Synthesis of a phenyl thio-ss-D-galactopyranoside library from 1,5-difluoro-2,4-dinitrobenzene: Discovery of efficient and selective monosaccharide inhibitors of galectin-7

Article abstract of DOI:10.1039/b502354h

The galectins are a family of β-galactoside-binding proteins that have been implicated in cancer and inflammation processes. Herein, we report the synthesis of a library of 28 compounds that was tested for binding to galectins-1, -3, -7, -8N and -9N. An aromatic nucleophilic substitution reaction between 1,5-difluoro-2,4-dinitrobenzene and a galacto thiol gave 5-fluoro-2,4-dinitrophenyl 2,3,4,6-tetra-O-acetyl-l-thio-β-D- galactopyranoside. This versatile intermediate was then modified in a two dimensional manner: either by further substitution of the second fluoride by amines or thiols, or by reduction of the nitro groups and acylation of the resulting amines, or both. Deacetylation then gave a library of aromatic β-galactosides that showed variable inhibitory activity against the different galectins, as shown by screening with a fluorescence-polarisation assay. Particularly efficient inhibitors were found against galectin-7, while less impressive enhancements of inhibitor affinity over methyl β-D-galactopyranoside were found for galectin-1, -3, -8N and -9N. The best inhibitors against galectin-7 showed significantly higher affinity (K _d as low as 140 μM) than both β-methyl galactoside (K _d 4.8 mM) and the unsubstituted β-phenyl thiogalactoside (non-inhibitory). The best inhibitors against galectin-7 were poor against the other galectins and thus have potential as structurally simple and selective tools for dissecting biological functions of galectin-7. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b502354h

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A R T I C L E
Synthesis of a phenyl thio-b-D-galactopyranoside library from
1,5-difluoro-2,4-dinitrobenzene: discovery of efficient and selective
monosaccharide inhibitors of galectin-7†
Ian Cumpstey,a Susanne Carlsson,b Hakon Lefflerb and Ulf J. Nilsson*a
a Organic Chemistry, Lund University, Box 124, SE-221 00, Lund, Sweden.
E-mail: ulf.nilsson@organic.lu.se; Fax: +46 46 2228218; Tel: +46 46 2228209
b Section MIG, Department of Laboratory Medicine, Lund University, Solvegatan 23, SE-223
62, Lund, Sweden
Received 15th February 2005, Accepted 23rd March 2005
First published as an Advance Article on the web 14th April 2005
The galectins are a family of b-galactoside-binding proteins that have been implicated in cancer and inflammation
processes. Herein, we report the synthesis of a library of 28 compounds that was tested for binding to galectins-1, -3,
-7, -8N and -9N. An aromatic nucleophilic substitution reaction between 1,5-difluoro-2,4-dinitrobenzene and a
galacto thiol gave 5-fluoro-2,4-dinitrophenyl 2,3,4,6-tetra-O-acetyl-1-thio-b-D-galactopyranoside. This versatile
intermediate was then modified in a two dimensional manner: either by further substitution of the second fluoride by
amines or thiols, or by reduction of the nitro groups and acylation of the resulting amines, or both. Deacetylation
then gave a library of aromatic b-galactosides that showed variable inhibitory activity against the different galectins,
as shown by screening with a fluorescence-polarisation assay. Particularly efficient inhibitors were found against
galectin-7, while less impressive enhancements of inhibitor affinity over methyl b-D-galactopyranoside were found for
galectin-1, -3, -8N and -9N. The best inhibitors against galectin-7 showed significantly higher affinity (Kd as low as
140 lM) than both b-methyl galactoside (Kd 4.8 mM) and the unsubstituted b-phenyl thiogalactoside
(non-inhibitory). The best inhibitors against galectin-7 were poor against the other galectins and thus have potential
as structurally simple and selective tools for dissecting biological functions of galectin-7.
binding, while the use of thioglycosides would confer stability
towards acidic and enzymatic hydrolysis. The synthesis of
thiogalactosides as disaccharide mimics via nucleophilic sub-
stitutions and conjugate additions of a galacto thiol has been
Introduction
The galectins are a family of carbohydrate-binding proteins
characterised by certain common amino acid sequences and a b-
galactoside recognition motif. To date, fourteen examples have
reported.5
been reported in mammals.1 The galectins occur in relatively
high concentrations in the body, localised in specific organs. It
Difluorodinitrobenzene 1 has two fluoro groups, which have
been successively substituted by amine nucleophiles. It has been
has been postulated that they may act as modulators of inter-
widely used as a bifunctional cross-linker since the early 1980s.6
cellular signalling, possibly by the regulation of multivalent
In addition, it was recently used as the basis for libraries of
interactions.2 In addition, evidence exists that the galectins
dialkylaminodinitrobenzenes7 or quinoxalinone heterocycles.8
play important roles in inflammation and cancer processes.3
We envisaged that nucleophilic monosubstitution of this aro-
However, the precise roles and mechanisms of action of the
matic fluoride by an anomeric thiol could lead to an aromatic
galectins remain unclear. The chemical synthesis of inhibitors
thiogalactoside9 that could act as a scaffold with plenty of scope
of these proteins can thus be seen as an important goal, both as
for further parallel structural modification. Substitution of the
useful tools in the ongoing investigation into the behaviour of
second fluoride by nucleophiles, and/or reduction of the nitro
the galectins, and also, in the somewhat longer term, as possible
drug-candidates.
Carbohydrate–protein interactions are notoriously weak. In
addition, carbohydrates are poor drugs, due to in vivo hydrolysis
groups and acylation of the resulting amines, would lead to a
broad range of compounds suitable for testing against galactose-
binding proteins, such as the galectins.
and their failure to cross membranes due to their high polarity.
The synthesis of stable, low-molecular-weight, high-affinity,
monovalent binders is an important and daunting challenge
Results and discussion
Anomerically pure 1-thio-b-D-galactopyranose 2 was synthe-
sised from galactose pentaacetate via the thiourea method.10
Reaction of 2 with 1,5-difluoro-2,4-dinitrobenzene 1 (1.1 equiv-
alents) in acetonitrile in the presence of 2,4,6-collidine as an
acid-scavenger afforded the desired thioglycoside 3 along with
a significant quantity of the disubstituted species 4 (3 : 4, 1.9
: 1). The formation of the disubstituted product 4 could be
minimised by the use of an excess (4.0 equivalents) of the
aromatic electrophile 1 (3 : 4, 4.8 : 1) (Scheme 1).
With the aromatic thioglycoside 3 in hand, our attention first
turned to a possible substitution of the second fluoride by other
nucleophiles. Thus, treatment of a pale yellow solution of 3 in
acetonitrile with various amines (in the presence of pyridine
or 2,4,6-collidine, except where excess amine was used as acid
scavenger) resulted, in most cases, in a rapid-to-instantaneous
for carbohydrate chemists. We recently reported the synthesis
of potent N-acetyllactosamine-based inhibitors of galectin-3, in
which modification at the 3-position of the galactose residue
led to high-affinity binding.4 In this paper, we present our
investigations into a complementary modification; replacement
of the N-acetyl glucosamine moiety by a non-carbohydrate
aglycon would render the resultant inhibitors more drug-like
and potentially of higher affinity towards the galectins. A
combinatorial investigation of structural modification of the
aglycon portion of the molecule could lead to high-affinity
† Electronic supplementary information (ESI) available: Proton spectra
of the final compounds 33–60. See http://www.rsc.org/suppdata/
ob/b5/b502354h/
1 9 2 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 2 2 – 1 9 3 2
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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Next, our attention turned to the application of thiols as nu-
cleophiles for substitutions of the second fluoride. Reactions of 3
with various thiols were investigated, and a striking difference in
reactivity compared to the amines was observed. Thiols required
much longer reaction times for the starting material 3 to be
consumed (Table 1). In the case of butanethiol, products other
than the desired 16 began to appear (as monitored by TLC)
before the consumption of starting material 3 was complete,
possibly arising from the substitution of the sugar moiety
by butanethiol. With ethanethiol, a large excess of the thiol
was added in order to drive the reaction to completion (17).
Nevertheless, sulfur-substituted aromatics 15–17 were isolated
in acceptable yields. For t-butyl thiol (attempted synthesis of 18),
though, no reaction was seen, even after extended reaction times,
heating, or after the addition of sodium hydride as a stronger
base.
With substituted compounds 5–13, 15–17 in hand, our
attention turned to the nitro groups, and a potential reduction–
acylation sequence as an entry to a second avenue of diversi-
fication of the library. Various conditions for the reduction of
aromatic nitro groups were tested, using 3 as a model compound,
including hydrogenation,11 zinc12 and tin(II) chloride13 (Table 2).
The resulting diamine (19) was difficult to isolate, so direct
acylation of the crude reaction products was favoured, either
with acetic anhydride to give diacetamide (20), or benzoyl
chloride to give dibenzamide (21). The best results were obtained
using tin(II) chloride as reducing agent, so these conditions were
applied to all the compounds 20–32. The crude products were
acylated directly with acetic anhydride and pyridine, and the
colourless acetamides 20–32 were isolated without incident.
Deacetylation of the fluorinated compounds 3 and 20, 21 was
accomplished by the use of sodium methoxide in methanol to
give 33 and 48, 49 (Table 3). However, in the case of the dinitro
compound 3, more than one equivalent of sodium methoxide
was required for successful deprotection, and we found that sub-
stitution of the fluoride by methoxide to give 33 had taken place.
In the cases of the nitrogen-substituted compounds 5–13 and 22–
29, deprotection by methanolic sodium methoxide was deemed
unfeasible, due to a potential incompatibility with an acidic
Scheme 1 Reagents and conditions: (i) 2,4,6-collidine, MeCN, 1,5-di-
fluoro-2,4-dinitrobenzene (1), 93% (3 : 4, 4.8 : 1).
colour change to deep yellow, due to the dinitroaniline products.
Work-up of the reactions, simply by concentration in vacuo, and
purification by column chromatography gave the desired anilines
5–13 (Table 1) in excellent yields, except for two cases. While
secondary amines with primary carbon substituents (giving 5
and 6) and a primary amine with a secondary carbon substituent
(giving 12) gave rapid, quantitative conversion to the desired
products, a secondary amine with secondary carbon substituents
(attempted synthesis of 14) failed to react at all, even after
prolonged reaction time, presumably on steric grounds. Aniline
required a prolonged reaction time for the complete conversion
of starting material, but still formed the product (13) in excellent
yield. The reaction of 3 with glycine methyl ester (to give 11) was
sluggish and low-yielding, possibly due to the low solubility
of the hydrochloride salt used. In general though, the reaction
of galactoside 3 with amines provides a rapid, efficient route,
easily followed by a colour change, and with facile work-up and
purification, to potentially large libraries of compounds.
Table 1 Substitution of 3 with amines and thiols to give 5–18
Table 2 Nitro-reductions and acylations to give 19–32
X
Conditionsa
Time
Yield (%)
Source
Conditionsa
Product
Y
Z
Yield (%)
5
NBu2
NEt2
B
A
C
40 min
20 min
35 min
2 h
20 min
10 min
68 h
Quant.
Quant.
Quant.
Quant.
Quant.
Quant.
59
Quant.
Quant.
No reaction
66
6
7
NHAll
NHBn
NHBu
NHEt
NHCH2CO2Me
NHiPr
NHPh
NiPr2
3
C
19
20
20
21
22
23
24
25
26
27
28
29
30
31
32
F
F
F
F
NH2
69b
26
58
40
75
80
54
62
28
67
58
96
41
57
90
8
B
3
A
D
B
NHAc
NHAc
NHBz
NHAc
NHAc
NHAc
NHAc
NHAc
NHAc
NHAc
NHAc
NHAc
NHAc
NHAc
9
A
Cb
D
C
A
B
3
10
11
12
13
14
15
16
17
18
3
5
D
D
D
D
D
D
D
D
D
D
D
NBu2
NEt2
NAcAll
NAcBn
NAcBu
NAcEt
NAciPr
NHPh
SBn
30 min
17 h
6
7
8
SBn
SBu
B
96 h
120 h
48 h
9
Bc
Ad
Ae
39
87
10
12
13
15
16
17
SEt
StBu
No reaction
a (A) XH (1 eq.), s-collidine (1.5 eq.), MeCN. (B) XH (1 eq.), pyridine (1.5
eq.), MeCN. (C) XH (2.5 eq.), MeCN. (D) XH·HCl (1.1 eq.), pyridine
(2.5 eq.), MeCN. b 70% Aqueous solution of EtNH2. c Formation of
other products and unreacted starting material according to TLC.
d Addition of large excess (40 eq.) of EtSH after 26 h. e Heating to 50 ◦C
gave no reaction. Addition of NaH gave no reaction.
SBu
SEt
a (A) (i) Zn, MeOH, AcOH; (ii) Ac2O, pyridine. (B) (i) H2, EtOH, EtOAc,
HCl(aq), Pd/C; (ii) Ac2O, pyridine. (C) SnCl2·2H2O, EtOH, 70 ◦C. (D)
(i) SnCl2·2H2O, EtOH, 70 ◦C; (ii) Ac2O, pyridine. b Crude yield.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 2 2 – 1 9 3 2
1 9 2 3

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ion-exchange resin used in the work-up.14 Therefore, the use of a
volatile amine (butylamine), in conjunction with methanol
(with the possible addition of THF as co-solvent), which could
simply be removed in vacuo as a work-up, was preferred.15 This
method gave the desired deprotected compounds 35–43 and
50–57 in generally excellent yields.
Table 3 Deacetylations to give 33–60
The nitro-containing sulfur-substituted compounds 15–17
again proved problematic, however, in the amine-mediated
deacetylation reactions. Both benzyl- and butyl-substituted
compounds 15 and 16 gave multiple products according to TLC,
and, in both cases, the formation of a deep yellow colour in
the reaction was observed. In the case of 16, in addition to
the desired, pale yellow species 45, the deep yellow butylamine
39, and the corresponding non-carbohydrate-containing dibutyl
compound were obtained, presumably via aromatic nucleophilic
substitution of the respective sulfide groups by butylamine. Thus,
the ethyl sulfide 17 was deprotected using methanolic sodium
methoxide to give 46. The reduced-acylated sulfur-substituted
compounds 30–32 were also deprotected without incident using
butylamine to give 58–60.
