Interactions of aromatic mannosyl disulfide derivatives with Concanavalin A: synthesis, thermodynamic and NMR spectroscopy studies

Article abstract of DOI:10.1016/j.carres.2009.06.008

α-d-Mannopyranosyl units were attached to an aromatic scaffold through disulfide linkages to obtain mono- to trivalent glycosylated ligands for lectin binding studies. Isothermal titration calorimetric (ITC) measurements indicated that binding affinities of these derivatives to Concanavalin A (Con A) were comparable to or slightly higher than that of methyl α-d-mannopyranoside (K_a values in the range of 10⁴ M^-1). The stoichiometries of the lectin-ligand complexes were in agreement with the formal valencies (1-3) of the respective ligands indicating cross-linking in interactions with the di- and trivalent derivatives. Multivalency effects could not, however, be observed with the latter. These ligands were shown to bind to the carbohydrate binding site of Con A using saturation transfer difference (STD) NMR competition experiments.

Full text of DOI:10.1016/j.carres.2009.06.008

Carbohydrate Research 344 (2009) 1758–1763
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Interactions of aromatic mannosyl disulfide derivatives with Concanavalin A:
synthesis, thermodynamic and NMR spectroscopy studies
a,
Bandaru Narasimha Murthy a, Sharmistha Sinha b, Avadhesha Surolia b, , Narayanaswamy Jayaraman
,
*
*
c
d,
*
László Szilágyi c, , Ildikó Szabó , Katalin E. Kövér
*
a Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India
b National Institute of Immunology, Aruna Asif Ali Marg, New Delhi 110 067, India
c Department of Organic Chemistry, University of Debrecen, H-4010 Debrecen, pf. 20, Hungary
d Department of Inorganic Chemistry, University of Debrecen, H-4010 Debrecen, pf. 21, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
a-D-Mannopyranosyl units were attached to an aromatic scaffold through disulfide linkages to obtain
mono- to trivalent glycosylated ligands for lectin binding studies. Isothermal titration calorimetric
(ITC) measurements indicated that binding affinities of these derivatives to Concanavalin A (Con A) were
Received 1 December 2008
Received in revised form 25 May 2009
Accepted 3 June 2009
comparable to or slightly higher than that of methyl
a-D-mannopyranoside (Ka values in the range of
Available online 6 June 2009
104 Mꢀ1). The stoichiometries of the lectin-ligand complexes were in agreement with the formal valen-
cies (1–3) of the respective ligands indicating cross-linking in interactions with the di- and trivalent
derivatives. Multivalency effects could not, however, be observed with the latter. These ligands were
shown to bind to the carbohydrate binding site of Con A using saturation transfer difference (STD)
NMR competition experiments.
Keywords:
Glycomimetics
Glycosyl disulfides
Isothermal titration calorimetry
Ligand–lectin interactions
Saturation transfer difference (STD) NMR
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
were clearly demonstrated in vivo on human tumor cell lines.
Based on these results disulfide-linked sugar derivatives were sug-
Lectins are carbohydrate-binding proteins involved in a multi-
tude of biological processes. To provide insight into the molecular
basis of their biological activity, the binding of synthetic carbohy-
drate derivatives or glycoconjugates is currently studied with var-
ious techniques (for a recent survey, see Ref. 1). Discovering novel
carbohydrate structures with lectin-binding properties may con-
tribute to our understanding of the structure, energetics, and
dynamics of lectin–saccharide complexes.
Replacing the interglycosidic oxygen in disaccharides by a
disulfide bond has been proposed as a promising approach to ob-
tain novel glycomimetics.2,3 The disulfide bridge, featuring a
three-bond distance between the anomeric carbon and the agly-
con, provides a larger conformational space than the natural,
two-bond glycosidic linkage,4 and differences in the stereoelec-
tronic properties between the O- and S-atoms may also play a role
in interactions with proteins. Such studies have not, however, been
reported until recently when it was shown4–6 that appropriately
positioned symmetric diglycosyl disulfides are capable to bind to
various lectins, as judged from competition binding experiments.
