10.7 Hz, H-4), 2.18,
=
The other component isolated from the mixture was 17 (14 mg,
2.15, 2.14, 2.13, 2.12, 2.07, 2.06, 2.05, 1.98, 1.97 (10 s, 30H,
1
3%); [α] 19,3 (c 0,5, CHCl3); H NMR (CDCl3, 500 MHz): δ 115 CH3CO), 1.26, 1.15 (2 d, 3H each, J = 6.2 Hz, (CH3)2CHO); 13C
5.37 (dd, 1H, J3’,4’ = 3.4, J4’, 5’ = 1.0 Hz, H-4’), 5.10 (t, 1H, J1’,2’
=
NMR (CDCl3, 125.7 MHz): δ 170.4 – 169.5 (CH3CO), 93.8 (C-
8
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 9 of 11
Organic & Biomolecular Chemistry
1), 82.4 (C-1’’), 81.1 (C-1’), 74.4 (C-5’), 74.1 (C-5’’), 73.4 (C-2), 60 (CH3)2CHO); 13C RMN (D2O, 125.7 MHz): δ 97.2 (C-1), 85.6
71.7, 71.6 (C-3’, 3’’), 70.8 (Me2CHO), 69.5 (C-5), 67.0, 66.9,
66.6 (C-2’’, 4’, 4’’), 66.6 (C-2’), 64.2 (C-6), 61.4, 61.0 (C-6’,
6’’), 45.3 (C-4), 44.1 (C-3), 23.2, 21.5 [(CH3)2CHO], 20.9 – 20.5
(CH3CO). Found: C, 49.29; H, 5.79. Calc. for C41H58O24S2: C,
(C-1’), 79.1 (C-5’), 73.8 (C-3’), 72.0 (Me2CHO), 71.8 (C-3), 69.7
(C-2’), 68.7 (C-4), 66.3 (C-5), 64.4 (C-2), 62.2 (C-6), 61.1 (C-6’),
47.2 (C-4), 22.4, 20.6 [(CH3)2CHO]. Found: C, 44.87; H, 7.38.
View Online
5
Calc. for C15H28O10S: C, 44.99; H, 7.05%. HRMS (ESI+): m/z
49.29; H, 5.85%. HRMS (ESI+): m/z found 1021.2693 65 found 423.1297 ([M+Na]+); calc. for C15H28NaO10S 423.1295.
([M+Na]+); calc. for C41H58NaO24S2 1021.2651.
2-Propyl
4-S-(β-D-galactopyranosyl)-4-thio-α-D-
General procedure for the O-deacylation of the
10 thiooligosaccharides
glucopyranoside (26)
70 Deacetylation of 10 (50 mg, 0.072 mmol) was performed in the
The O-acylated thiodisaccharides 8, 9, 10 or 15 or the
dithiotrisaccharides 20 and 23 were suspended in
MeOH/H2O/Et3N 4:5:1 (10 mL) was stirred at room temperature
15 for 2 h. The mixture was concentrated and the residue, dissolved
in water (1 mL) was eluted through a column filled with Dowex
MR-3C mixed bed ion-exchange resin. The deionized solutions
were concentrated and the free thiooligosaccharides were purified
by dissolution in water (1 mL) and filtration through an octadecyl
20 C18 minicolumn (Amprep, Amersham Biosciences). Evaporation
of the solvent afforded the fully deprotected thiooligosaccharides,
which monitored by TLC (2,5:1:1 n-BuOH/EtOH/H2O) exhibited
a single spot (Rf values are reported for each compound).