The collection of aromatic b-thiogalactosides 33–60 was
screened for inhibition of galectins-1, -3, -7, -8N (N-
terminal domain) and -9N (N-terminal domain), using a
fluorescence-polarisation assay.16,17 In addition, methyl b-D-
galactopyranoside 61, phenyl 1-thio-b-D-galactopyranoside 62
and methyl b-lactoside 63 were included as reference com-
pounds. We found that the potency of the inhibitors against
all five galectins varied across the library, with many inhibitors
being better than the simple monosaccharide 61 and the under-
ivatised thiophenyl galactoside 62 against all five galectins, while
others were discovered to be inactive. Furthermore, it seems that
the phenyl moiety of the inhibitors 33–60 interferes with binding
to all five of the galectins, as the phenyl thiogalactoside 62 was
found to be a worse inhibitor than the methyl galactoside 61 in
all cases. For each of the galectins, dissociation constants for a
selection of the better inhibitors are shown in Table 4.
From Conditionsa Product
X
Y
Yield (%)
3
Ab
A
B
C
B
B
B
B
A
B
B
Bc
Bc
A
C
A
A
B
B
B
B
B
B
B
B
B
B
B
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
OMe
GalS
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
70
4
Quant.
Quant.
Quant.
96
99
Quant.
96
5
NBu2
NEt2
NHAll
NHBn
NHBu
NHEt
6
7
8
9
10
11
12
13
15
16
17
19
20
21
22
23
24
25
26
27
28
29
30
31
32
NHCH2CO2Me NO2
69
95
NHiPr
NHPh
SBn
NO2
NO2
NO2
NO2
NO2
NH2
NHAc 99
NHBz 95
NHAc Quant.
NHAc 98
NHAc 76
NHAc Quant.
NHAc 71
NHAc Quant.
NHAc 96
NHAc Quant.
NHAc 83
NHAc Quant.
NHAc 99
Quant.
29
SBu
SEt
F
F
77d
78
44
F
NBu2
NEt2
NAcAll
NAcBn
NAcBu
NAcEt
NAciPr
NHPh
SBn
SBu
SEt
The results against galectin-1, -3, -8N and -9N were less
impressive, but nevertheless, inhibitors more potent than the
reference galactoside 61 were identified. Although the best
inhibitors of these four proteins turned out to be sub-millimolar
inhibitors, none were selective.
a (A) NaOMe (cat.), MeOH. (B) BuNH2, MeOH, THF. (C) BuNH2,
MeOH. b More than 1 equivalent of NaOMe was required. c Reaction
not clean according to TLC. d Impure product with the n-butylamine-
substituted compound 39 as a minor impurity.
Table 4 Kd (mM) values for selected inhibitors against galectins-1, -3, -7, -8N and -9N as measured in a competitive a fluorescence-polarisation
assay. The four best inhibitors against each galectin are shown in addition to the reference compounds methyl b-D-galactopyranoside 61, phenyl
1-thio-b-D-galactopyranoside 62 and methyl b-lactoside 63
Galectin-1
Galectin-3
Galectin-7
Galectin-8N
Galectin-9N
Top two galectin-1 inhibitors
52
53
0.49
0.41
1.8
1.5
1.3
0.68
1.8
1.4
0.8
1.5
Top three galectin-7 inhibitors
34
44
58
1.9
n.i.a
n.i.a
1.8
0.17
0.18
0.14
n.i.a
2.2
n.i.a
n.i.a
n.i.a
n.i.a
n.i.a
n.i.a
Top two galectin-3 and -8N inhibitors
38
39
0.64
0.72
0.77
0.75
0.83
1.0
0.45
0.55
1.0
1.0
Top two galectin-9N inhibitors
43
52
n.i.a
0.49
1.0
1.8
n.i.a
1.3
n.i.a
1.8
0.43
0.80
References
61
62
63
10
4.4
4.8
5.3
3.3
n.i.a
0.19
n.i.a
0.22
n.i.a
0.091
n.i.a
0.052
n.i.a
0.023
a Non-inhibitory.
1 9 2 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 2 2 – 1 9 3 2

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The inhibitor potency of 33–60 against galectin-7 varied
from inactive to Kd as low as 140 lM for the bis-N-acetyl-S-
benzyl derivative 58, which indicates that the substituted phenyl
moieties of 33–60 are situated close to and interact strongly the
protein surface. Three inhibitors (34, 44 and 58)were particularly
active with Kd values below 200 lM. Interestingly, two of the
best inhibitors (44 and 58) carry a benzyl sulfide moiety, which
suggests that the S-benzyl group makes favourable interactions
with galectin-7. Compound 34 carries two equivalent galactose
residues and, hence, is divalent. Even if galectin-7 is a protein
dimer,3b its two binding sites are too far apart, which makes
it unlikely that each binding site would interact with each of
the two galactosides on one molecule of 34. Compounds 34,
44 and 58 (Fig. 1) show high selectivity towards galectin-7
over the other galectins investigated, which demonstrates that
a combinatorial approach to galectin inhibitors can effectively
target the small differences in the binding pockets between the
different galectins. Such galectin selectivity is clearly important
when the inhibitors are used as chemical biology tools or
drug lead compounds targeting galectin-7. Compounds 34,
44 and 58 are as good inhibitors of galectin-7 as the best
natural saccharide reported18 (diLacNAc; Kd 135 lM) and the
best multivalent N-acetyllactosamine cluster19 (IC50 425 lM).
These results, preferably supported by structural information on
galectin-7-inhibitors complexes and by computational docking
experiments, will aid in the further development of high-affinity
monosaccharide-derived galectin-7-selective inhibitors.
Experimental
General methods
Melting points were recorded on a Kofler apparatus (Reichert)
and are uncorrected. Proton nuclear magnetic resonance (1H)
spectra were recorded on a Bruker DRX 400 (400 MHz) or
a Bruker ARX 300 (300 MHz) spectrometer; multiplicities are
quoted as singlet (s), doublet (d), doublet of doublets (dd), triplet
(t), apparent triplet (at) or apparent triplet of doublets (atd).
Carbon nuclear magnetic resonance (13C) spectra were recorded
on a Bruker DRX 400 (100.6 MHz) spectrometer, except for
44 and 58, for which 13C spectra were recorded on a Bruker
DRX 500 (125.1 MHz) spectrometer. Spectra were assigned
using COSY, HMQC and DEPT experiments. All chemical shifts
are quoted on the d-scale in parts per million (ppm). NMR
spectra of the di-N-substituted amides showed a mixture of two
rotamers and are therefore assignments are not reported. Copies
of proton spectra of the final compounds 33–60 are therefore
included as ESI.† Low- and high-resolution (HRMS) fast atom
bombardment mass spectra were recorded using a JEOL SX-120
instrument. Optical rotations were measured on a Perkin-Elmer
341 polarimeter with a path length of 1 dm; concentrations
are given in g per 100 mL. Thin layer chromatography (TLC)
was carried out on Merck Kieselgel sheets, pre-coated with
60F254 silica. Plates were developed using 10% sulfuric acid.
Flash column chromatography was carried out on silica (Matrex,
˚
60 A, 35–70 lm, Grace Amicon). Acetonitrile was distilled from
˚
calcium hydride and stored over 4 A molecular sieves.
5-Fluoro-2,4-dinitrophenyl 2,3,4,6-tetra-O-acetyl-1-thio-b-D-
galactopyranoside
3 and 1,5-Bis-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosylthio)-2,4-dinitrobenzene 4. S-(2,3,4,6-Tetra-
O-acetyl-b-D-galactopyranosyl) isothiouronium bromide10
(1.0 g, 2.05 mmol) was suspended in a mixture of dichloro-
methane (10 mL) and water (4 mL). Sodium metabisulfite
(1.0 g, 5.3 mmol) was added, and the mixture was stirred at
◦
40 C. After 1 h 30 min, the reaction mixture was poured into
water (100 mL) and extracted with dichloromethane (100 +
50 mL). The combined organic extracts were dried (MgSO4),
filtered and concentrated in vacuo to give 2,3,4,6-tetra-O-acetyl-
1-thio-b-D-galactopyranose 2 (820 mg) as a white foam, which
was used without further purification. Compound 2 (820 mg)
and s-collidine (0.41 mL, 3.08 mmol) were dissolved in distilled
acetonitrile (10 mL) and 1,5-difluoro-2,4-dinitrobenzene 1
(1.68 g, 8.21 mmol) was added. The reaction was stirred at RT
under N2. After 5 h, TLC (1 : 1 heptane–ethyl acetate) indicated
the complete consumption of starting material (Rf 0.27) and
the formation of major (Rf 0.33) and minor (Rf 0.1) products.
The reaction mixture was poured into H2SO4 (10% aqueous,
100 mL), and extracted with dichloromethane (100 + 50 mL).
The combined organic extracts were washed with saturated aq.
sodium bicarbonate (100 mL), dried (MgSO4), filtered, and
concentrated in vacuo. The residue was purified by flash column
chromatography (dichloromethane → 9 : 1, dichloromethane–
diethyl ether → 3 : 1, dichloromethane–diethyl ether) to afford
the monosubstituted compound 3 (864 mg, 77% over two steps)
Fig. 1 Structures and Kd values of the three best inhibitors against
galectin-7.
In conclusion, we have demonstrated the potential of 1,5-
difluoro-2,4-dinitrobenzene 1 as a scaffold for the synthesis
of combinatorial carbohydrate libraries. An advantage of this
approach is that there are several points in the molecule open to
diversification. Following initial substitution by a carbohydrate-
thiol, substitution of the second fluoride by a wide variety
of amines proceeded particularly efficiently. Reduction of the
two nitro groups and acylation with different acylating agents
added a second dimension to the library. We have applied the
methodology to the synthesis of a library of b-thiogalactosides
as potential inhibitors of galectins. Screening against galectins-1,
-3, -7, -8N and -9N gave promising results, with sub-millimolar
inhibitors of all galectins being discovered. In particular, se-
lective inhibitors of galectin-7 with affinity similar to those
of the best natural ligands were identified. The compounds
reported herein appear to be the most potent monovalent
monosaccharide galectin inhibitors reported to date. Future
work includes the further expansion of the library, in particular,
using the present results as a guide to alternative substitutions,
and beginning to explore more fully the potential of using
different acylating agents in the reduction-acylation step.
as pale yellow crystals, mp (EtOH) 140–141 ◦C; [a]D +7.5
20
(c, 0.5 in CHCl3); dH (400 MHz, CDCl3) 1.99, 2.02, 2.10, 2.22
(12H, 4 s, 4 COCH3), 4.12–4.23 (3H, m, H-5, H-6, H-6ꢀ), 4.87
(1H, d, J1,2 10.0 Hz, H-1), 5.13 (1H, dd, J2,3 9.9 Hz, J3,4 3.3 Hz,
H-3), 5.45 (1H, at, J 10.0 Hz, H-2), 5.54 (1H, d, J3,4 3.3 Hz,
H-4), 7.97 (1H, d, JH,F 12.0 Hz, Ar–H), 8.99 (1H, d, JH,F 7.2 Hz,
Ar–H); dC (100.6 MHz, CDCl3) 21.0 (q, 4 COCH3), 62.6 (t,
C-6), 66.0 (d, C-2), 67.5 (d, C-4), 71.9 (d, C-3), 76.1 (d, C-5),
83.8 (d, C-1), 119.0, 125.0 (2 dd, 2 ArH), 142.4 (s, Ar), 145.2 (d,
Ar), 155.8, 158.6 (2 s, 2 Ar), 169.8, 170.3, 170.4, 170.8 (4 s, 4
+
+
=
C O); m/z (FAB ) 571 (M + Na , 100), 331 (M − SAr, 77%)
(HRMS: Calc. for C20H21N2O13FSNa (M + Na+) 571.0646.
Found 571.0657).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 2 2 – 1 9 3 2
1 9 2 5

View Article Online
=
The disubstituted compound 4 (150 mg, 16% over two steps)
4 COCH3), 45.4 (t, NHCH2CH CH2), 61.3 (t, C-6), 66.1 (d, C-
2), 66.8 (d, C-4), 71.8 (d, C-3), 74.5 (d, C-5), 84.0 (d, C-1), 111.0
as pale yellow crystals was also obtained, mp (EtOH) 200–
204 ◦C; [a]D +66.4 (c, 0.5 in CHCl3); dH (400 MHz, CDCl3)
(d, ArH), 118.0 (t, NHCH2CH CH2), 126.4 (d, ArH), 128.9,
21
=
=
1.98, 2.04, 2.09, 2.19 (24H, 4 s, 8 COCH3), 4.08 (2H, dd, J5,6
135.0, 143.8, 146.6 (4 s, 4 Ar), 131.5 (d, NHCH2CH CH2),
+
+
=
ꢀ
5.8 Hz, J6,6 10.5 Hz, H-6), 4.19 (2H, atd, J 6.6 Hz, J4,5 1.1 Hz, H-
169.3, 170.2, 170.4 (3 s, 4 C O); m/z (FAB ) 608 (M + Na ,
100%) (HRMS: Calc. for C23H27N3O13SNa (M + Na+) 608.1162.
Found 608.1178).
5), 4.24 (2H, dd, J5,6 6.6 Hz, J6,6 10.5 Hz, H-6ꢀ), 5.08–5.12 (2H,
m, H-1), 5.25–5.32 (4H, m, H-2, H-3), 5.49 (2H, d, J3,4 1.5 Hz,
H-4), 8.04 (1H, s, Ar–H), 8.90 (1H, s, Ar–H); dC (100.6 MHz,
CDCl3) 20.7, 20.7, 20.8 (3 q, 8 COCH3), 61.6 (t, C-6), 66.9, 71.4
(2 d, C-2, C-3, C-4), 74.8 (d, C-5), 82.8 (d, C-1), 123.1, 130.5 (2
d, 2 ArH), 138.2, 145.4 (2 s, Ar), 169.3, 169.9, 170.1, 170.8 (4 s,
ꢀ
ꢀ
5-Benzylamino-2,4-dinitrophenyl
2,3,4,6-tetra-O-acetyl-1-
thio-b-D-galactopyranoside 8. Yellow crystals, mp (EtOH)
172–173 ◦C; [a]D −111 (c, 1.0 in CHCl3); dH (400 MHz,
22
CDCl3) 1.98, 2.01, 2.05, 2.14 (12H, 4 s, 4 COCH3), 3.51 (1H,
+
+
=
8 C O); m/z (FAB ) 915 (M + Na , 100%) (HRMS: Calc. for
ꢀ
atd, J4,5 1.0 Hz, J 6.6 Hz, H-5), 3.99 (1H, dd, J5,6 6.6 Hz, J6,6
C34H40N2O22S2Na (M + Na+) 915.1412. Found 915.1404).