Importantly, inhibitory activities against an endogenous lectin
gested ‘as new substance platform for lectin-directed drug de-
sign’.4 The binding affinities of the disulfide disaccharides with
manno configurations were found to be comparable to that of
methyl
a-D-mannopyranoside in experiments with Concanavalin
A.6 The structures and thermodynamics of Con A in complexes
with various mannopyranosides have previously been extensively
investigated in solution and in the solid state.7–12
In the present study, we have chosen to attach one to three
mannopyranosyl units to a benzene ring through disulfidomethyl-
ene linkages to prepare mono- to trivalent glycosyl disulfide deriv-
atives. It is known that the binding of aromatic glycosides to Con A
is stronger than aliphatic ones.13 We desired to study interactions
of these novel ligands with Con A to assess the binding affinities
and thermodynamic parameters, and also to probe multivalency
effects, if any.
2. Results and discussion
2.1. Syntheses of the ligands 1–6
The sodium salt of 1-thio-
from 2,3,4,6-tetra-O-acetyl-1-thio-
a
-
a
D
-mannopyranose, obtained
-D
-mannopyranose14,15 by
* Corresponding authors. Fax: +36 52 453836 (L.S.).
E-mail address: lszilagyi@tigris.klte.hu (L. Szilágyi).
treatment with sodium methoxide, was reacted directly with
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.06.008

B. N. Murthy et al. / Carbohydrate Research 344 (2009) 1758–1763
1759
methanethiosulfonatomethylenebenzenes I to IV (Scheme 1) fol-
lowing a modified procedure.16 The mannopyranosyl disulfide li-
gands 1–4 (Chart 1) were obtained after purification by column
chromatography (see Section 3).
Compounds 5 and 6 were prepared by standard procedures as
described in Section 3.
the enthalpy, and the entropy changes.17,18 The ITC measurements
were conducted at pH 7.4, in HEPES buffer and the ligand and lec-
tin concentrations ranged from 2.5 to 9 mM and from 0.2 to
0.4 mM, respectively. The Con A exists as a tetramer at this pH.19
Displayed in Figure 1 are representative raw and integrated
plots for the calorimetric titration of the monovalent sugar disul-
fide ligand 1 and the divalent ligand 3. The equilibrium con-
stants, thermodynamic parameters, and stoichiometries for the
interactions of ligands 1 to 6 with Con A are presented in Table
1. The binding parameters of the glycosyl disulfides were com-
2.2. ITC studies
The binding potencies of 1–6 (Chart 1) were assessed first by
ITC, which provides not only the binding constants but also the
associated thermodynamic parameters, the free energy changes,
pared with methyl
a-D-mannopyranoside (MeaMan), D-mannose
(Man), thioglycoside 5, and glycoside 6. Compounds 5 and 6
OAc
AcO
OH
HO
O
O
NaOMe, MeOH
AcO
AcO
HO
HO
SH
SNa
+
OH
O
HO
HO
HO
CH3SO2SCH2
S
S
CH2
n
n
I : n = 1 II, III, : n = 2 IV : n = 3
1
2 3
4
: n = 3
: n = 1
,
, : n = 2
Scheme 1. Synthesis of glycosyl disulfide ligands 1–4.
OH
HO
O
HO
HO
HO
OH
O
HO
HO
S
S
S
1
5
HO
HO
HO
OH
OH
OH
OH
OH
O
O
S
S
S
S
HO
HO
HO
OH
O
2
OH
OH
OH
OH
O
O
6
HO
OH
O
HO
HO
S
S
S
S
3
OH
OH
OH
OH
HO
HO
HO
OH
O
O
S
S
S
S
OH
OH
OH
OH
O
4
S
S
Chart 1. Structures of the mono-, di-, and trivalent mannopyranosyl ligands 1–6.

1760
B. N. Murthy et al. / Carbohydrate Research 344 (2009) 1758–1763
Time (min)
10 20 30 40 50 60 70 80 90 100
a
Time (min)
10 20 30 40 50 60 70 80 90
b
0
0
0
-2
-4
-6
0
-2
-4
-6
-8
0
-8
0
-2
-4
-6
-8
-2
-4
-6
1
3
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Molar Ratio
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Molar Ratio
Figure 1. Raw and integrated data for the calorimetric titration of Con A with (a) monovalent sugar disulfide 1 and (b) divalent sugar disulfide 3.
Table 1
2 and 3 to separate lectin binding sites. Similarly, the n-value of
Binding stoichiometries and thermodynamic parameters for various glycosyl disulfide
0.39 observed for 4 is an indication of approximate functional tri-
valency for this triglycoside-type ligand. In the absence of the pos-
sibility for an intramolecular complexation within the lectin
tetramer, due to distance constraints, the observed functional
valencies relate to intermolecular complexation, that is, the
cross-linking of the lectin tetramers by the ligands. While the
cross-linked complexes exist in solution at low molar ratios of li-
gand to lectin, at higher ratios the cross-linking between the ligand
and the lectin becomes dense. As a result, a visible precipitation of
the complex could be observed in the ITC cell when saturation of
the binding sites was completed.