same conditions describe above, to afford 26 (27 mg, 93%); Rf
1
0,57; [α]
+59.5 (c 0.8 in H2O); H RMN (D2O, 500 MHz): δ
5.07 (d, 1H, J1,2 = 3.9 Hz, H-1), 4.58 (d, 1H, J1’,2’ = 9.7 Hz, H-1’),
4.10 – 3.90 (m, 4H, H-5, 6’a, 6´b, Me2CHO), 3.77 (t, 1H, J2,3
=
75 J3,4 = 9.7 Hz, H-3), 3.75 – 3.62 (m, 6H, H-3, 6a, 6b, 3’, 4’, 5’),
3.57 (dd, J1,2 = 3.9, J2,3 = 9.7 Hz, H-2), 3.56 (t, 1H, J1’,2’ = J2’,3’
= 9.7, H-2’), 2.84 (t, 1H, J3,4 = J4,5 = 10.8 Hz, H-4), 1.23, 1.17 (2
d, 3H each, J = 6.3 Hz, (CH3)2CHO); 13C RMN (D2O, 125.7
MHz): δ 96.3 (C-1), 84.2 (C-1’), 79.1, 73.7, 72.3, 72.0, 70.7,
80 69.8, 69.7, 68.7 (C-2, 3, 5, 2’, 3’, 4’, 5’, Me2CHO), 61.4 (C-6),
61.2 (C-6’), 47.4 (C-4), 22.4, 20.4 [(CH3)2CHO]. HRMS (ESI+):
m/z found 423.1303 ([M+Na]+); calc. for C15H28NaO10S
423.1295.
25 2-Propyl
3-S-(β-D-galactopyranosyl)-3-thio-α-D-
glucopyranoside (24)
85 bis[2-Propyl
4-S-(β-D-galactopyranosyl)-3,4-dithio-α-D-
galactopyranosid-3-yl]-disulfide (27)
The general procedure for the O-deacylation applied to
compound 8 (0.36 g, 0.52 mmol) afforded 24 (192 mg, 92%); Rf
30 0.58; [α] +80.7 (c 1.04 in H2O); H RMN (D2O, 500 MHz): δ
Deacetylation of 15 (50 mg, 0.07 mmol) gave 27 (28 mg, 89%);
Rf 0.48; [α] +14.1 (c 1.7 in MeOH); 1H NMR (D2O, 500 MHz):
90 δ 5.01 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.57 (d, 1H, J1’,2’ = 9.8 Hz, H-
1’), 4.05 – 3.84 (m, 6H, H-5, 6a, 6b, Me2CHO, 4’, 5’), 3.73 (dd,
1H, J1,2 = 3.6, J2,3 = 10.1 Hz, H-2), 3.70 – 3.54 (m, 3H, H-3’, 6’a,
6’b), 3.47 (t, 1H, J1’,2’ = J2’,3’ = 9.6 Hz, H-2’), 3.13 – 3.02 (m, 2H,
H-3, 4), 1.16, 1.11 (2 d, 3H each, J = 6.2 Hz, (CH3)2CHO); 13C
95 NMR (D2O, 125.7 MHz): δ 96.2 (C-1), 83.8 (C-1’), 78.9, 73.8,
70.9, 69.8, 69.6, 69.58, 68.5 (C-2, 5, 2’, 3’, 4’, 5’, Me2CHO),
62.0 (C-6), 60.9 (C-6’), 46.6, 45.2 (C-3, 4), 22.5, 20.6
[(CH3)2CHO]. HRMS (ESI+): m/z found 853.2112 ([M+Na]+);
calc. for C30H54NaO18S4 853.2091.
1
4.92 (d, 1H, J1,2 = 3.5 Hz, H-1), 4.51 (d, 1H J1’,2’ = 9.6 Hz, H-1’),
3.89 (m, 1H, J = 6.2 Hz, Me2CHO), 3.86 (d, 1H, J3’,4’ = 3.0 Hz,
H-4’), 3.75 (dd, 1H, J6a,6b = 10.3 Hz, H-6a), 3.69 – 3.59 (m, 5H,
H-5, 5’, 6b, 6’a, 6’b), 3.56 (dd, 1H, J1,2 = 3.5, J2,3 = 10.7 Hz, H-
35 2), 3.54 (dd, 1H, J2’,3’ = 9.4, J3’,4’ = 3.0 Hz, H-3’), 3.53 (t, 1H,
J1’,2’ = J2’,3’ = 9.5 Hz, H-2’), 3.32 (t, 1H, J3,4 = J4,5 = 8.9 Hz, H-4),
3,01 (t, 1H, J2,3 = J3,4 = 10.7 Hz, H-3), 1.16, 1.08 (2 d, 3H each, J
= 6.2 Hz, (CH3)2CHO); 13C RMN (D2O, 125.7 MHz): δ 95.5 (C-
1), 85.4 (C-1’), 79.1 (C-5 ó 5’), 74.2 (C-3’), 72.8 (C-5 ó 5’), 70.3
40 (C-2), 70.2 (Me2CHO), 70.0 (C-2’), 68.9 (C-4’), 67.7 (C-4), 61.1
× 2 (C-6, C-6’), 51.9 (C-3), 22.3, 20.4 [(CH3)2CHO]. Found: C,
44.98; H, 7.29. Calc. for C15H28O10S: C, 44.99%; H, 7.05. HRMS
(ESI+): m/z found 423.1301 ([M+Na]+); calc. for C15H28NaO10S
423.1295.