11.5 Hz, H-6), 4.03 (1H, dd, J5,6 6.6 Hz, J6,6 11.5 Hz, H-6ꢀ),
4.49 (1H, d, J1,2 10.1 Hz, H-1), 4.70 (1H, dd, Jgem 16.0 Hz, JCH,NH
5.4 Hz, PhCHHꢀ), 4.79 (1H, dd, Jgem 16.0 Hz, JCH,NH 5.9 Hz,
PhCHHꢀ), 4.87 (1H, dd, J2,3 10.0 Hz, J3,4 3.3 Hz, H-3), 5.32
(1H, at, J 10.0 Hz, H-2), 5.36 (1H, dd, J3,4 3.3 Hz, J4,5 1.0 Hz,
H-4), 6.88 (1H, s, Ar–H), 7.31–7.47 (5H, m, Ph–H), 8.84 (1H,
at, J 5.5 Hz, NH), 9.18 (1H, s, Ar–H); dC (100.6 MHz, CDCl3)
20.7, 20.8, 20.9 (3 q, 4 COCH3), 47.1 (t, PhCH2), 61.5 (t, C-6),
66.0 (d, C-2), 66.8 (d, C-4), 71.7 (d, C-3), 74.3 (d, C-5), 83.9
(d, C-1), 111.1 (d, ArH), 126.4, 126.7, 128.6, 129.5 (4 d, ArH),
135.3, 144.2, 146.6 (3 s, Ar), 169.2, 170.1, 170.2, 170.2 (4 s, 4
ꢀ
ꢀ
Typical procedure for substitution with amines
Monofluoro derivative 3 (50 mg, 0.091 mmol) and pyridine
(1.5 equivalents) were dissolved in distilled acetonitrile (2 mL),
and the amine (1.1 equivalents) was added. After TLC (1 : 1,
heptane–ethyl acetate) indicated the complete consumption of
starting material (Rf 0.3), the reaction mixture was concentrated
in vacuo and the residue was purified by flash column chro-
matography (typically 1 : 1, heptane–ethyl acetate, containing
5% triethylamine) to give the aniline 5–13.
+
+
=
C O); m/z (FAB ) 658 (M + Na , 100%) (HRMS: Calc. for
5-Dibutylamino-2,4-dinitrophenyl
2,3,4,6-tetra-O-acetyl-1-
22
C27H29N3O13SNa (M + Na+) 658.1319. Found 658.1317).
thio-b-D-galactopyranoside 5. A yellow oil; [a]D −193 (c,
1.0 in CHCl3); dH (400 MHz, CDCl3) 0.90 (6H, t, J 7.4 Hz,
N(CH2CH2CH2CH3)2), 1.26–1.35 (4H, m, N(CH2CH2CH2-
CH3)2), 1.57–1.64 (4H, m, N(CH2CH2CH2CH3)2), 1.99,
2.03, 2.04, 2.18 (12H, 4 s, 4 COCH3), 3.23–3.32 (4H, m,
N(CH2CH2CH2CH3)2), 4.01 (1H, atd, J4,5 0.8 Hz, J 6.6 Hz,
5-Butylamino-2,4-dinitrophenyl 2,3,4,6-tetra-O-acetyl-1-thio-
b-D-galactopyranoside 9. Yellow crystals, mp (Et2O/heptane)
128–129 ◦C; [a]D −73.7 (c, 1.0 in CHCl3); dH (400 MHz,
22
CDCl3) 1.02 (3H, t, J 7.4 Hz, NHCH2CH2CH2CH3), 1.49–
1.55 (2H, m, NHCH2CH2CH2CH3), 1.77–1.82 (2H, m,
NHCH2CH2CH2CH3), 1.99, 2.03, 2.03, 2.18 (12H, 4 s, 4
COCH3), 3.41–3.46 (2H, m, NHCH2), 4.05–4.11 (2H, m, H-
ꢀ
ꢀ
H-5), 4.06–4.11 (1H, m, H-6), 4.22 (1H, dd, J5,6 5.7 Hz, J6,6
11.1 Hz, H-6ꢀ), 4.88 (1H, d, J1,2 10.1 Hz, H-1), 5.12 (1H, dd, J2,3
9.9 Hz, J3,4 3.3 Hz, H-3), 5.39 (1H, at, J 10.0 Hz, H-2), 5.49 (1H,
dd, J3,4 3.3 Hz, J4,5 0.8 Hz, H-4), 7.18, 8.69 (2H, 2 s, 2 Ar–H);
dC (100.6 MHz, CDCl3) 13.9 (q, N(CH2CH2CH2CH3)2), 20.2
(t, N(CH2CH2CH2CH3)2), 21.0, 21.0, 21.1 (3 q, 4 COCH3),
29.6 (t, N(CH2CH2CH2CH3)2), 52.1 (t, N(CH2CH2CH2CH3)2),
61.0 (t, C-6), 66.2 (d, C-2), 66.7 (d, C-4), 71.8 (d, C-3), 74.5
(d, C-5), 84.5 (d, C-1), 116.9, 126.2 (2 d, 2 ArH), 135.5, 136.0,
140.5, 147.5 (4 s, 4 Ar), 169.3, 170.2, 170.2, 170.3 (4 s, 4
5, H-6), 4.27 (1H, dd, J5,6 9.2 Hz, J6,6 14.3 Hz, H-6ꢀ), 4.94 (1H,
d, J1,2 10.0 Hz, H-1), 5.15 (1H, dd, J2,3 9.9 Hz, J3,4 3.3 Hz, H-3),
5.41 (1H, at, J 10.0 Hz, H-2), 5.50 (1H, d, J3,4 3.3 Hz, H-4),
7.00 (1H, s, Ar–H), 8.40 (1H, t, J 4.8 Hz, NH), 9.11 (1H, s,
Ar–H); dC (100.6 MHz, CDCl3) 13.9 (q, NHCH2CH2CH2CH3),
20.3 (t, NHCH2CH2CH2CH3), 20.6, 20.7 (2 q, 4 COCH3), 30.7
(t, NHCH2CH2CH2CH3), 43.4 (t, NHCH2), 61.3 (t, C-6), 66.1
(d, C-2), 66.9 (d, C-4), 71.8 (d, C-3), 74.6 (d, C-5), 84.0 (d, C-1),
110.7, 126.4 (2 d, 2 ArH), 128.7, 134.8, 143.6, 146.7 (4 s, 4 Ar),
ꢀ
ꢀ
+
+
=
C O); m/z (FAB ) 680 (M + Na , 100%) (HRMS: Calc. for
C28H39N3O13SNa (M + Na+) 680.2101. Found 680.2092).
169.3, 170.1, 170.2 (3 s, 4 C O); m/z (FAB ) 624 (M + Na ,
100), 602 (M + H+, 9%) (HRMS: Calc. for C24H31N3O13SNa
(M + Na+) 624.1475. Found 624.1487).
+
+
=
5-Diethylamino-2,4-dinitrophenyl
2,3,4,6-tetra-O-acetyl-1-
thio-b-D-galactopyranoside 6. Yellow crystals, mp (Et2O)
144–146 ◦C; [a]D −104 (c, 1.0 in CHCl3); dH (400 MHz,
5-Ethylamino-2,4-dinitrophenyl 2,3,4,6-tetra-O-acetyl-1-thio-
b-D-galactopyranoside 10. Yellow crystals, mp (Et2O) 164–
22
CDCl3) 1.27 (6H, t, J 7.1 Hz, N(CH2CH3)2), 1.99, 2.02, 2.04,
2.18 (12H, 4 s, 4 COCH3), 3.31–3.42 (4H, m, N(CH2CH3)2),
165 ◦C; [a]D −78.0 (c, 1.0 in CHCl3); dH (400 MHz, CDCl3)
22
ꢀ
ꢀ
4.04–4.11 (2H, m, H-5, H-6), 4.27 (1H, dd, J5,6 5.2 Hz, J6,6
1.48 (3H, t, J 7.2 Hz, NHCH2CH3), 1.99, 2.03, 2.05, 2.19 (12H,
4 s, 4 COCH3), 3.47–3.52 (2H, m, NHCH2CH3), 4.04 (1H, dd,
14.2 Hz, H-6ꢀ), 4.90 (1H, d, J1,2 10.0 Hz, H-1), 5.13 (1H, dd,
J2,3 10.0 Hz, J3,4 3.3 Hz, H-3), 5.39 (1H, at, J 10.0 Hz, H-2),
5.48 (1H, d, J3,4 3.3 Hz, H-4), 7.18 (1H, s, Ar–H), 8.66 (1H, s,
Ar–H); dC (100.6 MHz, CDCl3) 12.6 (q, N(CH2CH3)2), 20.7,
20.8 (2 q, 4 COCH3), 46.5 (t, N(CH2CH3)2), 61.2 (t, C-6), 66.2
(d, C-2), 66.8 (d, C-4), 71.8 (d, C-3), 74.5 (d, C-5), 84.5 (d, C-1),
116.1, 126.1 (2 d, 2 ArH), 135.4, 135.7, 140.4, 146.7 (4 s, 4 Ar),
ꢀ
J5,6 6.9 Hz, J6,6 10.8 Hz, H-6), 4.10 (1H, atd, J4,5 0.8 Hz, J
6.3 Hz, H-5), 4.30 (1H, dd, J5,6 5.7 Hz, J6,6 10.8 Hz, H-6ꢀ), 4.94
(1H, d, J1,2 10.0 Hz, H-1), 5.14 (1H, dd, J2,3 9.9 Hz, J3,4 3.3 Hz,
H-3), 5.42 (1H, at, J 10.0 Hz, H-2), 5.50 (1H, dd, J3,4 3.3 Hz, J4,5
0.8 Hz, H-4), 7.02 (1H, s, Ar–H), 8.35 (1H, t, J 4.7 Hz, NH), 9.12
(1H, s, Ar–H); dC (100.6 MHz, CDCl3) 14.2 (q, NHCH2CH3),
20.7, 20.8 (2 q, 4 COCH3), 38.5 (t, NHCH2CH3), 61.3 (t, C-6),
66.1 (d, C-2), 66.9 (d, C-4), 71.8 (d, C-3), 74.6 (d, C-5), 84.1 (d,
ꢀ
ꢀ
+
+
=
169.3, 170.1, 170.4 (3 s, 4 C O); m/z (FAB ) 624 (M + Na ,
100), 602 (M + H+, 12%) (HRMS: Calc. for C24H31N3O13SNa
(M + Na+) 624.1475. Found 624.1471).
C-1), 110.6, 126.4 (2 d, 2 ArH), 128.7, 134.7, 143.7, 146.5 (4 s,
+
=
4 Ar), 169.3, 170.1, 170.4 (3 s, 4 C O); m/z (FAB ) 574 (M +
5-Allylamino-2,4-dinitrophenyl 2,3,4,6-tetra-O-acetyl-1-thio-
b-D-galactopyranoside 7. Yellow crystals, mp (Et2O) 117–
H+, 100%) (HRMS: Calc. for C22H28N3O13S (MH+) 574.1343.
Found 574.1350).
119 ◦C; [a]D −9.2 (c, 1.0 in CHCl3); dH (400 MHz, CDCl3)
22
1.99, 2.03, 2.05, 2.19 (12H, 4 s, 4 COCH3), 4.03–4.09 (2H, m, H-
5-(2-Methoxycarbonylmethylamino)-2,4-dinitrophenyl 2,3,4,6-
=
ꢀ
5, H-6), 4.14–4.17 (2H, m, NHCH2CH CH2), 4.28 (1H, dd, J5,6
tetra-O-acetyl-1-thio-b-D-galactopyranoside 11. Yellow crys-
tals, mp (EtOH) 164–166 ◦C; [a]D −78.9 (c, 1.0 in CHCl3); dH
20
9.0 Hz, J6,6 14.2 Hz, H-6ꢀ), 4.88 (1H, d, J1,2 10.0 Hz, H-1), 5.11
(1H, dd, J2,3 9.9 Hz, J3,4 3.3 Hz, H-3), 5.32–5.43 (3H, m, H-2,
ꢀ
(400 MHz, CDCl3) 2.00, 2.03, 2.04, 2.19 (12H, 4 s, 4 OCOCH3),
=
ꢀ
NHCH2CH CH2), 5.49 (1H, d, J3,4 3.3 Hz, H-4), 6.02 (1H, m,
NHCH2CH CH2), 6.95 (1H, s, Ar–H), 8.60 (1H, at, J 5.5 Hz,
NH), 9.14 (1H, s, Ar–H); dC (100.6 MHz, CDCl3) 20.7, 20.8 (2 q,
3.86 (3H, s, COOCH3), 4.10 (1H, dd, J5,6 5.9 Hz, J6,6 11.0 Hz,
=
H-6), 4.19 (1H, atd, J4,5 0.9 Hz, J 6.1 Hz, H-5), 4.26–4.30 (3H,
m, H-6ꢀ, NHCH2), 4.92 (1H, d, J1,2 10.1 Hz, H-1), 5.15 (1H,
1 9 2 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 2 2 – 1 9 3 2

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dd, J2,3 9.9 Hz, J3,4 3.3 Hz, H-3), 5.41 (1H, at, J 10.0 Hz, H-2),
5.53 (1H, dd, J3,4 3.3 Hz, J4,5 0.9 Hz, H-4), 7.00 (1H, s, Ar–H),
8.84 (1H, t, J 5.4 Hz, NH), 9.16 (1H, s, Ar–H); dC (100.6 MHz,
CDCl3) 20.7, 20.8 (2 q, 4 OCOCH3), 45.1 (t, NHCH2), 61.9 (t,
C-6), 66.1 (d, C-2), 67.3 (d, C-4), 71.8 (d, C-3), 74.8 (d, C-5),
83.8 (d, C-1), 111.2, 126.3 (2 d, 2 ArH), 129.5, 135.8, 144.0, 145.7
75.0 (d, C-5), 83.4 (d, C-1), 123.8, 125.7 (2 d, 2 ArH), 128.7,
129.2, 129.4 (3 d, Ph–CH), 133.3, 140.3, 142.4, 142.7, 144.9
+
=
(5 s, 5 Ar), 169.3, 170.1, 170.1, 170.3 (4 s, 4 C O); m/z (FAB )
653 (M + H+, 29%) (HRMS: Calc. for C27H29N2O13S2 (MH+)
653.1111. Found 653.1116).
5-Butylsulfanyl-2,4-dinitrophenyl
thio-b-D-galactopyranoside 16. Monofluoro derivative
2,3,4,6-tetra-O-acetyl-1-
=
(4 s, 4 Ar), 168.8, 169.3, 170.1, 170.2, 170.3 (5 s, 5 C O); m/z
3
(FAB+) 618 (M + H+, 100%) (HRMS: Calc. for C23H28N3O15S
(MH+) 618.1241. Found 618.1248).