In order to elucidate whether the binding of disulfide ligands
occurs at the carbohydrate binding site of the lectin, as opposed
to non-specific binding, competition experiments were run, using
saturation transfer difference (STD) NMR spectroscopy.23,24 This
method has found widespread application in the study of carbohy-
drate–protein recognition phenomena25 and is suitable to investi-
gate competition between two ligands for the same binding site on
the protein. Changes in the STD signal intensities upon titration of
a protein–ligand A complex with ligand B are indicative of a com-
petition process between A and B.24
ligands–lectin interactionsa
Ligand
N
Ka
D
G
D
H
TDS
1
2
3
4
5
6
0.96
0.56
0.54
0.39
1.1
1.03
1.04
1.0
(9.4 0.3) ꢁ 103
(2.11 0.2) ꢁ 104
(3.71 0.2) ꢁ 104
(3.16 0.2) ꢁ 104
(8.6 0.08) ꢁ 103
(2.23 0.02) ꢁ 104
(7.9 0.04) ꢁ 103
(2.21 0.05) ꢁ 103
ꢀ5.41
ꢀ5.9
ꢀ5.83 0.1
ꢀ8.73 0.2
ꢀ10.25 0.1
ꢀ12.7 0.4
ꢀ7.21 0.04
ꢀ9.32 0.06
ꢀ7.83 0.12
ꢀ3.92 0.2
ꢀ0.42
ꢀ2.83
ꢀ4.02
ꢀ6.53
ꢀ1.86
ꢀ3.37
ꢀ2.56
0.67
ꢀ6.23
ꢀ6.17
ꢀ5.35
ꢀ5.95
ꢀ5.27
ꢀ4.42
MeaMan
Man
a
Thermodynamic parameters were derived from one-site binding model; Ka is in
G, H, and T
S are in the units of kcal molꢀ1. Errors in
G are ꢂ1–
S are in the range of 1–3%.
the unit of Mꢀ1
5%. Errors in T
;
D
D
D
D
D
were included to investigate the role, if any, of the disulfide
bond on the binding.
The c parameter, which is the product of the macromolecular
concentration and the binding constant,20,21 was above 1 for all
the ligand–lectin complexations. As shown in Table 1, the
gurated ligands 1–4 exhibited binding affinities comparable to or
slightly better than the monovalent glycosides 5, 6, or Me Man.
The association constant (Ka) values for the monovalent disulfide-
and thioglycoside derivatives 1 and 5, respectively, are practically
a-confi-
a
An example is shown in Figure 2. Traces (a) and (b) are the reg-
ular and STD 1H NMR spectra, respectively, of 3 in the presence of
Con A. The appearance of the ligand signals in (b) is indicative of
the binding of ligand 3 to Con A. Saturation transfer resonances
in spectrum (b) reveal contacts of sugar ring protons 2–6 (3.5–
3.9 ppm) and aromatic protons (7.3–7.5 ppm) to the protein. The
anomeric signal (4.7 ppm) is suppressed by the water suppression
sequence which also reduces the intensities of the SCH2 resonances
identical with that of MeaMan. Interestingly, the O-glycoside 6
binds ca. three times stronger. The Ka enhancements observed for
the di- and trivalent ligands 2 and 4, respectively, may be ascribed
to the binding of individual mannose units in these derivatives to
independent lectin binding sites (vide infra). The binding of the
divalent ligand 3 is, however, stronger than expected on a valen-
cy-corrected basis alone.
at 4.12 and 4.24 ppm. Addition of increasing amounts of MeaMan
to the sample causes to decrease the signal intensities of 3. When
The binding process is favored by large enthalpic changes with
all the ligands. The binding stoichiometry was close to 1 for all the
monovalent derivatives, whereas it was close to 0.5 for the divalent
ligands 2 and 3. This is an indication of the functional divalency22
of these derivatives, that is, binding of each of the sugar residues in
signal intensities are plotted as a function of the concentration of
the MeaMan competitor added to the Con A–3 complex, displace-
ment of 3 by Me
a
Man becomes evident (Fig. 3). This is an evidence
to indicate that the binding of 3 takes place at the carbohydrate
binding site of Con A.