100
2-Propyl
4-S-(β-D-galactopyranosyl)-3,4-dithio-α-D-
glucopyranoside (28)
45
Compound 27 (85 mg, 0.10 mmol) was treated with tris(2-
2-Propyl
4-S-(β-D-galactopyranosyl)-4-thio-α-D-
105 carboxyethyl)phosphine hydrochloride (TCEP, 300 mg, 1.0
mmol) in H2O (4.3 mL) at rt for 1 h, when TLC analysis (2.5:1:1
n-BuOH/EtOH/H2O) showed the complete conversion of 27 into
a faster moving spot of Rf 0.64. The solution was passed through
a column filled with Dowex MR-3C mixed bed ion-exchange
110 resin and the resulting deionized solution was concentrated. The
residue was dissolved in water (1 mL) and eluted through an
octadecyl C18 minicolumn. Upon evaporation of water was
obtained 28 (38 mg, 92%); 1H NMR (D2O, 500 MHz): δ 4.96 (d,
1H, J1,2 = 3.6 Hz, H-1), 4.50 (d, 1H, J1’,2’ = 9.8 Hz, H-1’), 3.98 –
115 3.58 (m, 9H, H-5, 6a, 6b, Me2CHO, 3’, 4’, 5’, 6’a, 6’b), 3.56 (dd,
1H, J1,2 = 3.6, J2,3 = 10.6 Hz, H-2), 3.49 (t, 1H, J1’,2’ = J2’,3’ = 9.6
Hz, H-2’), 3.29 (t, 1H, J2,3 = J3,4 = 11.1 Hz, H-3), 2.85 (t, 1H, J3,4
gulopyranoside (25)
Deacetylation of 9 (0.13 g, 0.19 mmol) gave syrupy 25 (72 mg,
50 95%); Rf 0,55; [α]
MHz): δ 4.88 (d, 1H, J1,2 = 4.0 Hz, H-1), 4.41 (d, 1H, J1’,2’ = 9.8
Hz, H-1’), 4.36 (ddd, 1H, J4,5 = 2.1, J5,6a = 5.1, J5,6b = 7.1 Hz , H-
5), 4.01 (t, 1H, J2,3 = J3,4 = 3.6 Hz, H-3), 3.95 (t, 1H, J1,2 = J2,3
3.8 Hz, H-2), 3.82 (d, 1H, J3’,4’ = 2.8 Hz, H-4’), 3.82 (m, 1H, J =
55 6.3 Hz, Me2CHO), 3,69 (dd, 1H, J5,6a = 5.1, J6a,6b = 12.0 Hz, H-
6a), 3.64 (dd, 1H, J5,6b = 7.1, J6a,6b = 12.1 Hz , H-6b), 3.59 – 3.54
(m, 3H, H-5’, 6’a, 6’b), 3.49 (dd, 1H, J2’,3’ = 9.5, J3’,4’ = 3.3 Hz,
H-3’), 3.40 (t, 1H, J1’,2’ = J2’,3’ = 9.6, H-2’), 3.21 (dd, 1H, J3,4
3.0, J4,5 = 2.6 Hz, H-4), 1.11, 1.04 (2 d, 3H each, J = 6.2 Hz,
1
+51.7 (c 1.1 in H2O); H RMN (D2O, 500
=
=
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 9

Organic & Biomolecular Chemistry
Page 10 of 11
= J4,5 = 11.1 Hz, H-4), 1.17, 1.11 (2 d, 3H each, J = 6.2 Hz, 60 0.2 M aqueous sodium borate buffer (4.0 mL, pH 10.0). The
(CH3)2CHO); 13C NMR (D2O, 125.7 MHz): δ 95.9 (C-1), 83.7
(C-1’), 78.9, 73.8, 73.1, 72.5, 70.8, 69.6, 68.6 (C-2, 5, 2’, 3’, 4’,
5’, Me2CHO), 61.9 (C-6), 61.0 (C-6’), 48.8 (C-4), 43.0 (C-3),
22.4, 20.6 [(CH3)2CHO]. HRMS (ESI+): m/z found 439.1059
([M+Na]+); calc. for C15H28NaO9S2 439.1067.