(50 mg, 0.091 mmol) and pyridine (11 ll, 0.14 mmol) were
dissolved in distilled acetonitrile (2 mL) and 1,4-butanethiol
(29 ll, 0.27 mmol) was added. After 120 h, TLC (1 : 1,
heptane–ethyl acetate) showed the presence of starting material
(Rf 0.3) and the formation of a product. The reaction mixture
concentrated in vacuo, and the residue was purified by flash
column chromatography (40 : 1, dichloromethane–diethyl
ether) to give the thiobutyl compound 16 (22 mg, 39%) as
2,4-Dinitro-5-isopropylaminophenyl 2,3,4,6-tetra-O-acetyl-1-
thio-b-D-galactopyranoside 12. Yellow crystals, mp (Et2O) 149–
150 ◦C; [a]D −125 (c, 1.0 in CHCl3); dH (400 MHz, CDCl3) 1.44
22
(6H, t, J 6.0 Hz, NHCH(CH3)2), 2.00, 2.03, 2.05, 2.19 (12H, 4 s,
4 COCH3), 3.91–3.96 (1H, m, NHCH(CH3)2), 4.00–4.09 (2H,
m, H-5, H-6), 4.32 (1H, dd, J5,6 5.0 Hz, J6,6 10.3 Hz, H-6ꢀ), 4.92
(1H, d, J1,2 10.0 Hz, H-1), 5.13 (1H, dd, J2,3 9.9 Hz, J3,4 3.3 Hz,
H-3), 5.41 (1H, at, J 10.0 Hz, H-2), 5.49 (1H, d, J3,4 3.3 Hz, H-
4), 7.00 (1H, s, Ar–H), 8.39 (1H, d, J 7.1 Hz, NH), 9.12 (1H, s,
Ar–H); dC (100.6 MHz, CDCl3) 20.7, 20.8, 20.8 (3 q, 4 COCH3),
22.2, 22.8 (2 q, NHCH(CH3)2), 45.2 (d, NHCH(CH3)2), 60.9 (t,
C-6), 66.1 (d, C-2), 66.7 (d, C-4), 71.8 (d, C-3), 74.3 (d, C-5),
84.3 (d, C-1), 110.7, 126.7 (2 d, 2 ArH), 128.6, 134.4, 143.7,
ꢀ
ꢀ
pale yellow crystals, mp (EtOH) 137–138 ◦C; [a]D +44.0 (c,
21
1.0 in CHCl3); dH (400 MHz, CDCl3) 1.00 (3H, t, J 7.3 Hz,
SCH2CH2CH2CH3), 1.52–1.61 (2H, m, SCH2CH2CH2CH3),
1.77–1.85 (2H, m, SCH2CH2CH2CH3), 1.99, 2.02, 2.05, 2.19
(12H, 4 s, 4 COCH3), 3.03–3.16 (2H, m, SCH2), 4.06–4.15 (2H,
m, H-5, H-6), 4.23 (1H, dd, J5,6 6.6 Hz, J6,6 11.1 Hz, H-6ꢀ), 4.93
(1H, d, J1,2 10.0 Hz, H-1), 5.13 (1H, dd, J2,3 9.9 Hz, J3,4 3.3 Hz,
H-3), 5.33 (1H, at, J 10.0 Hz, H-2), 5.50 (1H, d, J3,4 3.3 Hz,
H-4), 7.72, 9.04 (2H, 2 s, 2 Ar–H); dC (100.6 MHz, CDCl3) 13.9
(q, SCH2CH2CH2CH3), 20.7, 20.7, 20.7 (3 q, 4 COCH3), 22.4
(t, SCH2CH2CH2CH3), 29.3 (t, SCH2CH2CH2CH3), 32.6 (t,
SCH2CH2CH2CH3), 61.5 (t, C-6), 66.0 (d, C-2), 66.9 (d, C-4),
71.7 (d, C-3), 75.1 (d, C-5), 83.8 (d, C-1), 123.7, 125.7 (2 d, 2
ArH), 139.7, 142.4, 142.7, 145.8 (4 s, 4 Ar), 169.3, 170.1, 170.1,
ꢀ
ꢀ
=
145.8 (4 s, 4 Ar), 169.3, 170.1, 170.2, 170.4 (4 s, 4 C O); m/z
(FAB+) 610 (M + Na+, 100), 588 (M + H+, 12%) (HRMS: Calc.
for C23H29N3O13SNa (M + Na+) 610.1319. Found 610.1320).
2,4-Dinitro-5-phenylaminophenyl
2,3,4,6-tetra-O-acetyl-1-
thio-b-D-galactopyranoside 13. Yellow crystals, mp (Et2O)
209–213 ◦C; [a]D −70.2 (c, 1.0 in CHCl3); dH (400 MHz,
22
+
+
=
CDCl3) 1.96, 2.00, 2.02, 2.09 (12H, 4 s, 4 COCH3), 3.31 (1H,
170.2 (4 s, 4 C O); m/z (FAB ) 619 (M + H , 100%) (HRMS:
dd, J5,6 5.6 Hz, J5,6 8.7 Hz, H-5), 3.41 (1H, dd, J6,6 10.9 Hz,
Calc. for C24H31N2O13S2 (MH+) 619.1268. Found 619.1261).
ꢀ
ꢀ
H-6), 3.90 (1H, dd, J5,6 8.7 Hz, J6,6 10.9 Hz, H-6ꢀ), 4.62 (1H, d,
J1,2 10.1 Hz, H-1), 4.98 (1H, dd, J2,3 9.9 Hz, J3,4 3.3 Hz, H-3),
5.29 (1H, at, J 10.0 Hz, H-2), 5.35 (1H, d, J3,4 3.3 Hz, H-4), 7.10
(1H, s, Ar–H), 7.36–7.40 (3H, m, Ph–H), 7.52–7.56 (2H, m,
Ph–H), 9.16 (1H, s, Ar–H), 9.84 (1H, s, NH); dC (100.6 MHz,
CDCl3) 20.6, 20.6, 20.7 (3 q, 4 COCH3), 60.0 (t, C-6), 66.1,
66.2 (2 d, C-2, C-4), 71.6 (d, C-3), 74.0 (d, C-5), 83.2 (d, C-1),
111.8, 126.3 (2 d, 2 ArH), 126.1, 128.1, 130.5 (3 d, Ph–CH),
129.1, 135.5, 137.0, 143.8, 145.6 (5 s, Ph–C, 4 Ar), 169.1, 169.8,
ꢀ
ꢀ
5-Ethylsulfanyl-2,4-dinitrophenyl
thio-b-D-galactopyranoside 17. Monofluoro derivative
2,3,4,6-tetra-O-acetyl-1-
3
(50 mg, 0.091 mmol) and collidine (18 ll, 0.14 mmol) were
dissolved in distilled acetonitrile (2 mL) and ethanethiol (7 ll,
0.091 mmol) was added. After 26 h, TLC (1 : 1 heptane–ethyl
acetate) showed the presence of starting material (Rf 0.3) and
the formation of a small quantity of a product (Rf 0.2). Further
ethanethiol (0.3 mL, 4.1 mmol) was added, and the reaction was
stirred further. After a further 22 h, TLC (1 : 1, heptane–ethyl
acetate) showed the complete consumption of starting material
(Rf 0.3), and the formation of a single product (Rf 0.2). The
reaction mixture was diluted with dichloromethane (30 mL)
and washed with sodium bicarbonate (30 mL of a saturated
aqueous solution). The aqueous phase was re-extracted with
dichloromethane (15 mL), and the combined organic extracts
were washed with H2SO4 (10% aqueous, 30 mL), dried (MgSO4),
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography (1 : 1, heptane–ethyl acetate) to
give the thioethyl compound 17 (47 mg, 87%) as pale yellow
crystals, mp (EtOH) 177–178 ◦C; [a]D22 +27.0 (c, 1.0 in CHCl3);
dH (400 MHz, CDCl3) 1.51 (3H, t, J 7.4 Hz, SCH2CH3),
1.99, 2.02, 2.06, 2.19 (12H, 4 s, 4 COCH3), 3.09–3.19 (2H, m,
SCH2CH3), 4.07–4.12 (2H, m, H-5, H-6), 4.25–4.31 (1H, m,
H-6ꢀ), 4.92 (1H, d, J1,2 10.0 Hz, H-1), 5.13 (1H, dd, J2,3 10.0 Hz,
J3,4 3.3 Hz, H-3), 5.34 (1H, at, J 10.0 Hz, H-2), 5.50 (1H, d,
J3,4 3.3 Hz, H-4), 7.75 (1H, s, Ar–H), 9.06 (1H, s, Ar–H); dC
(100.6 MHz, CDCl3) 12.4 (q, SCH2CH3), 20.6, 20.7, 20.7 (3 q,
4 COCH3), 26.9 (t, SCH2CH3), 61.5 (t, C-6), 66.0 (d, C-2), 66.9
(d, C-4), 71.6 (d, C-3), 75.0 (d, C-5), 84.0 (d, C-1), 123.8, 125.4
(2 d, 2 ArH), 140.0, 142.2, 142.6, 145.6 (4 s, 4 Ar), 169.3, 170.0,
+
+
=
170.1, 170.1 (4 s, 4 C O); m/z (FAB ) 644 (M + Na , 100), 602
(M + H+, 9%) (HRMS: Calc. for C26H27N3O13SNa (M + Na+)
644.1162. Found 644.1167).
5-Benzylsulfanyl-2,4-dinitrophenyl
1-thio-b-D-galactopyranoside 15. Monofluoro derivative
2,3,4,6-tetra-O-acetyl-
3
(50 mg, 0.091 mmol) and pyridine (15 ll, 0.18 mmol) were
dissolved in distilled acetonitrile (2 mL) and benzyl thiol
(21 mL, 0.18 mmol) was added. After 96 h, TLC (1 : 1,
heptane–ethyl acetate) showed the presence of starting material
(Rf 0.3) and a yellow product with the same Rf. The reaction
mixture was diluted with diethyl ether (30 mL) and washed with
sodium bicarbonate (30 mL of a saturated aqueous solution)
and H2SO4 (10% aqueous, 30 mL), dried (MgSO4), filtered
and concentrated in vacuo. The residue was purified by flash
column chromatography (1 : 1, heptane–ethyl acetate) to give
a mixture of the starting material 3 and the title compound 15
(56 mg, 4 : 1). Separation by flash column chromatography (40 :
1, dichloromethane–diethyl ether) gave the pure thiobenzyl
compound 15 (39 mg, 66%) as pale yellow crystals, mp
(Et2O–heptane) 129–131 ◦C; [a]D −26.7 (c, 1.0 in CHCl3);
21
dH (400 MHz, CDCl3) 1.99 (3H, s, COCH3), 2.03 (6H, s, 2
+
+
=
170.1, 170.3 (4 s, 4 C O); m/z (FAB ) 613 (M + Na , 100%)
(HRMS: Calc. for C22H26N2O13S2Na (M + Na+) 613.0774.
Found 613.0780).
COCH3), 2.17 (3H, s, COCH3), 3.81 (1H, m, H-5), 4.07 (1H,
ꢀ
ꢀ
ꢀ
dd, J5,6 5.8 Hz, J6,6 11.7 Hz, H-6), 4.19 (1H, dd, J5,6 7.1 Hz, J6,6
11.7 Hz, H-6ꢀ), 4.37 (2H, s, PhCH2), 4.70 (1H, d, J1,2 10.0 Hz,
H-1), 5.00 (1H, dd, J2,3 10.0 Hz, J3,4 3.3 Hz, H-3), 5.31 (1H, at,
J 10.0 Hz, H-2), 5.42 (1H, dd, J3,4 3.3 Hz, J4,5 1.0 Hz, H-4),
7.37–7.43 (5H, m, Ph–H), 7.69, 9.07 (2H, 2 s, 2 Ar–H); dC
(100.6 MHz, CDCl3) 20.7, 20.7, 20.8 (3 q, 4 COCH3), 37.6 (t,
PhCH2), 61.8 (t, C-6), 66.1 (d, C-2), 67.0 (d, C-4), 71.6 (d, C-3),
Typical procedure for reduction using tin(II) chloride hydrate and
subsequent acylation
Dinitro compound (3, 5–10, 12, 13, 15–17, 0.05 mmol) was
suspended in ethanol (2 mL) at 70 ◦C. Tin(II) chloride dihydrate
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 2 2 – 1 9 3 2
1 9 2 7

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(10 equivalents) was added, and the mixture was stirred for
3 h. After this time, the reaction mixture was poured onto
ice-water (30 mL) with the addition of further ethanol (ca. 2
mL). The mixture was brought up to pH 7 by the addition of
sodium bicarbonate (ca. 3 mL of a saturated aqueous solution),
and extracted with ethyl acetate (2 25 mL). The combined
organic extracts were washed with sodium bicarbonate (30 mL
of a saturated aqueous solution), dried (MgSO4), filtered, and
concentrated in vacuo.
C-3), 75.0 (d, C-5), 87.4 (d, C-1), 111.4, 132.2 (2 d, 2 ArH), 139.2,
+
=
139.5 (2 s, Ar), 169.5, 170.1, 170.1, 170.5 (4 s, C O); m/z (FAB )
648 (M + Na+, 100%) (HRMS: Calc. for C28H39N3O11SNa
(M + Na+) 648.2203. Found 648.2208).
2,4-Diacetamido-5-(N-allylacetamido)phenyl 2,3,4,6-tetra-O-
21
acetyl-1-thio-b-D-galactopyranoside 24. A colourless oil; [a]D
−1.8 (c, 1.0 in CHCl3); m/z (FAB+) 652 (M + H+, 100%)
(HRMS: Calc. for C29H38N3O12S (MH+) 652.2176. Found
652.2182).
The residue was dissolved in a mixture of acetic anhydride
(1 mL) and pyridine (1 mL) and stirred at RT for 16 h. After this
time, TLC (4 : 1, ethyl acetate–heptane) showed the formation
of a single product. The mixture was diluted with ethyl acetate
(30 mL) and washed with H2SO4 (10% aqueous, 30 mL) and
sodium bicarbonate (30 mL of a saturated aqueous solution).
The organic phase was dried (MgSO4), filtered, and concentrated
in vacuo. The residue was purified by flash column chromatog-
raphy (typically ethyl acetate → 9 : 1, ethyl acetate–methanol)
to afford the di- or tri-acetamide 20, 22–32.
2,4-Diacetamido-5-(N-benzylacetamido)phenyl 2,3,4,6-tetra-
O-acetyl-1-thio-b-D-galactopyranoside 25. A colourless oil;
[a]D −7.9 (c, 1.0 in CHCl3); m/z (FAB+) 724 (M + Na+, 100%)
22
(HRMS: Calc. for C33H39N3O12SNa (M + Na+) 724.2152. Found
724.2151).
2,4-Diacetamido-5-(N-butylacetamido)phenyl 2,3,4,6-tetra-
O-acetyl-1-thio-b-D-galactopyranoside 26. A colourless oil;
[a]D −1.5 (c, 1.0 in CHCl3); m/z (FAB+) 690 (M + Na+, 100%)
22
(HRMS: Calc. for C30H41N3O12SNa (M + Na+) 690.2309.