B. N. Murthy et al. / Carbohydrate Research 344 (2009) 1758–1763
1761
(b)
(a)
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
ppm
Figure 2. Binding of ligand 3 to Con A as seen in a 1H NMR STD experiment. Sample conditions: Con A (80
l
M), NH4OAc buffer (100 mM, pH 7.2), NaCl (150 mM), CaCl2
(2 mM) in 500
l
L D2O at 300 K. (a) ligand 3 (4 mM), standard 1H NMR spectrum; (b) same sample, STD 1H NMR spectrum. For the STD measurement conditions, see: Section 3.
sugar-disulfide-based approach especially in view of increasing
evidence that ligand efficiency does not depend only on valency
but on a multitude of other factors such as the topology and archi-
80
70
60
50
40
30
20
tecture of the epitopes, their density, and/or the nature of the scaf-
,12,26–30
folds.
On the other hand, the disulfide linkage is easily
established and offers remarkable possibilities to manipulate the
interactions via mild chemical transformations of this bond.31–33
3. Experimental
3.1. General methods
Chemicals were purchased from commercial sources and were
used without further purifications. Solvents were dried and dis-
tilled according to literature procedures. Analytical TLC was per-
formed on commercial Merck plates coated with Silica Gel GF254
(0.25 mm). Silica Gel Merck (100–200 mesh) was used for column
chromatography. High-resolution mass spectra were recorded on a
Bruker micrOTOF-Q instrument by electrospray ionization (ESI)
technique. Lectin Con A (salt-free lyophilized powder) was pur-
chased from Sigma.
10
2.5
3
3.5
4
log[MeαMan]
Figure 3. Titration data for the experiment in Figure 2. Changes of the aromatic
signal intensities (ꢂ7.4 ppm) in the STD 1H NMR spectra of ligand 3 as a function of
the added MeaMan. The concentration (log[MeaMan] in lM) of the added MeaMan
competitor was increased from 0.4 to 4 mM.
3.2. ITC and NMR studies
In summary, we have constructed novel glycosyl disulfide
derivatives characterized by attachment of one to three -man-
Isothermal titrations were performed using a microcalorimeter
Microcal VP-ITC. Aqueous solutions were prepared from doubly
a-D
nopyranosyl units to a benzene ring through disulfidomethylene
linkages. ITC measurements indicated binding of these derivatives
to the lectin Con A with binding affinities comparable to or slightly
distilled water purified through a Milli Q-plus system to 18.2 MX
resistance. All ligand–lectin binding experiments were performed
in HEPES buffer (10 mM) (pH 7.4) containing NaCl (150 mM), CaCl2
higher than that of MeaMan. The stoichiometries of the lectin–li-
(1 mM), and MnCl2 (1 mM). Buffer solution was filtered (0.2 lm)
gand complexes were in agreement with the formal valencies (1–
3) of the respective ligands therefore, cross-linking is likely to oc-
cur in interactions with the di- and trivalent derivatives (2–4).
Multivalency (or cluster glycoside) effects could not, however, be
observed with the latter. Saturation transfer difference (STD)
NMR competition experiments indicated binding to the carbohy-
drate binding site of Con A. The present derivatives, together with
recently reported diglycosyl-disulfides4–6 represent novel carbohy-
drate structures with lectin-binding properties to study lectin–car-
bohydrate interactions. Further studies will be needed to assess the
and thoroughly degassed. The concentration of Con A was deter-
mined spectrophotometrically at 280 nm using A1% 1cm = 13.7 at
pH 7.4 and expressed in terms of monomer (Mr = 25, 600).34 In
individual titrations, injection of 6–10
lL of ligand was added from
the computer-controlled 300 L microsyringe at intervals of 3 min
l
into the Con A solution dissolved in the same buffer as the ligand;
the microsyringe stirring at 300 rpm. All measurements were
We thank a referee for raising these points.

