Synthesis and binding affinity analysis of positional thiol analogs of mannopyranose for the elucidation of sulfur in different position

Article abstract of DOI:10.1016/j.tet.2015.04.060

Synthetic routes towards thio-α/β-d-mannose derivatives are presented. Double parallel or double serial inversion was successfully applied in the efficient synthesis of 2-thio- or 2,4-di-thio-mannoside derivatives. The protein recognition properties of th

Full text of DOI:10.1016/j.tet.2015.04.060

Tetrahedron 71 (2015) 4023e4030
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and binding affinity analysis of positional thiol analogs of
mannopyranose for the elucidation of sulfur in different position
,
a,
Bin Wu a, Jiantao Ge a, Bo Ren a, Zhichao Pei b , Hai Dong
*
*
a Key Laboratory for Large-Format Battery Materials and System, Ministry Education, School of Chemistry & Chemical Engineering, Huazhong
University of Science & Technology, Luoyu Road 1037, Wuhan, 430074, PR China
b State Key Laboratory of Crop Stress Biology in Arid Areas and College of Science, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthetic routes towards thio-a/b-D-mannose derivatives are presented. Double parallel or double serial
Received 17 March 2015
Received in revised form 14 April 2015
Accepted 18 April 2015
inversion was successfully applied in the efficient synthesis of 2-thio- or 2,4-di-thio-mannoside de-
rivatives. The protein recognition properties of the synthesized positional thiol analogs of mannose were
then evaluated in a competition binding assay with the model lectin Concanavalin A (Con A), in order to
investigate the roles of thiol group in the different position of the mannopyranose ring in binding affinity.
Available online 24 April 2015
Though the substitution of oxygen atom with sulfur atom in the methyl
displayed low or no binding affinity towards Con A, it was a surprise finding that the methyl 2-thio-
mannoside displayed four times higher inhibition than methyl -mannoside, indicating the particular
importance of 2-position for modification of -mannoside. Methyl 3-thio- -mannoside also dis-
a-D-mannoside ring usually
Keywords:
Sulfur-containing mannosides
Double inversion
Binding affinity
Concanavalin A
a-D-
a-D
a
-D
a-D
played inhibition towards Con A, indicating that the O-atom at the C-3 position is less important in the
binding site.
Quartz crystal microbalance
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
generating dynamic carbohydrate libraries based on easy oxidation
of thiols to give disulfides.27e31
Carbohydrateeprotein recognitions are of great significance in
many fundamental cellular processes such as adhesion, trafficking,
communication, proliferation, and cell death.1e5 The recognitions
are involved in cancer and in the early stages of infection, playing
roles of cell surface receptors enabling adherence of bacteria, par-
asites, and viruses.6e9 New therapies and drugs would be able to be
developed if these interactions are thoroughly understood and
controlled. Thus, well-defined carbohydrate ligands need to be
synthesized and further studied on their interactions to the cor-
responding proteins. Sulfur-containing glycosides are often applied
in the synthesis of thio-oligosaccharides.10e16 In comparison with
nature O-linked ligands, studies have shown that various thio-
oligosaccharides display increased conformational flexibility.17e19
Generally these sulfur-containing analogs are more stable due to
lower rates of both acid-catalyzed and enzymatic hydrolysis, and
thus are potential candidates as glycosidase inhibitors and are of
special interests in enzyme-inhibition studies.20e26 In addition,
sulfur-containing carbohydrates have special advantages in
The lectins are another class of carbohydrate-specific proteins
besides enzymes and antibodies. Carbohydrate-lectin interactions
play important roles in cell-cell recognition.32e34 Concanavalin A
(Con A) is the most extensively studied member of the lectins and
has a strong binding affinity to the a
-linked mannose.35e37 This
binding to the mannose moiety of glycoproteins on cell surface is
relate to the cell growth and death. For example, Con A has been
found as potential anti-hepatoma therapeutic recently.38 In early
studies, by the analysis of affinity of various derivatives of D-man-
nose and other saccharides to Con A, Goldstein et al. suggested that
O-atoms of the C-1, C-2 and C-3 hydroxyl groups and H-atoms of
the C-4 and C-6 are involved in the H-bonding to the protein.39e42
Recently, by the analysis of methyl a-mannoside-Con A complex
crystal structure, it was suggested that 3-, 4- and 6-OHs of the
mannoside provide the main contribution to affinity through H-
bonding, whereas 2-OH and 1-OMe extend into solvent.36,37
However, the complex in real solvent might not be exactly the
same as its crystal structure. For example, as derivatives of 1- or 6-
thio mannose are easy to synthesize, the affinities of 1-thio-
mannose and methyl 6-thio- -mannoside towards Con A have
been measured.30 Methyl 6-thio-
-mannoside shows no activity
as expected. However, 1-thio- -mannose shows lower activity
compared to methyl -mannoside, suggesting that 1-OMe group
a-D-
a-D
a-D
a-D
* Corresponding authors. E-mail addresses: peizc@nwafu.edu.cn (Z. Pei), hdong@
mail.hust.edu.cn (H. Dong).