Found 690.2301).
2,4-Diacetamido-5-fluorophenyl 2,3,4,6-tetra-O-acetyl-1-thio-
22
b-D-galactopyranoside 20. A pale yellow oil; [a]D +20.0 (c,
2,4-Diacetamido-5-(N-ethylacetamido)phenyl 2,3,4,6-tetra-O-
0.25 in CHCl3); dH (400 MHz, CDCl3) 1.97, 2.03, 2.11, 2.14
(12H, 4 s, 4 OCOCH3), 2.17, 2.21 (6H, 2 s, 2 NHCOCH3), 3.88
22
acetyl-1-thio-b-D-galactopyranoside 27. A colourless oil; [a]D
+0.4 (c, 1.0 in CHCl3); m/z (FAB+) 662 (M + Na+, 23%)
(HRMS: Calc. for C28H37N3O12SNa (M + Na+) 662.1996. Found
662.1985).
ꢀ
(1H, at, J 6.2 Hz, H-5), 4.04 (1H, dd, J5,6 7.4 Hz, J6,6 11.6 Hz,
H-6), 4.13 (1H, dd, J5,6 5.2 Hz, J6,6 11.6 Hz, H-6ꢀ), 4.54 (1H, d,
1,2 10.0 Hz, H-1), 5.00 (1H, dd, J2,3 9.9 Hz, J3,4 3.3 Hz, H-3), 5.18
ꢀ
ꢀ
J
2,4-Diacetamido-5-(N-isopropylacetamido)phenyl
2,3,4,6-
(1H, at, J 9.9 Hz, H-2), 5.38 (1H, d, J3,4 3.3 Hz, H-4), 7.27 (1H,
d, JH,F 8.9 Hz, Ar–H), 7.56, 8.48 (2H, 2 s, 2 NH), 8.96 (1H, d,
tetra-O-acetyl-1-thio-b-D-galactopyranoside 28. A colourless
22
oil; [a]D −3.2 (c, 1.0 in CHCl3); m/z (FAB+) 676 (M + Na+,
J
H,F 5.5 Hz, Ar–H); dC (100.6 MHz, CDCl3) 20.7, 20.7, 20.7, 21.0
100), 654 (M + H+, 12%) (HRMS: Calc. for C29H39N3O12SNa
(M + Na+) 676.2152. Found 676.2162).
(4 q, 4 OCOCH3), 24.6 (s, NHCOCH3), 61.9 (t, C-6), 67.2 (d,
C-2, C-4), 71.8 (d, C-3), 75.1 (d, C-5), 88.0 (d, C-1), 116.1, 122.9
=
(2 d, 2 ArH), 128.6, 138.0 (2 s, 2 Ar), 168.3 (s, NC O), 169.6,
2,4-Diacetamido-5-phenylaminophenyl 2,3,4,6-tetra-O-acetyl-
+
+
=
170.0, 170.1, 170.6 (4 s, 4 OC O); m/z (FAB ) 595 (M + Na ,
100), 573 (M + H+, 14%) (HRMS: Calc. for C24H29N2O11FSNa
(M + Na+) 595.1374. Found 595.1375).
1-thio-b-D-galactopyranoside 29.
A pale yellow oil; dH
(400 MHz, CDCl3) 1.97, 2.00, 2.02, 2.03 (12H, 4 s, 4 OCOCH3),
2.16, 2.18 (6H, 2 s, 2 NCOCH3), 3.85 (1H, at, J 6.3 Hz, H-5), 4.00
ꢀ
ꢀ
(1H, dd, J5,6 7.3 Hz, J6,6 11.5 Hz, H-6), 4.10 (1H, dd, J5,6 5.4 Hz,
2,4-Diacetamido-5-dibutylaminophenyl 2,3,4,6-tetra-O-acetyl-
J6,6 11.5 Hz, H-6ꢀ), 4.51 (1H, d, J1,2 10.1 Hz, H-1), 4.98 (1H, dd,
ꢀ
22
1-thio-b-D-galactopyranoside 22. A colourless oil; [a]D −8.8
J
2,3 10.0 Hz, J3,4 3.4 Hz, H-3), 5.18 (1H, at, J 10.0 Hz, H-2), 5.38
(c, 1.0 in CHCl3); dH (400 MHz, CDCl3) 0.88 (6H,
(1H, d, J3,4 3.4 Hz, H-4), 6.13 (1H, br s, NH), 6.82–6.90 (3H,
m, Ph–H), 7.20–7.24 (2H, m, Ph–H), 7.48 (1H, s, Ar–H), 8.11,
8.46 (2H, 2 br s, 2 NH), 8.61 (1H, br s, Ar–H); dC (100.6 MHz,
CDCl3) 20.7, 20.7, 20.8, 20.9 (4 q, 4 OCOCH3), 24.2, 24.7 (2 q, 2
NCOCH3), 61.8 (t, C-6), 67.3, 67.4 (2 d, C-2, C-4), 71.7 (d, C-3),
75.0 (d, C-5), 88.8 (d, C-1), 116.9, 120.7, 129.6 (3 d, Ph–CH),
t,
J 6.9 Hz, N(CH2CH2CH2CH3)2), 1.25–1.36 (8H, m,
N(CH2CH2CH2CH3)2), 1.96, 2.03, 2.04, 2.15 (12H, 4 s, 4
OCOCH3), 2.17 (6H, s, 2 NCOCH3), 2.77–2.87 (4H, m,
N(CH2CH2CH2CH3)2), 3.89 (1H, at, J 6.2 Hz, H-5), 3.96 (1H,
ꢀ
ꢀ
dd, J5,6 7.1 Hz, J6,6 11.3 Hz, H-6), 4.16 (1H, dd, J5,6 5.4 Hz,
J6,6 11.3 Hz, H-6ꢀ), 4.51 (1H, d, J1,2 9.8 Hz, H-1), 5.00 (1H, dd,
ꢀ
117.0, 130.2 (2 d, 2 ArH), 132.4, 132.6, 136.3, 144.0 (4 s, Ar),
J2,3 9.9 Hz, J3,4 3.2 Hz, H-3), 5.16 (1H, at, J 9.8 Hz, H-2), 5.34
(1H, d, J3,4 3.2 Hz, H-4), 7.31 (1H, s, Ar–H), 8.59, 8.83 (2H,
2 br s, 2 NH), 9.18 (1H, br s, Ar–H); dC (100.6 MHz, CDCl3)
14.1 (q, N(CH2CH2CH2CH3)2), 20.7 (t, N(CH2CH2CH2CH3)2),
20.7, 20.8, 21.1 (3 q, 4 OCOCH3), 24.7, 25.0 (2 q, 2 NCOCH3),
29.9 (t, N(CH2CH2CH2CH3)2), 55.7 (t, N(CH2CH2CH2CH3)2),
61.9 (t, C-6), 67.2, 67.2 (2 d, C-2, C-4), 71.9 (d, C-3), 75.0 (d, C-
5), 87.5 (d, C-1), 112.3, 132.3 (2 d, 2 ArH), 139.6 (s, Ar), 169.4,
+
=
168.6, 169.7, 170.0, 170.2, 170.5 (5 s, C O); m/z (FAB ) 646
(M + H+, 100), 645 (M+, 99%) (HRMS: Calc. for C30H35N3O11S
(M + Na+) 645.1992. Found 645.1996).
2,4-Diacetamido-5-benzylsulfanylphenyl 2,3,4,6-tetra-O-acetyl-
21
1-thio-b-D-galactopyranoside 30. A colourless oil; [a]D −19.0
(c, 0.5 in CHCl3); dH (400 MHz, CDCl3) 1.96, 2.18 (6H, 2 s, 2
NCOCH3), 1.98, 2.03, 2.10, 2.15 (12H, 4 s, 4 OCOCH3), 3.83–
+
+
=
170.0, 170.5 (3 s, C O); m/z (FAB ) 704 (M + Na , 100%)
(HRMS: Calc. for C32H47N3O11SNa (M + Na+) 704.2829. Found
704.2825).
ꢀ
3.90 (3H, m, H-5, PhCH2), 4.00 (1H, dd, J5,6 7.4 Hz, J6,6 11.5 Hz,
H-6), 4.13 (1H, dd, J5,6 5.3 Hz, J6,6 11.5 Hz, H-6ꢀ), 4.43 (1H,
d, J1,2 9.9 Hz, H-1), 5.01 (1H, dd, J2,3 9.9 Hz, J3,4 3.3 Hz, H-
3), 5.17 (1H, at, J 9.9 Hz, H-2), 5.38 (1H, d, J3,4 3.3 Hz, H-4),
7.06–7.09 (2H, m, Ph–H), 7.23–7.25 (3H, m, Ph–H), 7.55 (1H, s,
Ar–H), 8.06, 8.67 (2H, 2 br s, 2 NH), 9.26 (1H, br s, Ar–H); dC
(100.6 MHz, CDCl3) 20.7, 20.8, 20.8, 21.1 (4 q, 4 OCOCH3),
24.8 (2 q, 2 NCOCH3), 41.8 (t, PhCH2), 61.9 (t, C-6), 67.2, 67.3
(2 d, C-2, C-4), 71.8 (d, C-3), 75.1 (d, C-5), 88.5 (d, C-1), 112.0
(br d, ArH), 127.8, 128.8, 128.9 (3 d, Ph–CH), 138.1 (s, Ph–C),
143.2, 143.5 (2 s, Ar), 145.4 (d, ArH), 169.6, 170.1, 170.2, 170.6
ꢀ
ꢀ
2,4-Diacetamido-5-diethylaminophenyl 2,3,4,6-tetra-O-acetyl-
22
1-thio-b-D-galactopyranoside 23. A colourless oil; [a]D −12.0
(c, 1.0 in CHCl3); dH (400 MHz, CDCl3) 0.93–0.96 (6H, m,
N(CH2CH3)2), 1.95, 2.03, 2.14, 2.17, 2.18 (18H, 5 s, 4 OCOCH3,
2 NCOCH3), 2.85–2.97 (4H, m, N(CH2CH3)2), 3.88 (1H, at, J
ꢀ
6.2 Hz, H-5), 3.96 (1H, dd, J5,6 7.0 Hz, J6,6 11.3 Hz, H-6), 4.14
(1H, dd, J5,6 5.5 Hz, J6,6 11.3 Hz, H-6ꢀ), 4.51 (1H, d, J1,2 9.9 Hz,
H-1), 5.00 (1H, dd, J2,3 9.9 Hz, J3,4 3.3 Hz, H-3), 5.16 (1H, at,
J 9.9 Hz, H-2), 5.34 (1H, dd, J3,4 3.3 Hz, J4,5 0.8 Hz, H-4), 7.29
(1H, s, Ar–H), 8.56, 8.78 (2H, 2 s, 2 NH), 9.18 (1H, s, Ar–H);
dC (100.6 MHz, CDCl3) 13.0 (q, N(CH2CH3)2), 20.6, 20.7, 20.8,
21.1 (4 q, 4 OCOCH3), 24.7, 25.0 (2 q, 2 NCOCH3), 49.6 (t,
N(CH2CH3)2), 61.8 (t, C-6), 67.2, 67.3 (2 d, C-2, C-4), 71.9 (d,
ꢀ
ꢀ
+
+
=
(4 s, C O); m/z (FAB ) 677 (M + H , 100%) (HRMS: Calc. for
C31H37N2O11S2 (MH+) 677.1839. Found 677.1849).
2,4-Diacetamido-5-butylsulfanylphenyl 2,3,4,6-tetra-O-acetyl-
20
1-thio-b-D-galactopyranoside 31. A colourless oil; [a]D +26.4
(c, 0.5 in CHCl3); dH (400 MHz, CDCl3) 0.90 (3H, t, J 7.3 Hz,
1 9 2 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 2 2 – 1 9 3 2

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SCH2CH2CH2CH3), 1.37–1.57 (4H, m, SCH2CH2CH2CH3),
1.97, 2.03, 2.10, 2.16 (12H, 4 s, 4 OCOCH3), 2.19, 2.23 (6H,
2 s, 2 NCOCH3), 2.69–2.77 (2H, m, SCH2CH2CH2CH3), 3.87
719 (M + Na+, 100%) (HRMS: Calc. for C34H33N2O11FSNa
(M + Na+) 719.1687. Found 719.1688).
5-Methoxy-2,4-dinitrophenyl 1-thio-b-D-galactopyranoside 33.
Peracetylated compound 3 (40 mg, 0.073 mmol) was dissolved
in methanol (2 mL). Sodium (0.5 mg, 0.02 mmol) was dissolved
in methanol (0.5 mL) and added to the sugar solution. The
mixture was stirred at RT and after 30 min, TLC (4 : 1, ethyl
acetate–methanol) showed no deprotected product formation
(the only carbohydrate-containing species present had Rf 0.9).
Further sodium (2 mg) in methanol (1 mL) was added, and
the mixture was stirred for a further 40 min. After this time,
TLC (9 : 1, ethyl acetate–methanol) showed the formation of a
single product (Rf 0.2). The mixture was concentrated in vacuo
and the residue was purified by flash column chromatography
(9 : 1, ethyl acetate–methanol) to afford the methoxy-substituted
compound 33 (20 mg, 70%) as a white solid; dH (400 MHz,
DMSO-d6) 3.42–3.51 (2H, m, H-3, H-6), 3.54–3.59 (1H, m, H-
6ꢀ), 3.61–3.68 (1H, m, H-2), 3.71–3.74 (2H, m, H-4, H-5), 4.15
(3H, s, OCH3), 4.70 (1H, d, JOH,4 4.5 Hz, OH-4), 4.82 (1H, at,
J 5.5 Hz, OH-6), 5.03 (1H, d, J1,2 9.7 Hz, H-1), 5.09 (1H, d,
ꢀ
(1H, at, J 6.4 Hz, H-5), 4.01 (1H, dd, J5,6 7.3 Hz, J6,6 11.5 Hz,
H-6), 4.14 (1H, dd, J5,6 5.3 Hz, J6,6 11.5 Hz, H-6ꢀ), 4.52 (1H,
d, J1,2 10.0 Hz, H-1), 5.00 (1H, dd, J2,3 10.0 Hz, J3,4 3.3 Hz,
H-3), 5.18 (1H, at, J 10.0 Hz, H-2), 5.37 (1H, dd, J3,4 3.3 Hz,
J4,5 0.8 Hz, H-4), 7.68 (1H, s, Ar–H), 8.46, 8.67 (2H, 2 br s, 2
NH), 9.29 (1H, br s, Ar–H); dC (100.6 MHz, CDCl3) 13.7 (q,
SCH2CH2CH2CH3), 20.7, 20.7, 20.8, 21.0 (4 q, 4 OCOCH3),
21.9 (t, SCH2CH2CH2CH3), 24.9, 25.1 (2 q, 2 NCOCH3), 31.6
(t, SCH2CH2CH2CH3), 36.6 (t, SCH2CH2CH2CH3), 61.9 (t, C-
6), 67.3, 67.3 (2 d, C-2, C-4), 71.8 (d, C-3), 75.1 (d, C-5), 88.3
(d, C-1), 112.5 (br d, ArH), 142.8 (s, Ar), 144.0 (d, ArH), 169.5,
ꢀ
ꢀ
+
+
=
170.1, 170.2, 170.6 (4 s, C O); m/z (FAB ) 665 (M + Na ,
58%) (HRMS: Calc. for C28H38N2O11S2Na (M + Na+) 665.1815.