1762
B. N. Murthy et al. / Carbohydrate Research 344 (2009) 1758–1763
made at 25 °C. Control experiments were performed by injecting
the ligand into a cell containing buffer with no protein, and the
heats of dilution subtracted from those measured in the presence
of Con A. The initial injection was discarded in order to remove
the effect of titrant diffusion across the syringe tip during the
equilibration process. The experimental data were fitted to a one
site binding model, using a nonlinear least-squares procedure,20
3.3.4. 1,3-Bis(
a-D-mannopyranosyldithiomethylene)benzene
(2)
White solid, 1.103 g (76%), mp 76–78 °C, ½a D22
ꢃ
+114.7 (c 0.22,
MeOH); 1H NMR (CD3OD, 500 MHz): d 7.27–7.38 (m, 4H, Aryl-H),
5.11 (d, 2H, J1,2 1.6 Hz, H-1), 4.11 (d, 2H, J
12.3 Hz, S–CH2a),
S-CH2a;b
4.02 (d, 2H, S–CH2b), 3.96 (dd, 2H, J2,3 3.3 Hz, H-2), 3.88 (dd, 2H,
J5,6a 2.3 Hz, J6a,6b 11.6 Hz, H-6a), 3.84 (m, 2H, H-5), 3.77 (dd, 2H,
J5,6b 5.7 Hz, H-6b), 3.67 (t, 2H, J4,5 9.5 Hz, H-4), 3.58 (dd, 2H, J3,4
9.5 Hz, H-3); 13C NMR (D2O, 125 MHz): d 138.6, 131.7, 130.3,
129.9 (Aryl-C), 93.0 (C-1), 75.6 (C-5), 72.4 (C-2, C-3), 67.8 (C-4),
61.9 (C-6), 43.9 (S–CH2); HRESIMS: calcd for C20H30O10S4
[M+Na]+: 581.0614; found: m/z 581.0630.
with
DH, Ka (association constant), and n (number of binding sites
for monomer), as adjustable parameters.
1H and 13C NMR spectral analyses were performed using a Bru-
ker Avance DRX-500 spectrometer operating at 500 and 125 MHz,
respectively, and equipped with a 5 mm z-gradient multinuclear
proton detection (bbi) probe head. The residual solvent signal
was used as the internal standard. For STD measurements the
3.3.5. 1,2-Bis(a-D-mannopyranosyldithiomethylene)benzene
duration of the 1H 90° pulse was 15.5
ls and semi-selective irradi-
(3)
ation of Con A resonances was achieved by a train of Gaussian 90°
pulses of 50 ms each. The residual water signal was suppressed by
a WATERGATE sequence. All spectra were processed with XWINNMR
2.6. The samples for STD experiments contained 4 mM carbohy-
drate ligand in complex with 80 lM Con A in 500 lL D2O with
150 mM NaCl and 2 mM CaCl2 added in a 100 mM NH4OAc buffer
Pale yellowish solid, 1.147 g (79%), mp 134–136 °C, ½a D22
ꢃ
+63.9
(c 0.5, MeOH); 1H NMR (CD3OD, 500 MHz): d 7.24–7.36 (m, 4H,
Aryl-H), 5.13 (d, 2H, J1,2 1.5 Hz, H-1), 4.36 (d, 2H, JS-CH
12.2 Hz,
2a;b
S-CH2a), 4.18 (d, 2H, S-CH2b), 3.95 (dd, 2H, J2,3 3.2 Hz, H-2), 3.87
(dd, 2H, J5,6a 2.2 Hz, J6a,6b 11.5 Hz, H-6a), 3.83 (m, 2H, H-5), 3.79
(dd, 2H, J5,6b 5.1 Hz, H-6b), 3.69 (t, 2H, J4,5 9.4 Hz, H-4), 3.60 (dd,
2H, J3,4 9.4 Hz, H-3); 13C NMR (D2O, 125 MHz) d 136.2, 132.7,
129.5 (Aryl-C), 93.2 (C-1), 75.5 (C-5), 72.4 (C-2), 72.3 (C-3), 67.7
(C-4), 61.9 (C-6), 42.1 (S–CH2); HRESIMS: calcd for C20H30O10S4
[M+Na]+: 581.0614; found: m/z 581.0620.
of pH 7.2 at 300 K.
3.3. Synthetic procedures
3.3.1. General procedure for the preparation of aromatic
methanethiosulfonates I–IV
3.3.6. 1,3,5-Tris(a-D-mannopyranosyldithiomethylene)benzene
Sodium methanethiosulfonate was prepared from sodium sul-
fide and mesyl chloride as described.35 Aromatic methanethiosulf-
onates I, II, and III were synthesized via reaction of sodium
methanethiosulfonate with commercially available mono- and
bis(bromomethyl)benzenes according to literature procedures.36,37
1,3,5-Tris(methanethiosulfonatomethylene)benzene IV was ob-
tained via an analogous reaction starting from 1,3,5-tris(bromo-
methyl)benzene (89%), mp 100–102 °C; HRESIMS: calcd for
C12H18O6S6 [M+Na]+: 472.932; found: m/z 472.936. 1H NMR (CDCl3,
500 MHz) d 7.41 (s, 3H, Aryl-H), 4.38 (s, 6H, SCH2), 3.12 (s, 9H,
CH3); 13C NMR (CDCl3 125 MHz) d 137.3 (C-1,-3,-5), 129.5 (C-2,-
4,-6), 51.1 (CH3), 39.9 (SCH2).