concentration of the released o-nitrophenol ( 21300) was
measured by visible absorption spectroscopy at 410 nm. The Ki
and Km values were determined from de Lineweaver-Burk plot.
View Online
5
Acknowledgments
65 We are indebted to the University of Buenos Aires (Project
UBACyT 01/W526, 2011-2014), the National Research Council
of República Argentina (CONICET, Project PIP 0064) and the
National Agency for Promotion of Science and Technology
(ANPCyT, Project PICT 2007-00291) for financial support. O. V.
70 and M. L. U. are Research Members of CONICET. We thank
Alejandro J. Cagnoni for conducting part of the kinetics.
2-Propyl
glucopyranoside (29)
3,4-di-S-(β-D-galactopyranosyl)-3,4-dithio-α-D-
10
Dithiotrisaccharide 20 was deacetylated under the conditions
described above, to afford free 29 (22 mg, 95%), Rf 0.45; [α]
+18.1 (c 0.8 in H2O); 1H NMR (D2O, 500 MHz): δ 4.98 (d, 1H,
J1,2 = 3.7 Hz, H-1), 4.62 (d, 1H, J1’,2’ = 9.6 Hz, H-1’), 4.51 (d, 1H,
15 J1’’,2’’ = 9.6 Hz, H-1’’), 4.01 – 3.84 y 3.67 – 3.53 (m, 12H, H-5,
6a, 6b, 3’, 4’, 5’, 6’a, 6’b, 3’’, 4’’, 5’’, 6’’a, 6’’b), 3.75 (dd, 1H,
J1,2 = 3.6, J2,3 = 10.6 Hz, H-2), 3.51 (t, 1H, J1’,2’ = J2’,3’ = 9.6, H-
Notes and references
2’), 3.50 (t, 1H, J1’’,2’’ = J2’’,3’’ = 9.6, H-2’’), 3.19 (dd, 1H, J2,3
10.7, J3,4 = 11.8 Hz, H-3), 2.93 (dd, 1H, J3,4 = 11.7, J4,5 = 11.2
20 Hz, H-4), 1.15, 1.09 (2 d, 3H each, J = 6.2 Hz, (CH3)2CHO); 13
NMR (D2O, 125.7 MHz): δ 95.7 (C-1), 84.0, 83.6 (C-1’,1’’),
78.9, 78.8, 73.7, 72.7, 71.8, 70.6, 69.9, 69.7, 68.7, 68.6 (C-2, 5,
2’, 3’, 4’, 5’, 2’’, 3’’, 4’’, 5’’, Me2CHO), 62.0 (C-6) , 61.1, 61.0
(C-6’, 6’’), 47.9, 44.0 (C-3, 4) 22.4, 20.4 [(CH3)2CHO]. HRMS
25 (ESI+): m/z found 601.1580 ([M+Na]+); calc. for C21H38NaO14S2
601.1595.
=
CIHIDECAR-CONICET-UBA, Departamento de Química Orgánica,
75 FCEN, Universidad de Buenos Aires, Pabellón II, Ciudad Universitaria,
1428-Buenos Aires, Argentina. Tel/Fax: +005411 4576 3352; E-mail:
varela@qo.fcen.uba.ar
C
† Electronic Supplementary Information (ESI) available: 1H and 13C
NMR spectra for the thiodisaccharides 8-13, 24-26, 28; disulfides 16, 17,
80 27 and dithiotrisaccharides 20, 29, 30. See DOI: 10.1039/b000000x/
1
2
3
(a) H. Yuasa, M. Izumi and H. Hashimoto, Curr. Topics Med. Chem.,
2009, 9, 76–86; (b) Y. W. Kim, H. M. Chen, J. H. Kim, J. Mullegger,
D. Mahuran, and S. G. Withers, Chembiochem., 2007, 8, 1495–1499;
(c) Z. J. Witczak and J. M. Culhane, Appl. Microbiol. Biotechnol.,
2005, 69, 237–244; (d) P. M. Dey and Z. J. Witczak, Minirev. Med.