Found 665.1809).
2,4-Diacetamido-5-ethylsulfanylphenyl 2,3,4,6-tetra-O-acetyl-
22
1-thio-b-D-galactopyranoside 32. A colourless oil; [a]D +25.3
(c, 1.0 in CHCl3); dH (400 MHz, CDCl3) 1.22 (3H, t, J 7.3 Hz,
SCH2CH3), 1.96, 2.02, 2.09, 2.15 (12H, 4 s, 4 OCOCH3), 2.18,
2.22 (6H, 2 s, 2 NCOCH3), 2.69–2.77 (2H, m, SCH2CH3), 3.87
J
OH,3 5.7 Hz, OH-3), 5.58 (1H, d, JOH,2 5.7 Hz, OH-2), 7.60, 8.80
(2H, 2 s, 2 Ar–H); dC (100.6 MHz, DMSO-d6) 57.8 (q, OCH3),
61.3 (t, C-6), 68.4, 68.8, 74.4, 79.9, 84.6 (5 d, C-1, C-2, C-3, C-
4, C-5), 112.7, 124.0, 136.9, 145.0, 155.2 (Ar); m/z (FAB+) 415
(M + Na+, 7%) (HRMS: Calc. for C13H16N2O10SNa (M + Na+)
415.0423. Found 415.0434).
ꢀ
(1H, at, J 6.3 Hz, H-5), 4.00 (1H, dd, J5,6 7.3 Hz, J6,6 11.5 Hz,
H-6), 4.11–4.15 (1H, m, H-6ꢀ), 4.52 (1H, d, J1,2 10.0 Hz, H-1),
4.99 (1H, dd, J2,3 10.0 Hz, J3,4 3.3 Hz, H-3), 5.17 (1H, at, J
10.0 Hz, H-2), 5.36 (1H, dd, J3,4 3.3 Hz, J4,5 0.8 Hz, H-4), 7.67
(1H, s, Ar–H), 8.45, 8.66 (2H, 2 s, 2 NH), 9.28 (1H, s, Ar–H); dC
(100.6 MHz, CDCl3) 15.0 (q, SCH2CH3), 20.7, 20.7, 20.8, 21.1 (4
q, 4 OCOCH3), 24.9, 25.0 (2 q, 2 NCOCH3), 30.7 (t, SCH2CH3),
61.9 (t, C-6), 67.2, 67.3 (2 d, C-2, C-4), 71.8 (d, C-3), 75.0 (d,
C-5), 88.2 (d, C-1), 112.7 (br d, ArH), 142.5, 142.9 (2 s, Ar),
144.1 (d, ArH), 168.1 (s, NCOCH3), 169.5, 170.1, 170.1, 170.6
Typical procedure for deprotection using sodium methoxide
Peracetylated compound (4, 17, 20, 21, 0.04 mmol) was dissolved
in methanol (2 mL). Sodium (0.5 mg, 0.02 mmol) was dissolved
in methanol (0.5 mL) and added to the sugar solution. The
mixture was stirred at RT and after 1 h 30 min, TLC (4 : 1, ethyl
acetate–methanol) showed the formation of a single product.
Duolite C436 was added and the mixture was stirred for a further
30 min, after which time it was filtered and concentrated in
vacuo. The residue was purified by flash column chromatography
(typically 7 : 1, ethyl acetate–methanol) to afford the deprotected
compound 34, 46, 48, 49.
+
+
=
(4 s, C O); m/z (FAB ) 637 (M + Na , 100%) (HRMS: Calc.
for C26H34N2O11S2Na (M + Na+) 637.1502. Found 637.1498).
2,4-Dibenzamido-5-fluorophenyl 2,3,4,6-tetra-O-acetyl-1-thio-
b-D-galactopyranoside 21. Dinitro compound 3 (50 mg, 0.091
mmol) was dissolved in a mixture of ethanol (5 mL) and ethyl
acetate (2 mL). Palladium (10% on carbon, 8 mg) was added,
and the mixture was stirred under H2. After 4 h, TLC (1 : 1,
heptane–ethyl acetate) indicated the complete consumption of
starting material (Rf 0.3) and the formation of a single product
(Rf 0.1). The mixture was stirred for a further 24 h, during which
time no change was detected by TLC. The product had Rf 0.7
on TLC (9 : 1, ethyl acetate–methanol). The reaction mixture
was filtered through Celite and concentrated in vacuo.
1,5-Bis(b-D-galactopyranosylthio)-2,4-dinitrobenzene 34.
A
21
yellow solid; [a]D −149 (c, 0.1 in H2O); dH (300 MHz, D2O)
3.63–3.74 (8H, m, H-2, H-3, H-6, H-6ꢀ), 3.86 (2H, at, J 5.8 Hz,
H-5), 3.93 (2H, d, J3,4 1.6 Hz, H-4), 5.16–5.23 (2H, m, H-1), 7.69
(1H, s, Ar–H), 9.02 (1H, s, Ar–H); dC (100.6 MHz, D2O) 61.6 (t,
C-6), 69.0, 69.2, 73.9, 80.2, 83.3 (5 d, C-1, C-2, C-3, C-4, C-5),
124.7, 125.9 (2 d, 2 ArH), 141.6, 142.6 (2 s, Ar); m/z (FAB+)
579 (M + Na+, 52%) (HRMS: Calc. for C18H24N2O14S2Na (M +
Na+) 579.0567. Found 579.0593).
The residue was dissolved in dichloromethane (5 mL) and
pyridine (1.5 mL) and benzoyl chloride (0.5 mL) were added.
The mixture was stirred at RT for 4 h. After this time, TLC
(ethyl acetate) showed the formation of a single product (Rf 0.8).
The mixture was diluted with ethyl acetate (50 mL) and washed
with H2SO4 (10% aqueous, 2 25 mL) and sodium bicarbonate
(25 mL of a saturated aqueous solution). The organic phase
was dried (MgSO4), filtered, and concentrated in vacuo. The
residue was purified by flash column chromatography (3 : 2,
ethyl acetate–heptane) to afford the dibenzamide 21 (26 mg,
5-Ethylsulfanyl-2,4-dinitrophenyl
1-thio-b-D-galactopyrano-
21
side 46. A pale yellow solid; [a]D −185 (c, 0.2 in MeOH–
CHCl3, 1 : 1); dH (400 MHz, DMSO-d6) 1.34 (3H, t, J 7.4 Hz,
SCH2CH3), 3.28–3.39 (2H, m (obs), SCH2CH3), 3.43–3.49 (1H,
m, H-3), 3.54 (2H, at, J 5.5 Hz, H-6, H-6ꢀ), 3.59–3.66 (1H,
m, H-2), 3.68 (1H, at, J 5.9 Hz, H-5), 3.75 (1H, at, J 3.6 Hz,
H-4), 4.71 (1H, d, JOH,4 4.4 Hz, OH-4), 4.81 (1H, at, J 5.3 Hz,
OH-6), 5.02 (1H, d, J1,2 9.7 Hz, H-1), 5.08 (1H, d, JOH,3 5.7 Hz,
OH-3), 5.55 (1H, d, JOH,2 5.8 Hz, OH-2), 7.75, 8.93 (2H, 2 s,
2 Ar–H); m/z (FAB+) 423 (M + H+, 84%) (HRMS: Calc. for
C14H19N2O9S2 (MH+) 423.0532. Found 423.0541).
22
40%) as an off-white solid; [a]D +39.0 (c, 1.0 in CHCl3); dH
(400 MHz, CDCl3) 1.87, 1.97, 2.09, 2.13 (12H, 4 s, 4 COCH3),
ꢀ
3.66 (1H, dd, J5,6 7.0 Hz, J6,6 11.0 Hz, H-6), 3.83 (1H, at, J
6.3 Hz, H-5), 3.89 (1H, dd, J5,6 5.3 Hz, J6,6 11.0 Hz, H-6ꢀ), 4.62
(1H, d, J1,2 10.0 Hz, H-1), 5.01 (1H, dd, J2,3 9.9 Hz, J3,4 3.3 Hz,
H-3), 5.26 (1H, at, J 10.0 Hz, H-2), 5.34 (1H, d, J3,4 3.3 Hz,
H-4), 7.42 (1H, d, JH,F 10.2 Hz, Ar–H), 7.47–8.01 (10H, m, Bz–
H), 8.12 (1H, d, JH,F 2.8 Hz, NH), 9.39 (1H, s, NH), 9.60 (1H,
d, JH,F 7.9 Hz, Ar–H); dC (100.6 MHz, CDCl3) 20.6, 20.7, 20.7,
20.9 (4 q, 4 OCOCH3), 61.6 (t, C-6), 67.3, 67.4 (2 d, C-2, C-4),
71.8 (d, C-3), 75.0 (d, C-5), 88.4 (d, C-1), 115.1 (d, ArH), 123.0
(dd, JC,F 21.3 Hz, ArH), 127.4, 127.6, 128.8, 129.0, 132.2, 132.5
ꢀ
ꢀ
2,4-Diacetamido-5-fluorophenyl 1-thio-b-D-galactopyranoside
22
48. A colourless oil; [a]D −17 (c, 0.5 in MeOH); dH (300 MHz,
D2O) 2.19 (6H, s, 2 COCH3), 3.53–3.78 (5H, m, H-2, H-3, H-5,
H-6, H-6ꢀ), 3.95 (1H, d, J3,4 3.1 Hz, H-4), 4.63 (1H, d, J1,2 9.4 Hz,
H-1), 7.55 (1H, d, JH,F 10.7 Hz, Ar–H), 7.87 (1H, d, JH,F 7.4 Hz,
Ar–H); dC (100.6 MHz, D2O) 22.7, 23.0 (2 q, 2 COCH3), 61.3
(6 d, Bz–CH), 134.2, 134.7, 138.7 (3 s, Ar), 164.9, 165.2 (2 s, 2
(t, C-6), 69.0, 69.6, 74.3, 79.5 (4 d, C-2, C-3, C-4, C-5), 88.3 (d,
+
+
=
=
=
NC O), 169.6, 170.0, 170.1, 170.3 (4 s, 4 OC O); m/z (FAB )
C-1), 122.8, 133.6 (Ar), 173.7, 173.9 (2 s, 2 C O); m/z (FAB )
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 2 2 – 1 9 3 2
1 9 2 9

View Article Online
427 (M + Na+, 64%) (HRMS: Calc. for C16H21N2O7FSNa (M +
Na+) 427.0951. Found 427.0956).
m, NHCH2CH2CH2CH3), 1.60–1.70 (2H, m, NHCH2CH2-
CH2CH3), 3.42–3.47 (1H, m), 3.51–3.66 (6H, m), 3.74–3.75 (1H,
m, H-4), 4.67 (1H, d, JOH,4 4.5 Hz, OH-4), 4.79 (1H, at, J 5.1 Hz,
OH-6), 4.85 (1H, d, J1,2 9.7 Hz, H-1), 5.06 (1H, d, JOH,3 5.5 Hz,
OH-3), 5.48 (1H, d, JOH,2 6.1 Hz, OH-2), 7.14 (1H, s, Ar–H),
8.60 (1H, t, J 5.7 Hz, NH), 8.93 (1H, s, Ar–H); m/z (FAB+)
456 (M + Na+, 100%) (HRMS: Calc. for C16H23N3O9SNa (M +
Na+) 456.1053. Found 456.1054).
2,4-Dibenzamido-5-fluorophenyl 1-thio-b-D-galactopyranoside
49. A white solid; [a]D22 +15.2 (c, 1.1 in MeOH); dH (300 MHz,
MeOH-d4) 3.47–3.63 (5H, m, H-2, H-3, H-5, H-6, H-6ꢀ), 3.87
(1H, d, J3,4 2.9 Hz, H-4), 4.51 (1H, d, J1,2 9.1 Hz, H-1), 7.50–
7.64 (6H, m, Ph–H), 7.69 (1H, d, JH,F 10.3 Hz, Ar–H), 7.91–8.08
(4H, m, Ar–H), 8.65 (1H, d, JH,F 7.6 Hz, Ar–H); dC (100.6 MHz,
D2O) 62.2 (t, C-6), 70.2, 70.7, 76.3, 81.0 (4 d, C-2, C-3, C-4, C-
5), 90.7 (d, C-1), 121.3, 124.5, 128.8, 128.9, 129.7, 129.8, 133.8,
135.4 (Ar); m/z (FAB+) 551 (M + Na+, 100%) (HRMS: Calc.
for C26H25N2O7FSNa (M + Na+) 551.1264. Found 551.1252).
5-Ethylamino-2,4-dinitrophenyl 1-thio-b-D-galactopyranoside
40. A yellow solid; dH (400 MHz, DMSO-d6) 1.25 (3H, t, J
7.1 Hz, NHCH2CH3), 3.42–3.47 (1H, m, H-3), 3.52–3.65 (6H,
m, H-2, H-5, H-6, H-6ꢀ, NHCH2CH3), 3.75 (1H, at, J 3.6 Hz,
H-4), 4.66 (1H, d, JOH,4 4.5 Hz, OH-4), 4.79 (1H, at, J 5.4 Hz,
OH-6), 4.84 (1H, d, J1,2 9.7 Hz, H-1), 5.05 (1H, d, JOH,3 5.8 Hz,
OH-3), 5.47 (1H, d, JOH,2 6.0 Hz, OH-2), 7.15 (1H, s, Ar–H),
8.64 (1H, t, J 5.7 Hz, NH), 8.93 (1H, s, Ar–H); m/z (FAB+) 428
(M + Na+, 53), 406 (M + H+, 76%) (HRMS: Calc. for
C14H20N3O9S (MH+) 406.0920. Found 406.0918).
Typical procedure for deprotection using butylamine
Peracetylated compound 5–13, 15, 16, 19, 22–32 (0.03 mmol)
was dissolved in a mixture of methanol (2 mL) and butylamine
(0.3 mL), with the optional addition of THF (1 mL), and
stirred at RT. After 16 h, TLC (4 : 1, ethyl acetate–methanol)
showed the formation of a single product. The mixture was
concentrated in vacuo, and the residue was purified by flash
column chromatography (typically 7 : 1 ethyl acetate–methanol)
to afford the deprotected compound 35–45, 47, 50–60.