(4)
White solid, 1.190 g (58%), mp 153–155 °C, ½a D22
ꢃ
+31.6 (c 0.5,
MeOH); 1H NMR (CD3OD, 500 MHz): d 7.29 (br s, 3H, Aryl-H),
5.12 (d, 3H, J1,2 1.5 Hz, H-1), 4.11 (d, 3H, J
12.6 Hz, S–CH2a),
S-CH2a;b
4.03 (d, 3H, S–CH2b), 3.96 (dd, 3H, J2,3 3.2 Hz, H-2), 3.90 (dd, 3H,
J5,6a 2.3 Hz, J6a,6b 11.8 Hz, H-6a), 3.85 (m, 3H, H-5), 3.77 (dd, 3H,
J5,6b 5.9 Hz, H-6b), 3.65 (t, 3H, J4,5 9.5 Hz, H-4), 3.58 (dd, 3H, J3,4
9.5 Hz, H-3); 13C NMR (CD3OD 125 MHz): d 139.5, 130.8, (Aryl-C),
94.6 (C-1), 76.5 (C-5), 73.1 (C-2, C-3), 68.8 (C-4), 62.9 (C-6), 44.1
(S–CH2); HRESIMS: calcd for C27H42O15S6 [M+Na]+: 821.0740;
found: m/z 821.0755.
3.3.7. 2-Phenylethyl 1-thio-
This compound was prepared as described14 starting from
2,3,4,6-tetra-O-acetyl-1-thio- -mannopyranose. The syrup ob-
a-D-mannopyranoside (5)
3.3.2. General procedure for the syntheses of glycosyl disulfide
ligands 1–4
a-D
tained after deacetylation could not be crystallized. Overall yield
2,3,4,6-Tetra-O-acetyl-1-thio-a-D
-mannopyranose14,15 (0.910 g,
0.476 g (59%); ½a D22
ꢃ
+186 (c 0.4, MeOH), lit.14
½
a 2D7
+198 (c 0.9,
ꢃ
2.5 mmol) in dry MeOH (10 mL) was treated with 1 M sodium
methoxide in MeOH (2.6 mL, 2.6 mmol) at room temperature for
0.5 h. Water (3–4 mL) was added to dissolve the precipitated so-
MeOH). 1H NMR (Me2SO-d6, 500 MHz) d 7.34–7.12 (m, 5H, phe-
nyl-H), 5.18 (br s, 1H, H-1), 3.69 (dd, 1H, H-2), 3.64 (m, 1H, H-5),
3.36–3.52 (m, 4H, H-3, H-4, H-6a,b), 2.86–2.80 (m, 4H, 2 ꢁ CH2).
13C NMR (Me2SO-d6, 125 MHz) d 140.7, 128.7, 128.5, 126.3 (phe-
nyl), 85.0 (C-1), 74.6, 72.0, 71.7, 67.5 (C-2 to C-5), 61.3 (C-6),
35.7, 31.7 (2 ꢁ CH2). HRESIMS: calcd for C14H20O5S [M+Na]+:
323.0929; found: m/z 323.0922.
dium
D-mannopyranose 1-thiolate followed by the addition of
the calculated amounts of methanethiosulfonates I, II, III, or IV.
The reaction mixture was kept at rt until TLC (38:7:3 EtOAc–
MeOH–water) indicated disappearance of the starting materials
(ca. 1 h). After evaporation to dryness under diminished pressure,
the crude products were purified by column chromatography on
silica gel.