a-D
http://dx.doi.org/10.1016/j.tet.2015.04.060
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

4024
B. Wu et al. / Tetrahedron 71 (2015) 4023e4030
should be involved in pronounced interactions with the Con A
lectin. Thus, synthesis of various derivatives of -mannose modified
D
at the different positions and further measurement of their affinity
towards Con A in real solvent might provide additional information
for the binding mechanism. Synthesises of 1- or 6-thio glycosides
have been widely reported due to their distinctive reaction
selectivities.43e47 However, the synthesises of 2-, 3- or 4-thio gly-
cosides are rarely reported likely due to the requirements of com-
plicated multi-step reactions including selective protection/
deprotection and epimerization.48,49
Scheme 1. Synthesis of 1-thio-a/b-D-mannopyranose 2; (a) Ac2O, pyridine, rt, 4h; (b)
HOAc-HBr, BF3$Et2O, CH2Cl2, 0 ꢀC to rt, 2h, 90%; (c) TBASAc, toluene, 5h, 85%; (d) NaOH,
We have developed an efficient method for the synthesis of b-D-
MeOH, rt, 2h, then with Hþ exchange resin, 95%.
mannoside derivatives by double parallel or double serial in-
version.50 The strategy is based on multiple regioselective acylation
via the respective stannylene intermediates,51 followed by simulta-
neous inversion of both addressed hydroxyl groups or stepwise in-
version of the hydroxyl groups. The double parallel or double serial
inversion strategy was also applied in synthesis of orthogonally
protected galactosamine thioglycoside building blocks.52 Recently,
we developed a multiple regioselective acetylation method using
tetrabutylammonium acetate as a catalyst.53,54 This method is more
convenient and environmentally friendly than organotin method. In
the present study, we developed synthetic routes towards positional
Synthesis of methyl 6-thio-a-D-mannopyranoside 3 was re-
ported to go through compound 15 and 16 starting from free
methyl mannoside 14.30 In this method, tosylation of compound 14
formed compound 15, and compound 15 reacted with 5 equiv of
KSAc in DMF at 70 ꢀC to form compound 16 in 60% yield. After
careful scrutinize of this reaction, we found that compound 15
reacting with 1.5 equiv of KSAc at 35 ꢀC led to compound 17 in 70%
yield (Scheme 2). Treatment of compound 15 with 5 equiv of KSAc
at 35 ꢀC also formed compound 16. However the yield was im-
proved by 77% in this case.
thio-a/b-D-mannose derivatives (Fig. 1) using these methodologies.
Especially, 2-thio- and 2,4-dithio-mannoside derivatives were effi-
cient synthesized by the application of double parallel or double
serial inversion, so as to avoid complicated multi-step reactions. The
protein recognition properties of these positional thiol analogs of
mannose were then evaluated in a competition binding assay with
the model lectin Con A through Quartz Crystal Microbalance (QCM),
in order to investigate the roles of thiol group in the different po-
sition of the mannopyranose ring in binding affinity.