5-(2-Methoxycarbonylmethylamino)-2,4-dinitrophenyl 1-thio-
b-D-galactopyranoside 41. A yellow solid; dH (400 MHz,
DMSO-d6) 3.22–3.64 (6H, m (obs), H-2, H-3, H-4, H-5, H-
6, H-6ꢀ), 3.75 (3H, s, COOCH3), 4.49 (1H, dd, Jgem 18.5 Hz, J
5.7 Hz, NHCHHꢀ), 4.62 (1H, dd, J 5.5 Hz, NHCHHꢀ), 4.70
(1H, d, JOH,4 4.4 Hz, OH-4), 4.78 (1H, d, J1,2 9.9 Hz, H-1), 4.84
(1H, at, J 4.9 Hz, OH-6), 5.07 (1H, d, JOH,3 5.7 Hz, OH-3), 5.50
(1H, d, JOH,2 6.1 Hz, OH-2), 7.02 (1H, s, Ar–H), 8.90 (1H, at, J
5.5 Hz, NH), 8.95 (2H, 2 s, 2 Ar–H); m/z (FAB+) 450 (M + H+,
29%) (HRMS: Calc. for C15H20N3O11S (MH+) 450.0819. Found
450.0818).
5-Dibutylamino-2,4-dinitrophenyl 1-thio-b-D-galactopyrano-
side 35. A yellow oil; dH (400 MHz, DMSO-d6) 0.84
(6H, t, J 7.3 Hz, N(CH2CH2CH2CH3)2), 1.20–1.29 (4H, m,
N(CH2CH2CH2CH3)2), 1.49–1.56 (4H, m, N(CH2CH2-
CH2CH3)2), 3.23–3.38 (4H, m, N(CH2CH2CH2CH3)2), 3.41–
3.46 (1H, m, H-3), 3.52 (2H, at, J 5.3 Hz, H-6, H-6ꢀ), 3.57–3.64
(2H, m, H-2, H-5), 3.75 (1H, at, J 3.9 Hz, H-4), 4.65 (1H, d,
JOH,4 4.5 Hz, OH-4), 4.78 (1H, at, J 5.2 Hz, OH-6), 4.83 (1H, d,
J1,2 9.7 Hz, H-1), 5.05 (1H, d, JOH,3 5.7 Hz, OH-3), 5.47 (1H, d,
JOH,2 6.1 Hz, OH-2), 7.33, 8.62 (2H, 2 s, 2 Ar–H); m/z (FAB+)
490 (M + H+, 100%) (HRMS: Calc. for C20H32N3O9S (MH+)
490.1859. Found 490.1860).
2,4-Dinitro-5-isopropylaminophenyl 1-thio-b-D-galactopyrano-
22
side 42. A yellow solid; [a]D −174 (c, 1.8 in MeOH); dH
(400 MHz, DMSO-d6) 1.29–1.30 (6H, m, NHCH(CH3)2), 3.43–
3.47 (1H, m, H-3), 3.52–3.55 (2H, m, H-6, H-6ꢀ), 3.60–3.66
(2H, m, H-2, H-5), 3.76 (1H, m, H-4), 4.26–4.31 (1H, m,
NHCH(CH3)2), 4.69 (1H, d, JOH,4 4.3 Hz, OH-4), 4.79–4.84 (2H,
m, H-1, OH-6), 5.06 (1H, d, JOH,3 5.6 Hz, OH-3), 5.46 (1H, d,
JOH,2 6.0 Hz, OH-2), 7.19 (1H, s, Ar–H), 8.30 (1H, d, J 7.8 Hz,
NH), 8.92 (1H, s, Ar–H); m/z (FAB+) 442 (M + Na+, 17), 420
(M + H+, 100%) (HRMS: Calc. for C15H22N3O9S (MH+)
420.1077. Found 420.1070).
5-Diethylamino-2,4-dinitrophenyl 1-thio-b-D-galactopyrano-
side 36. A yellow oil; dH (300 MHz, D2O) 1.23 (6H, t, J 7.1 Hz,
N(CH2CH3)2), 3.33–3.53 (4H, m, N(CH2CH3)2), 3.58 (1H, dd,
J2,3 9.1 Hz, J3,4 3.2 Hz, H-3), 3.69–3.89 (4H, m, H-2, H-5, H-6,
H-6ꢀ), 3.95 (1H, d, J3,4 3.2 Hz, H-4), 4.78 (1H, d, J1,2 9.8 Hz,
H-1), 7.44, 8.63 (2H, 2 s, 2 Ar–H); m/z (FAB+) 456 (M + Na+,
100%) (HRMS: Calc. for C16H23N3O9SNa (M + Na+) 456.1053.
Found 456.1053).
2,4-Dinitro-5-phenylaminophenyl 1-thio-b-D-galactopyrano-
side 43. Yellow-orange crystals; dH (400 MHz, DMSO-d6)
2.83–2.86 (1H, m), 2.91–2.96 (1H, m), 3.18–3.22 (1H, m),
3.26–3.51 (m (obs)), 3.69 (1H, m, H-4), 4.34 (1H, d, J1,2 9.7 Hz,
H-1), 4.50 (1H, at, J 5.2 Hz, OH-6), 4.54 (1H, d, J 4.5 Hz,
OH), 5.01 (1H, d, J 5.7 Hz, OH), 5.43 (1H, d, J 6.4 Hz, OH),
7.08 (1H, s, Ar–H), 7.35–7.39 (5H, m, Ph–H), 8.98 (1H, s,
Ar–H) 10.07 (1H, s, NH); m/z (FAB+) 476 (M + Na+, 100%)
(HRMS: Calc. for C18H19N3O9SNa (M + Na+) 476.0740. Found
476.0747).
5-Allylamino-2,4-dinitrophenyl 1-thio-b-D-galactopyranoside
37. A yellow solid; dH (400 MHz, DMSO-d6) 3.36–3.43 (1H,
m, H-3), 3.50–3.62 (1H, m, H-2, H-5, H-6, H-6ꢀ), 3.75 (1H, at,
J 3.7 Hz, H-4), 4.24–4.27 (2H, m, NHCH2), 4.67 (1H, d, JOH,4
4.6 Hz, OH-4), 4.69 (1H, d, J1,2 9.8 Hz, H-1), 4.83 (1H, at, J
5.3 Hz, OH-6), 5.06 (1H, d, JOH,3 5.6 Hz, OH-3), 5.16–5.22 (2H,
=
m, NHCH2CH CH2), 5.49 (1H, d, JOH,2 6.1 Hz, OH-2), 5.95–
6.02 (1H, m, NHCH2CH CH2), 7.01 (1H, s, Ar–H), 8.87 (1H,
=
5-Benzylsulfanyl-2,4-dinitrophenyl 1-thio-b-D-galactopyrano-
t, J 5.8 Hz, NH), 8.94 (1H, s, Ar–H); m/z (FAB+) 418 (M + H+,
100%) (HRMS: Calc. for C15H20N3O9S (M + Na+) 418.0920.
Found 418.0921).
21
side 44. A pale yellow solid; [a]D −114 (c, 0.2 in MeOH); dH
(400 MHz, DMSO-d6) 3.40–3.72 (6H, m, H-2, H-3, H-4, H-5,
H-6, H-6ꢀ), 4.64, 4.77 (2H, ABq, JAB 11.0 Hz, PhCH2), 4.74 (1H,
d, JOH,4 4.4 Hz, OH-4), 4.91 (1H, at, J 5.0 Hz, OH-6), 5.07–5.10
(2H, m, H-1, OH-3), 5.61 (1H, d, JOH,2 5.5 Hz, OH-2), 7.33–7.47
(5H, m, Ph–H), 7.90, 8.96 (2H, 2 s, 2 Ar–H); m/z (FAB+) 485
(M + H+, 11%); dC (125.1 MHz, DMSO-d6) 36.2 (SCH2), 61.5 (C-
6), 68.4, 68.9, 74.2, 79.0 (C-2, C-3, C-4, C-5), 84.3 (C-1), 123.8,
124.8, 128.0, 128.9, 129.8 (5 ArH), 134.3 (ArC), 140.2, 140.9
(2 ArS), 142.9, 144.5 (2 ArN) (HRMS: Calc. for C19H21N2O9S2
(MH+) 485.0688. Found 485.0688).
5-Benzylamino-2,4-dinitrophenyl 1-thio-b-D-galactopyrano-
side 38. A yellow solid; dH (400 MHz, DMSO-d6) 3.10–3.15
(1H, m, H-3), 3.28–3.38 (1H, m (obs), H-5), 3.43–3.55 (3H, m,
H-2, H-6, H-6ꢀ), 3.66 (1H, m, H-4), 4.19 (1H, d, J1,2 9.7 Hz,
H-1), 4.66 (1H, d, JOH,4 4.5 Hz, OH-4), 4.85 (2H, d, J 5.6 Hz,
NHCH2Ph), 4.89 (1H, at, J 5.1 Hz, OH-6), 5.06 (1H, d, JOH,3
5.6 Hz, OH-3), 5.50 (1H, d, JOH,2 5.8 Hz, OH-2), 6.93 (1H, s,
Ar–H), 7.28–7.40 (5H, m, Ph–H), 8.96 (1H, s, Ar–H), 9.10 (1H,
t, NH); m/z (FAB+) 468 (M + H+, 28%) (HRMS: Calc. for
C19H22N3O9S (MH+) 468.1077. Found 468.1074).
5-Butylsulfanyl-2,4-dinitrophenyl 1-thio-b-D-galactopyrano-
side 45. A pale yellow solid; dH (400 MHz, DMSO-d6) 0.94
(3H, t, J 7.3 Hz, SCH2CH2CH2CH3), 1.47–1.55 (2H, m,
SCH2CH2CH2CH3), 1.65–1.70 (2H, m, SCH2CH2CH2CH3),
5-Butylamino-2,4-dinitrophenyl 1-thio-b-D-galactopyranoside
39.
A yellow solid; dH (300 MHz, DMSO-d6) 0.93
(3H, t, J 7.3 Hz, NHCH2CH2CH2CH3), 1.34–1.47 (2H,
3.28–3.68 (7H, m
(obs), H-2, H-3, H-5, H-6, H-6ꢀ,
1 9 3 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 2 2 – 1 9 3 2

View Article Online
SCH2CH2CH2CH3), 3.75 (1H, at, J 3.6 Hz, H-4), 4.70
(1H, d, JOH,4 4.4 Hz, OH-4), 4.79 (1H, at, J 5.2 Hz, OH-6), 5.05
(1H, d, J1,2 9.6 Hz, H-1), 5.08 (1H, d, JOH,3 5.7 Hz, OH-3), 5.57
(1H, d, JOH,2 5.8 Hz, OH-2), 7.75, 8.92 (2H, 2 s, 2 Ar–H); m/z
(FAB+) 451 (M + H+, 17%) (HRMS: Calc. for C16H23N2O9S2
(MH+) 451.0845. Found 451.0849).
H-1), 4.49 (1H, m, OH-4), 4.60 (1H, m, OH-6), 4.89 (1H, d,
J
OH,3 3.6 Hz, OH-3), 5.37 (1H, d, JOH,2 2.8 Hz, OH-2), 6.77–6.80
(1H, m, Ph–H), 6.90–6.92 (2H, m, Ph–H), 7.18–7.22 (2H, m,
Ph–H), 7.28, 7.48, 8.16, 9.38, 9.49 (5H, 5 s, 2 Ar–H, 3 NH); m/z
(FAB+) 478 (M + H+, 92), 477 (M+, 100%) (HRMS: Calc. for
C22H27N3O7S (M+) 477.1570. Found 477.1559).
1H NMR spectrum shows a mixture, 45 : 39, ca. 5 : 1.
2,4-Diacetamido-5-benzylsulfanyl-phenyl 1-thio-b-D-galacto-
22
pyranoside 58. A white solid; [a]D +5.0 (c, 0.2 in MeOH);
2,4-Diamino-5-fluorophenyl 1-thio-b-D-galactopyranoside 47.
A pale brown oil; dH (300 MHz, D2O) 3.46–3.53 (1H, m, H-
2), 3.58–3.76 (4H, m, H-3, H-5, H-6, H-6ꢀ), 3.91 (1H, d, J3,4
3.3 Hz, H-4), 4.33–4.37 (1H, m, H-1), 6.43 (1H, d, JH,F 8.3 Hz,
Ar–H), 7.20 (1H, d, JH,F 11.0 Hz, Ar–H); dC (100.6 MHz, D2O)
61.4 (t, C-6), 69.1, 69.6, 74.3, 79.5 (4 d, C-2, C-3, C-4, C-5), 89.8
(d, C-1), 105.2, 123.2, 137.7 (Ar); m/z (FAB+) 343 (M + Na+,
64), 321 (M + H+, 67%) (HRMS: Calc. for C12H17N2O5FSNa
(M + Na+) 343.0740. Found 343.0740).
dH (400 MHz, DMSO-d6) 2.00, 2.07 (6H, 2 s, 2 COCH3), 3.26–
3.56 (5H, m (obs), H-2, H-3, H-5, H-6, H-6ꢀ), 3.64 (1H, m, H-4),
4.10 (2H, s, PhCH2), 4.31 (1H, d, J1,2 8.8 Hz, H-1), 4.53 (1H, d,
JOH,4 4.5 Hz, OH-4), 4.68 (1H, at, J 5.3 Hz, OH-6), 4.93 (1H, d,
J
OH,3 5.2 Hz, OH-3), 5.41 (1H, d, JOH,2 3.2 Hz, OH-2), 7.21–7.31
(5H, m, Ph-H), 7.64 (1H, s, Ar–H), 8.23, 9.19, 9.44 (3H, 3 br s,
2 NH, Ar–H); dC (125.1 MHz, DMSO-d6) 23.5, 24.0 (2 CH3),
38.3 (SCH2), 60.8 (C-6), 68.4, 69.0, 74.5, 79.7 (C-2, C-3, C-4,
C-5), 88.8 (C-1), 127.2, 128.5, 139.2 (ArH), 137.3 (2 ArN), 138.2
(2 ArH, ArC), 139.2 (2 ArS), 168.3, 168.4 (2 CO); m/z (FAB+)
531 (M + Na+, 100), 509 (M + H+, 41%). (HRMS: Calc. for
C23H28N2O7S2Na (M + Na+) 531.1236. Found 531.1234; Calc.
for C23H29N2O7S2 (MH+) 509.1416. Found 509.1418).