3.3.8. 2-Phenylethyl
a-D-mannopyranoside (6)
Dry -mannose (1 g, 5.5 mM) and 2-phenylethanol (6.8 mL,
D
55 mM) were refluxed in dry MeCN (130 mL) in the presence of
Amberlyst cation exchange resin for 5 h. After filtration, the reac-
tion mixture was diluted with water, extracted with CH2Cl2, and
the aqueous phase concentrated to dryness. The desired phenyl-
3.3.3. (a-D-Mannopyranosyldithiomethylene)benzene (1)
White amorphous solid, 0.690 g (81%), ½a D22
ꢃ
+75.7 (c 0.34,
MeOH); 1H NMR (CD3OD, 500 MHz): d 7.45–7.48 (m, 5H, Aryl-
H), 5.09 (d, 1H, J1,2 1.7 Hz, H-1), 4.11 (d, 1H, JS-CH
13.3 Hz, S–
ethyl
a-mannoside was isolated from the anomeric mixture
2a;b
CH2a), 4.05 (d, 1H, S–CH2b), 3.96 (m, 1H, H-2), 3.90 (dd, 1H, J5,6a
2.4 Hz, J6a,6b 12.7 Hz, H-6a), 3.89 (m, 1H, H-5), 3.81 (dd, 1H, J5,6b
6.2 Hz, H-6b), 3.61–3.72 (m, 2H, H-3, H-4); 13C NMR (CD3OD
125 MHz): d 138.8, 130.6, 129.7, 128.6 (Aryl-C), 94.6 (C-1), 76.5
(C-5), 73.1 (C-2, C-3), 68.9 (C-4) 62.9 (C-6), 44.5 (S-CH2); HRE-
SIMS: calcd for C13H18O5S2 [M+Na]+: 341.0488; found: m/z
341.0500.
through silica gel column chromatography (8:1.5:1 EtOAc–
MeOH–water). Colorless glassy material, 0.392 g (23%),
½ ꢃ
a 2D2
+54.8 (c 0.5, MeOH). 1H NMR (D2O, 500 MHz): d 7.35–7.20 (m,
5H, phenyl-H), 4.76 (br s, 1H, H-1), 3.87 (m, 1H, OCH2(a)), 3.81
(dd, 1H, H-2), 3.76 (m, 1H, OCH2(b)), 3.65 (dd, 1H, H-6a), 3.59
(dd, 1H, H-3), 3.54 (br.d, 1H, H-6b), 3.53 (t, 1H, H-4), 3.13 (m,
1H, H-5), 2.89–2.78 (m, 2H, Ph-CH2). 13C NMR (D2O, 125 MHz): d

B. N. Murthy et al. / Carbohydrate Research 344 (2009) 1758–1763
1763
11. Dam, T. K.; Roy, R.; Page, D.; Brewer, C. F. Biochemistry 2002, 41, 1359–
1363.
12. Gouin, S. G.; Vanquelef, E.; Garcia Fernandez, J. M.; Ortiz Mellet, C.; Dupradeau,
F. Y.; Kovensky, J. J. Org. Chem. 2007, 72, 9032–9045.
13. Poretz, R. D.; Goldstein, I. J. Biochem. Pharmacol. 1971, 20, 2727.
14. Durette, P. L.; Shen, T. Y. Carbohydr. Res. 1980, 81, 261–274.
15. Haque, M. B.; Roberts, B. P.; Tocher, D. A. J. Chem. Soc., Perkin Trans. 1 1998,
2881–2889.
139.1, 128.9, 128.5, 126.4 (phenyl), 99.3 (C-1), 72.4 (C-5), 70.4 (C-
3), 69.9 (C-2), 67.9 (OCH2), 66.3 (C-4), 60.5 (C-6), 34.9 (Ph-CH2).
HRESIMS: calcd for C14H20O6 [M+Na]+: 307.1158; found: m/z
307.1146.
Acknowledgments
16. Rendle, P. M.; Seger, A.; Rodrigues, J.; Oldham, N. J.; Bott, R. R.; Jones, J. B.;
Cowan, M. M.; Davis, B. G. J. Am. Chem. Soc. 2004, 126, 4750–4751.
17. Dam, T. K.; Brewer, C. F. Chem. Rev. 2002, 102, 387–429.
18. Christensen, T.; Toone, E. J. In Recognition of Carbohydrates in Biological Systems
Part A: General Procedures, 2003, pp 486–504.
19. Loris, R. Biochim. Biophys. Acta Gen. Subj. 2002, 1572, 198–208.
20. Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L. N. Anal. Biochem. 1989, 179,
131–137.
Financial support from the Hungarian Science Research Fund
(OTKA T-48713 and NK-68578) and Department of Science and
Technology, New Delhi, is gratefully acknowledged. We also thank
the Indo-Hungarian scientific exchange program (TéT IND-10/03;
DST/INT/HUN/P-06/03) for travel support. The excellent technical
assistance of Sára Balla (Debrecen) in the synthetic procedures is
highly appreciated.