Scheme 2. Synthesis of methyl 6-thio-a C
-D-mannopyranoside 3; (a) TsCl, pyridine, 0 ꢀ
to rt, (70%); (b) KSAc, DMF, 35 ꢀC, 6h, 77%; (c) i: KSAc, DMF, 35 ꢀC, 6h; ii: KNO2, DMF,
35 ꢀC, 2h, 70%; (d) NaOH, MeOH, rt, 2h, then with Hþ exchange resin; DTT, H2O, rt, 12h,
90%.
Synthesis of methyl 3-thio-a-D-mannopyranoside 4 has never
been reported. The approach to this compound we developed is
showed in Scheme 3. Starting from free methyl mannoside 14,
compound 18 was easily synthesized in 70% total yield, through
organotin-mediated regioselective benzylation,57,58 pyridine-
mediated benzoylation and finally debenzylation by catalytic hy-
drogenation. However, compound 20 was only obtained in 33%
yield when inversing compound 18 using nitrite-mediated epi-
merization methods, owing to the neighboring group participa-
tion.55,56 Triflation of compound 20 followed by a substitution of
thioacetate group led to compound 21 in 78% yield.
Fig. 1. The positional thio-a/b-D-mannose derivatives.
2. Results and discussion
The 1-thio-
starting from a fully acetylated
lowed by a substitution of thioacetate group at the anomeric center,
in light of a reported method.30 Synthesis of the 1-thio-
-man-
a
-D-mannose compound 1 can be easily produced,
a-D-mannose compound 11 fol-
b-D
nose compound 2 has been reported in a quite low yield.46 In the
reported method, compound 11 was substituted by a bromide
group at the anomeric center to form compound 12, then treating
compound 12 with KSAc in DMPU to form compound 13 in 45%
yield. It is possible that the low yield of compound 13 was caused by
partial formation of the five-membered acyloxonium ring arising
from the 2-OAc group in the polar solvents.55,56 In our method
(Scheme 1), compound 12 was treated with TBASAc in no-polar
solvent toluene for avoiding the neighboring group participation,
leading to compound 13 in 85% yield.
Scheme 3. Synthesis of methyl 3-thio-a-D-mannopyranoside 4; (a) i: Bn2SnO, toluene,
reflux,4h; ii: BnBr, TBAB,100 ꢀC, 8h; iii: BzCl, pyridine, 0 ꢀC to rt, 12h; iiii: PdeC, H2,
EtOH/AcOH (2:1), overnight, 60% total yield; (b) i: Tf2O, pyridine, CH2Cl2, ꢁ30 to ꢁ10 ꢀC,
4h; ii: KNO2, DMF, 50 ꢀC, 6h, 55% of 19 and 33% of 20; (c) i: Tf2O, pyridine, CH2Cl2, ꢁ30
to ꢁ10 ꢀC, 4h; ii: TBASAc, toluene, rt, 5h, 78%; (d) NaOH, MeOH, rt, 4h, then with Hþ
exchange resin; DTT, H2O, rt, 12h, 90%.

B. Wu et al. / Tetrahedron 71 (2015) 4023e4030
4025
Methyl
synthesized through double parallel and double serial inversion
starting from methyl
-galactopyranoside.50 These results in-
spired us to further explore if methyl 2-thio-, 4-thio- and 2,4-di-
thio- -mannopyranosides can also be efficiently synthesized
b
-
D
-mannopyranoside derivatives had been efficiently
organotin-mediated method to form compound 33.51 Thus, com-
pound 33 was firstly obtained in 85% yield through this method,
and then followed by a nitrite-mediated inversion,55,59 to form
taloside 34 accompanied by a side product probably caused by
neighboring group participation.55,56 Though 34 was unable to
separate from the side product, triflation of the mixture followed by
substitution of thioacetate group led to desired compound 35 in
33% yield over four steps after purification.