2,4-Diacetamido-5-dibutylaminophenyl 1-thio-b-D-galactopy-
22
ranoside 50.
A
colourless oil; [a]D
+10 (c, 0.65 in
MeOH); dH (400 MHz, DMSO-d6) 0.84–0.86 (6H, m,
N(CH2CH2CH2CH3)2), 1.24–1.33 (8H, m, N(CH2CH2CH2-
CH3)2), 2.05, 2.10 (6H, 2 s, 2 COCH3), 2.80–2.83 (2H, m,
N(CH2CH2CH2CH3)2), 3.30–3.54 (5H, m (obs), H-2, H-3, H-5,
H-6, H-6ꢀ), 3.67 (1H, m, H-4), 4.30 (1H, d, J1,2 8.6 Hz, H-1), 4.43
(1H, d, JOH,4 4.2 Hz, OH-4), 4.65 (1H, at, J 5.3 Hz, OH-6), 4.87
(1H, d, JOH,3 5.4 Hz, OH-3), 5.26 (1H, d, JOH,2 5.1 Hz, OH-2),
7.44 (1H, s, Ar–H), 8.55, 8.89, 9.38 (3H, 2 s, 2 NH, Ar–H); m/z
(FAB+) 536 (M + Na+, 100), 514 (M + H+, 46%) (HRMS: Calc.
for C24H39N3O7SNa (M + Na+) 536.2406. Found 536.2406; Calc.
for C24H40N3O7S (MH+) 514.2587. Found 514.2584).
2,4-Diacetamido-5-butylsulfanyl-phenyl 1-thio-b-D-galacto-
21
pyranoside 59. A colourless oil; [a]D −4.2 (c, 0.5 in
MeOH); dH (400 MHz, DMSO-d6) 0.87 (3H, t, J 7.3 Hz,
SCH2CH2CH2CH3), 1.35–1.53 (4H, m, SCH2CH2CH2CH3),
2.06, 2.07 (6H,
2 s, 2 COCH3), 2.84–2.87 (2H, m,
SCH2CH2CH2CH3), 3.26–3.38 (2H, m (obs), H-2, H-3),
3.43–3.56 (3H, m, H-5, H-6, H-6ꢀ), 3.66 (1H, at, J 3.3 Hz, H-4),
4.34 (1H, d, J1,2 8.8 Hz, H-1), 4.49 (1H, d, JOH,4 4.2 Hz, OH-4),
4.65 (1H, at, J 5.2 Hz, OH-6), 4.91 (1H, d, JOH,3 5.5 Hz, OH-3),
5.40 (1H, d, JOH,2 5.1 Hz, OH-2), 7.61 (1H, s, Ar–H), 8.19, 9.32,
9.44 (3H, 2 s, 2 NH, Ar–H); m/z (FAB+) 497 (M + Na+, 100),
475 (M + H+, 12%) (HRMS: Calc. for C20H31N2O7S2 (MH+)
475.1573. Found 475.1579).
2,4-Diacetamido-5-diethylaminophenyl 1-thio-b-D-galactopy-
22
ranoside 51. A white solid; [a]D −9.1 (c, 1.1 in MeOH); dH
(400 MHz, DMSO-d6) 0.90 (6H, t, J 7.1 Hz, N(CH2CH3)2),
2.05, 2.12 (6H, 2 s, 2 COCH3), 2.87–2.92 (2H, m, N(CH2CH3)2),
3.27–3.53 (5H, m, H-2, H-3, H-5, H-6, H-6ꢀ), 3.66 (1H, m, H-4),
4.31 (1H, d, J1,2 9.0 Hz, H-1), 4.43 (1H, d, JOH,4 4.1 Hz, OH-4),
4.64 (1H, at, J 5.3 Hz, OH-6), 4.87 (1H, d, JOH,3 5.4 Hz, OH-3),
5.23 (1H, d, JOH,2 5.1 Hz, OH-2), 7.41 (1H, s, Ar–H), 8.56, 8.95,
9.36 (3H, 2 s, 2 NH, Ar–H); m/z (FAB+) 480 (M + Na+, 42),
458 (M + H+, 100%) (HRMS: Calc. for C20H32N3O7S (MH+)
458.1961. Found 458.1953).
2,4-Diacetamido-5-ethylsulfanylphenyl
1-thio-b-D-galacto-
pyranoside 60. A white solid; dH (400 MHz, DMSO-d6) 1.18
(3H, t, J 7.3 Hz, SCH2CH3), 2.07 (6H, s, 2 COCH3), 2.85–2.90
(2H, m, SCH2CH3), 3.28–3.50 (5H, m (obs), H-2, H-3, H-5,
H-6, H-6ꢀ), 3.65 (1H, m, H-4), 4.36 (1H, d, J1,2 9.6 Hz, H-1),
4.49 (1H, m, OH-4), 4.64 (1H, m, OH-6), 4.91 (1H, d, JOH,3
5.5 Hz, OH-3), 5.39 (1H, m, OH-2), 7.61 (1H, s, Ar–H), 8.19,
9.31, 9.43 (3H, 2 s, 2 NH, Ar–H); m/z (FAB+) 447 (M +
H+, 100%) (HRMS: Calc. for C18H27N2O7S2 (MH+) 447.1260.
Found 447.1261).
2,4-Diacetamido-5-(N-allylacetamido)phenyl 1-thio-b-D-gala-
22
ctopyranoside 52. A white solid; [a]D −3.3 (c, 1.0 in MeOH);
m/z (FAB+) 506 (M + Na+, 42), 484 (M + H+, 100%) (HRMS:
Calc. for C21H30N3O8S (MH+) 484.1754. Found 484.1757).
Fluorescence-polarisation experiments and Kd determinations
for 33–60 against galectin-1. The Kd values were determined as
previously described16,17 with galectin-1 at 10 lM, the fluorescent
2,4-Diacetamido-5-(N-benzylacetamido)phenyl
1-thio-b-D-
21
galactopyranoside 53. A white solid; [a]D +12 (c, 0.7 in
MeOH); m/z (FAB+) 534 (M + H+, 100%) (HRMS: Calc. for
C25H32N3O8S (MH+) 534.1910. Found 534.1912).
probe
2-(fluorescein-5/6-ylthiourea)ethyl
b-D-galacto-
pyranosyl(1→4)-2-acetamido-2-deoxy-b-D-glucopyranoside17
at 0.1 lM, and 33–63 at either 2, 1 or 0.5 mM.
2,4-Diacetamido-5-(N-butylacetamido)phenyl
1-thio-b-D-
Fluorescence-polarisation experiments and Kd determinations
for 33–60 against galectin-3. The Kd values were determined
as previously described16,17 with human galectin-3 at 1 lM, the
fluorescent probe 2-(fluorescein-5/6-ylcarbonylamino)ethyl 3-
(4-methoxybenzyl)-b-D-galactopyranosyl(1→4)-2-acetamido-
2-deoxy-b-D-glucopyranoside4b at 0.1 lM, and 33–63 at either
2, 1 or 0.5 mM.
22
galactopyranoside 54. A white solid; [a]D −31 (c, 0.45 in
MeOH); m/z (FAB+) 522 (M + Na+, 26), 500 (M + H+, 100%)
(HRMS: Calc. for C22H34N3O8S (MH+) 500.2067. Found
500.2064).
2,4-Diacetamido-5-(N-ethylacetamido)phenyl
1-thio-b-D-
21
galacto-pyranoside 55. A colourless oil; [a]D +12 (c, 0.4 in
MeOH); m/z (FAB+) 472 (M + H+, 100%) (HRMS: Calc. for
C20H30N3O8S (MH+) 472.1754. Found 472.1759).
Fluorescence-polarisation experiments and Kd determinations
for 33–60 against galectin-7. The Kd values were deter-
mined as previously described16,17 with mouse galectin-7-
GST fusion protein at 3 lM, the fluorescent probe b-D-
galactopyranosyl(1→4)-2-acetamido-2-deoxy-b-D-glucopyra-
nosyl(1→3)-b-D-galactopyranosyl(1→4)-(N1-fluorescein-5-yl-
carbonylaminomethylcarbonyl)-b-D-glucopyranosylamine20 at
0.1 lM, and 33–63 at either 2, 1 or 0.2 mM. The protein
was obtained from E. coli BL21 Star (Invitrogen) containing
the pGEX-T2 expression plasmid (Pharmacia, Uppsala,
Sweden) with the full coding sequence of mouse galectin-7
2,4-Diacetamido-5-(N-isopropylacetamido)phenyl 1-thio-b-D-
21
galactopyranoside 56. A white solid; [a]D +18 (c, 0.4 in
MeOH); m/z (FAB+) 486 (M + H+, 100%) (HRMS: Calc. for
C21H32N3O8S (MH+) 486.1910. Found 486.1917).
2,4-Diacetamido-5-phenylaminophenyl
1-thio-b-D-galacto-
pyranoside 57. A white solid; dH (400 MHz, DMSO-d6) 2.05,
2.06 (6H, 2 s, 2 COCH3), 3.23–3.50 (5H, m (obs), H-2, H-3,
H-5, H-6, H-6ꢀ), 3.66 (1H, m, H-4), 4.30 (1H, d, J1,2 8.5 Hz,
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 2 2 – 1 9 3 2
1 9 3 1

View Article Online
inserted between the BamH1 and Sma1 sites (according to
manufacturers instructions) downstream of the GST-coding
sequence. The construct was kindly provided by Dr Francoise
Poirier and co-workers.21 Culture conditions and purification of
the galectin was essentially as described for other galectins.16,20
2 C. F. Brewer, M. C. Miceli and L. G. Baum, Curr. Opin. Struct. Biol.,
2002, 12, 616–623.
3 (a) H. Leffler (Editor), Glycoconjugate J., 2004, 19, 433–629; (b) H.
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ChemBioChem, 2002, 3, 183–189; (b) P. So¨rme, B. Kahl-Knutson, U.
Wellmar, B.-G. Magnusson, H. Leffler and U. J. Nilsson, Methods
Enzymol., 2003, 363, 157–169; (c) P. So¨rme, P. Arnoux, B. Kahl-
Knutsson, H. Leffler, J. M. Rini and U. J. Nilsson, J. Am. Chem.
Soc., 2005, 127, 1737–1743.
5 (a) U. J. Nilsson, E. J.-L. Fournier and O. Hindsgaul, Bioorg. Med.
Chem., 1998, 6, 1563–1575; (b) U. J. Nilsson, E. J.-L. Fournier, E. J.
Fryz and O. Hindsgaul, Comb. Chem. High Throughput Screening,
1999, 2, 337–355.
Fluorescence-polarisation experiments and Kd determinations
for 33–60 against galectin-8N. The Kd values were deter-
mined as previously described16,17 with a thioredoxin-galectin-
8N fusion protein20,22 at 0.5 lM, the fluorescent probe 2-(fluor-
escein-5-ylcarbonylamino)ethyl b-D-galactopyranosyl(1→4)-
2-acetamido-2-deoxy-b-D-glucopyranosyl(1→3)-b-D-galacto-
pyranosyl(1→4)-b-D-glucopyranoside22 at 0.1 lM, and 33–63
at 1 mM.
6 S. M. Deyev, G. A. Afanasenko and O. L. Polyanovsky, Biochim.
Biophys. Acta, 1978, 534, 358–367.
Fluorescence-polarisation experiments and Kd determinations
for 33–60 against galectin-9N. The Kd values were determined
as previously described16,17 with a thioredoxin-galectin-9N
fusion protein at 1 lM, the fluorescent probe 2-(fluorescein-5-
yl-carbonylamino)ethyl b-D-galactopyranosyl(1→4)-2-aceta-
mido-2-deoxy-b-D-glucopyranosyl(1→3)-b-D-galactopyrano-
syl(1→4)-b-D-glucopyranoside22 at 0.1 lM, and 33–63 at either
1 or 0.2 mM. The protein containing thioredoxin fused via a
linker at the N-terminus of the first 170 amino acids of human
galectin-9 was produced using the same expression system as
for galectin-8N (pET-32 vector, Novagen) in E. coli BL21 Star
and purified essentially as for other galectins.16,20
7 S. R. Nagy, G. Liu, K. S. Lam and M. S. Denison, Biochemistry,
2002, 41, 861–868; G. Liu, Y. Fan, J. R. Carlson, Z.-G. Zhao and
K. S. Lam, J. Comb. Chem., 2000, 2, 467–474.
8 A. Mazurov, Tetrahedron Lett., 2000, 41, 7–10.
9 H. Driguez and W. Szeja, Synthesis, 1994, 1413–1414.
10 B. D. Johnston and B. M. Pinto, J. Org. Chem., 2000, 65, 4607–
4617.
11 M. D. Ennis and N. B. Ghazal, Tetrahedron Lett., 1992, 33, 6287–
6290.
12 S. Cao and R. Roy, Tetrahedron Lett., 1996, 37, 3421–3424.
13 S. Cao, F. Hernandez-Mateo and R. Roy, J. Carbohydr. Chem., 1998,
17, 609–631.
14 In fact, the dinitroanilines 5–13 may be non-basic, as, for example,
9 remained in the organic (ethyl acetate) phase during an H2SO4
aqueous extraction. In addition, attempts to acylate 9 with acetic
anhydride and pyridine failed.
15 L. Yang and L. Guo, Tetrahedron Lett., 1996, 37, 5041–5044.
16 P. So¨rme, B. Kahl-Knutson, M. Huflejt, U. J. Nilsson and H. Leffler,
Anal. Biochem., 2004, 334, 36–47.
17 P. So¨rme, B. Kahl-Knutson, U. Wellmar, U. J. Nilsson and H. Leffler,
Methods Enzymol., 2003, 362, 504–512.
18 N Ahmad, H.-J. Gabius, H. Kaltner, S. Andre´, I. Kuwabara, F.-T.
Liu, S. Oscarson, T. Norberg and C. F. Brewer, Can. J. Chem., 2002,
80, 1096–1104.
Acknowledgements
This work was supported by the Swedish Research Council
and the program “Glycoconjugates in Biological Systems”,
sponsored by the Swedish Strategic Research Foundation.
A 2-azidoethyl glycoside precursor for the probe used for
galectins-8N and -9N was provided by the Consortium for
Functional Glycomics (grant number NIH GM62116). We are
grateful to Barbro Kahl-Knutson and Anneli Edstro¨m for
help with galectin production and fluorescence polarization
measurements.
19 S. Andre´, H. Kaltner, T. Furuike, S.-I. Nishimura and H.-J. Gabius,
Bioconjugate Chem., 2004, 15, 87–98.
¨
20 C. T. Oberg, S. Carlsson, E. Fillion, H. Leffler and U. J. Nilsson,
Bioconjugate Chem., 2003, 14, 1289–1297.
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O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 2 2 – 1 9 3 2