21. Turnbull, W. B.; Daranas, A. H. J. Am. Chem. Soc. 2003, 125, 14859–14866.
22. Dimick, S. M.; Powell, S. C.; McMahon, S. A.; Moothoo, D. N.; Naismith, J. H.;
Toone, E. J. J. Am. Chem. Soc. 1999, 121, 10286–10296.
23. Mayer, M.; Meyer, B. Angew. Chem., Int. Ed. 1999, 38, 1784–1788.
24. Mayer, M.; Meyer, B. J. Am. Chem. Soc. 2001, 123, 6108–6117.
25. Johnson, M. A.; Pinto, B. M. Carbohydr. Res. 2004, 339, 907–928.
26. Cairo, C. W.; Gestwicki, J. E.; Kanai, M.; Kiessling, L. L. J. Am. Chem. Soc. 2002,
124, 1615–1619.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2009.06.008.
27. Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling, L. L. J. Am.
Chem. Soc. 2002, 124, 14922–14933.
28. Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E. Angew. Chem., Int. Ed. 2006, 45,
2348–2368.
References
29. Smiljanic, N.; Moreau, V.; Yockot, D.; Benito, J. M.; Garcia Fernandez, J. M.;
Djedaini-Pilard, F. Angew. Chem., Int. Ed. 2006, 45, 5465–5468.
30. Gomez-Garcia, M.; Benito, J. M.; Rodriguez-Lucena, D.; Yu, J. X.; Chmurski, K.;
Ortiz Mellet, C.; Gallego, R. G.; Maestre, A.; Defaye, J.; Garcia Fernandez, J. M. J.
Am. Chem. Soc. 2005, 127, 7970–7971.
31. Ramström, O.; Lehn, J.-M. ChemBioChem 2000, 1, 41–48.
32. Ramström, O.; Lohmann, S.; Bunyapaiboonsri, T.; Lehn, J. M. Chem. Eur. 2004,
10, 1711–1715.
1. Lectins Analytical Technologies; Nilsson, C. L., Ed., 1st ed.; Elsevier, 2007.
2. Szilágyi, L.; Illyés, T. Z.; Herczegh, P. Tetrahedron Lett. 2001, 42, 3901–3903.
3. Szilágyi, L.; Varela, O. Curr. Org. Chem. 2006, 10, 1745–1770.
4. André, S.; Pei, Z. C.; Siebert, H. C.; Ramström, O.; Gabius, H. J. Bioorg. Med. Chem.
2006, 14, 6314–6326.
5. Pei, Z. C.; Aastrup, T.; Anderson, H.; Ramström, O. Bioorg. Med. Chem. Lett. 2005,
15, 2707–2710.
6. Pei, Z. C.; Larsson, R.; Aastrup, T.; Anderson, H.; Lehn, J. M.; Ramström, O.
Biosens. Bioelectron. 2006, 22, 42–48.
7. Swaminathan, C. P.; Surolia, N.; Surolia, A. J. Am. Chem. Soc. 1998, 120, 5153–5159.
8. Derewenda, Z.; Yariv, J.; Helliwell, J. R.; Kalb, A. J.; Dodson, E. J.; Papiz, M. Z.;
Wan, T.; Campbell, J. EMBO J. 1989, 8, 2189–2193.
9. Schwarz, F. P.; Puri, K. D.; Bhat, R. G.; Surolia, A. J. Biol. Chem. 1993, 268, 7668–
7677.
33. McDonald, R. E.; Hughes, D. J.; Davis, B. G. Angew. Chem., Int. Ed. 2004, 43,
3025–3329.
34. The Lectins: Properties, Functions and Applications in Biology and Medicine;
Goldstein, I. J., Poretz, R. D., Eds.; Academic Press: New York, 1998.
35. Shaked, Z.; Szajewski, R. P.; Whitesides, G. M. Biochemistry 1980, 19, 4156–
4166.
36. Dunbar, J. E. Dow Chemical Co., U.S. Patent No. 3,282,976, 1966, p 3.
37. Macke, J. D.; Field, L. J. Org. Chem. 1988, 53, 396–402.
10. Debenham, S. D.; Snyder, P. W.; Toone, E. J. J. Org. Chem. 2003, 68, 5805–5811.