Chem., 2003, 3, 271–280.
(a) T. M. Gloster and G. J. Davies, Org. Biomol. Chem., 2010, 8,
305–320; (b) D. C. Koester, A. Holkenbrink and D. B. Werz,
Syntesis, 2010, 3217-3242; (c) T. D. Butters, R. A. Dwek and F. M.
Platt, Curr. Top. Med. Chem., 2003, 3, 561–574; (d) N. Asano, R. J.
Nash, R. J. Molyneux and G. W. J. Fleet, Tetrahedron: Asymmetry,
2000, 11, 1645–1680.
85
90
2-Propyl
3-S-(β-D-galactofuranosyl)-4-S-(β-D-
galactopyranosyl)-3,4-dithio-α-D-glucopyranoside (30)
30
Compound 23 (100 mg, 0.08 mmol) was deacetylated following
the general procedure to afford 30 (40 mg, 83%); Rf 0.57 (n-
BuOH/EtOH/H2O 2.5:1:1); [α] +12.1 (c 1.4 in MeOH); 1H
NMR (D2O, 500 MHz): δ 5.34 (d, 1H, J1’,2’ = 3.7 Hz, H-1’), 4.95
35 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.48 (d, 1H, J1’’,2’’ = 9.6 Hz, H-1’’),
4.00–3.48 (m, 17H, H-2, 5, 6a, 6b, Me2CHO, 2’, 3’, 4’, 5’, 6’a,
(a) H. Driguez, Chembiochem., 2001, 2, 311–318; (b) H. Driguez,
Top. Curr. Chem., 1997, 187, 85–116.
95 4 (a) X. Wen, Y. Yuan, D. A. Kuntz, D. R. Rose and B. M. Pinto,
Biochemistry, 2005, 44, 6729–6737; (b) M. R. Wormald, A. J.
Petrescu, Y. -L. Pao, A. Glithero, T. Elliott and R. A. Dwek, Chem.
Rev., 2002, 102, 371–386; (c) G. E. Ritchie, B. E. Moffatt, R. B. Sim,
B. P. Morgan, R. A. Dwek and P. M. Rudd, Chem. Rev., 2002, 102,
6’b, 2’’, 3’’, 4’’, 5’’, 6’’a, 6’’b), 3.05 (dd, 1H, J2,3 = 10.5, J3,4
=
12.0 Hz, H-3), 2.96 (dd, 1H, J3,4 = 12.0, J4,5 = 10.6 Hz, H-4),
1.11, 1.07 (2 d, 3H each, J = 6.3 Hz, (CH3)2CHO); 13C NMR
40 (D2O, 125.7 MHz): δ 95.7 (C-1), 87.5 (C-1’), 83.9 (C-1’’), 81.8,
81.7 (C-2’,4’), 79.0 (C-3’), 76.5 (C-5’), 73.8, 73.0, 72.2, 70.6,
70.5, 69.7, 68.7 (C-2, 5, 2’’, 3’’, 4’’, 5’’, Me2CHO), 62.8 (C-6),
62.1, 61.0 (C-6’, 6’’), 48.5 (C-3), 43.4 (C-4), 22.4, 20.6
[(CH3)2CHO]. Found: C, 43.21; H, 6.42. Calc. for C21H38O14S2:
45 C, 43.59; H, 6.62%. HRMS (ESI+): m/z found 601.1589
([M+Na]+); calc. for C21H38NaO14S2 601.1595.
100
305–319; (d) H. Yuasa, C. Saotome and O. Kanie, Trends Glycosci.
Glycotech., 2002, 14, 231–254.
5
(a) A. J. Krentz and C. J.; Bailey, Drugs, 2005, 65, 385–411; (b) B.
Göke and C. Herrmann-Rinke, Diabetes Metab. Rev., 1998, 14, S31–
S38.
105 6 (a) G. H.-F. Yam, N. Bosshard, C. Zuber, B. Steinmann and J. Roth,
Am. J. Physiol.: Cell Physiol., 2006, 290, C1076–C1082; (b) A. R.
Sawkar, W.-C. Cheng, E. Beutler, C. -H. Wong, W. E. Balch and J.
W.; Kelly, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 15428–15433;
(c) J.-Q. Fan, S. Ishii, N. Asano and Y. Suzuki, Nat. Med., 1999, 5,
110
112–115.
Enzymatic Assay. Inhibition of the -galactosidase from E.
coli
7
A. Mehta, N. Zitzmann, P. M. Rudd, T. M. Block and R. A. Dwek,
FEBS Lett., 1998, 430, 17–22.
50
8 (a) M. von Itzstein, Nat. Rev. Drug Discovery, 2007, 6, 967–974; (b) J.
Zhang and W. Xu, Mini-Rev. Med. Chem., 2006, 6, 428–448; (c) E.
De Clerq, Nat. Rev. Drug Discovery, 2006, 59, 521–549; (d) N.
Asano, Glycobiology, 2003, 13, 93R-104R.
The E. coli -galactosidase (EC 3.2.1.23, grade VIII, >500 U/mg
protein) was purchased from Sigma. The enzyme (0.3 U; 1U = 1
enzyme unit hydrolyses 1 μmol of the nitrophenyl glycoside per
minute) was incubated with o-nitrophenyl -D-galactopyranoside
55 (concentration range: 0.4 to 2.5 mM) in sodium phosphate buffer
(100 mM, pH 7.3, MgCl2 1.2 mM, 2-mercaptoethanol 100 mM)
in the absence (control) or presence of the thioglycomimetics
(concentrations from 0.05 to 5.0 mM); the final volume was 0.50
mL. After 10 min at 37 ºC, the reaction was quenched by adding
115
9
M.-J. Papandréou, R. Barbouche, R. Guieu, M. P. Kieny and E.
Fenouillet, Mol. Pharmacol., 2002, 61, 186–193.
10 H. Laube, Clin. Drug Invest., 2002, 22, 141-156.
120 11 A. Moscona, New Engl. J. Med., 2005, 353, 1363-1373.
12 (a) S. Gerber-Lemaire and L. Juillerat-Jeanneret, Mini-Rev. Med.
Chem., 2006, 6, 1043–1052; (b) H. Paulsen and I. Brockhausen,
10 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 11 of 11
Organic & Biomolecular Chemistry
Glycoconjugate J., 2001, 18, 867–870; (c) P. E. Goss, M. A. Baker, J.
P. Caarver and J. W. Dennis, Clin. Cancer Res., 1995, 1, 935–944.
13 K. Pachamuthu and R. R. Schmidt, Chem. Rev., 2006, 106, 160–187.
14 L. Szilágyi and O. Varela, Curr. Org. Chem., 2006, 10, 1745–1770.
5 15 (a) D. -J. Namdjou, H. -M. Chen, E. Vinogradov, S. G. Withers and
W. W. Wakarchuk, ChemBioChem, 2008, 9, 1632–1640; (b) Y. -W.
Kim, H. -M. Chen, J. H. Kim, J. Müllegger, D. Mahuran, S. G.
Withers, ChemBioChem, 2007, 8, 1495–1499.
View Online
16 M. L. Uhrig, V. E. Manzano and O. Varela, Eur. J. Org. Chem.,
10
2006, 162–168.
17 A. J. Cagnoni, M. L. Uhrig and O. Varela, Bioorg. Med. Chem.,
2009, 17, 6203–6212.
18 E. Repetto, C. Marino, M. L. Uhrig and O. Varela, Eur. J. Org.
Chem., 2008, 3, 540–547.
15 19 E. Repetto, C. M. Marino, M. L. Uhrig and O. Varela, Bioorg. Med.
Chem., 2009, 17, 2703–2711.
20 M. L. Uhrig, L. Szilágyi, K. E. Kovér and Varela, O. Carbohydr.
Res., 2007, 342, 1841–1849.
21 V. E. Manzano, M. L. Uhrig and O. Varela, J. Org. Chem., 2008, 73,
20
25
30
7224–7235.
22 E. Repetto, V. E. Manzano, M. L. Uhrig and O. Varela, J. Org.
Chem., 2012, 77, 253–265.
23 (a) T. Kajimoto and M. Node, Curr. Top. in Med. Chem., 2009, 9,
13–33; (b) B. W. Matthews, C.R. Biol., 2005, 328, 549–556; (c) J. P.
Richard, Biochemistry, 1998, 37, 4305–4309; (d) H. W. Griesser, B.
Müller-Hill and P. Overath, Eur. J. Biochem., 1983, 137, 567–572.
24 G. Sutendra, S. Wong, M. E. Fraser and R. E. Huber, Biochem.
Biophys. Res. Commun., 2007, 352, 566–570.
25 (a) E. Montero, A. García-Herrero, J. L. Asensio, K. Hirai, S. Ogawa,
F. Santoyo-González, F. J. Cañada and J. Jiménez-Barbero, Eur. J.
Org. Chem., 2000, 1945–1952; (b) A. García-Herrero, E. Montero, J.
L. Muñoz, J. F. Espinosa, A. Vián, J. L. García, J. L. Asensio, F. J.
Cañada and J. Jiménez-Barbero, J. Am. Chem. Soc., 2002, 124, 4804–
4810.
35 26 M. Cerný, Adv. Carbohydr. Chem. Biochem., 2003, 58, 122–198.
27 L. A. Reed III and L. Goodman, Carbohydr. Res., 1981, 94, 91–99.
28 H. Bredereck, G. Höschele and K. Ruck, Chem. Ber., 1953, 86,
1277−1286.
29 (a) D. J. Cline, S. E. Redding, S. G. Brohawn, J. N. Psathas, J. P.
40
45
50
55
Schneider and C. Thorpe, Biochemistry, 2004, 43, 15195–15203; (b)
J. A. Burns, J. C. Butler, J. Moran and G. M. Whitesides, J. Org.
Chem., 1991, 56, 2648–2650.
30 J. L. Asensio, J. Jiménez-Barbero, Biopolymers, 1995, 35, 55–73.
31 S. André, Z. Pei, H.–C. Siebert, O. Ramström and H.–J. Gabius,
Bioorg. Med. Chem., 2006, 14, 6314−6326.
32 S. Martín-Santamaría, S. André, E. Buzamet, R. Caraballo, G.
Fernández-Cureses, M. Morando, J. P. Ribeiro, K. Ramírez-Gualito,
B. Pascual-Teresa, F. J. Cañada, M. Menéndez, O. Ramström, J.
Jiménez-Barbero, D. Solís and H.–J. Gabius, Org. Biomol. Chem.,
2011, 9, 5445−5455.
33 M. C. Fotia and R. J. Amorati, Pharm. Pharmacol., 2009, 61,
1435−1448.
34 G. K. Glantzounis, W. Yang, R. S. Koti, D. P. Mikhailidis, A. M.
Seifalian and B. R. Davidson, Curr. Pharm. Des., 2006, 12,
2891−2901.
35 P. Peltier, R. Euzen, R. Daniellou, C. Nugier-Chauvin and V.
Ferrières, Carbohydr. Res., 2008, 343, 1897–1923.
36 G. A. Nores, T. Dohi, M. Taniguchi and S.–I. Hakomori, J. Inmunol.,
1987, 139, 3171–3176.
60 37 M. A. Earle, S. Manku, P. G. Hultin, H. Li and M. M. Palcic,
Carbohydr. Res., 1997, 301, 1–4.
38 L. P. Harding, V. M. Marshall, Y. Hernandez, Y. Gu, M. Maqsood,
N. McLay and A. P. Laws, Carbohydr. Res., 2005, 340, 1107–1111.
39 (a) M. P. Bevilaqua, S. Stengelin, M. A. Gimbrone Jr. and B. Seed,
65
70
Science, 1989, 243, 1160−1165; (b) L. A. Lasky, Science, 1992, 258,
964−969.
40 A. Asnani and F.−I. Auzanneau, Carbohydr. Res., 2008, 343, 1653–
1664.
41 (a) R. R. Schmidt and M. Stumpp, Liebigs Ann. Chem., 1983, 1249–
1256; (b) P. –H. Amvam-Zollo and P. Sinaÿ, Carbohydr. Res., 1986,
150, 199–212.
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 11