b-D
a/b-D
by the application of the double parallel and double serial in-
version. The method was proven efficient for the synthesis of 2-
thio-, and 2,4-thio-
a
/b-
D-mannopyranosides (Scheme 4) but in-
efficient for the synthesis of 4-thio-a/b-D-mannopyranosides. 3,6-
di-OAc galactosides 23 and 30 were synthesized starting from
free galactosides 22 and 29 in high yields using acetate-mediated
methods.54 Triflation of compound 23 and 30 followed by sub-
stitution of thioacetate group afforded compound 25 and 31 in 63%
and 81% yields, respectively. In order to obtain products where only
the 2-positions were substituted by thioacetate group, the triflation
intermediates of compound 23 and 30 were allowed to react with
1.1 equiv of KOAc followed by substitution of thioacetate group. As
a result, compound 27 and 32 were obtained in 70% and 78% yields,
respectively. The triflation intermediates of compound 23 and 30
reacting with 1.1 equiv of KSAc led to intermediates where only the
4-positions were substituted by thioacetate group in high yields.
Scheme 5. Synthesis of methyl 4-thio-a-D-mannopyranoside; (a) i: Bu2SnO, MeOH,
reflux, 2h; ii: Ac2O, MeCN, 0 ꢀC to rt, 12h, 85%; (b) i: Tf2O, pyridine, CH2Cl2, ꢁ30 to
ꢁ10 ꢀC, 4h; ii: TBANO2, toluene, 50 ꢀC, 5h; (c) i: Tf2O, pyridine, CH2Cl2, ꢁ30 to ꢁ10 ꢀC,
4h; ii: TBASAc, toluene, rt, 6h., 35, 33% over 4 steps; (d) i: NaOH, MeOH, rt, 4h, then
with Hþ exchange resin; ii: DTT, H2O, rt, 12h, 88%.
The generation of 4-thio-a/b-D-mannopyranoside derivatives were
expected through following substitution by either acetate or nitrite.
However, this double serial inversion didn’t give any desired
products where the equatorial 2-position was supposed to be
inversed to an axial position. Therefore, in order to obtain 4-thio-
a-
Deacylation of these acylated sulfur-containing mannosides can
afford the desired free thio-mannosides. We recently demonstrated
that using NaOH and NaOMe in methanol for deacylation are
identical.60 Thus, in this study, all the deacylation were performed
in methanol using NaOH as a catalyst (Table S1). As the formed
sulfhydryl group appeared much stronger acidity than hydroxyl
group and would neutralize the base catalyst, a little more than
a stoichiometric amount of the base is necessary for each thio-
acetate group. The results proved NaOH as a catalyst efficient in the
deacylation of acylated sulfur-containing carbohydrates.
D-mannopyranoside 9, we have to design a conventional strategy
though it is much more complicated (Scheme 5). It was reported
that compound 14 could be regioselectively acetylated using
We have obtained positional thio-a/b-D-mannose derivatives
1e9. Their relative binding affinities towards the target lectin Con A
were then determined using a QCM flow-through instrumentation
and mannose-functionalized sensor chips. The polystyrene surfaces
were functionalized with PFPA-derivatized a-D-monomannoside by
a Photo-Click functionalization methodology (Fig. S1),61 which is
because a larger binding capacity of Con A was showed towards this
functionalized surface.14 In order to maintain reducing conditions
while analyzing the thiol monomers, a minimal amount of dithio-
threitol (DTT), showing no influence on the binding itself, was
added to the samples. The results from the binding study are pre-
sented in Fig. 2 and Table 1. The stable surfaces allowed for re-
producible binding over time thus generating inhibitory response
curves, which fitted well to the binding data. The EC50-value of
tested thio-mannosides compared with mannoside 14 and man-
nose are showed in Table 1.
It is well known that anomeric configuration is critical and
conformers play important roles for the mannose/Con A binding.
Thus methyl -mannoside 14 displays much stronger binding
towards Con A than mannose since mannose usually is a mixture of
and -conformer. It can be seen that the measured EC50 values by
us are 1.6 mM for methyl -mannoside 14 and 8.6 mM for
-conformer including 1-thio- -mannose 2,
-mannoside 7 and methyl-2,4-di-thio- -man-
a-
a-D
a
b
a-D
mannose. All
methyl-2-thio-
b
b-D
b
-D
b-D
noside 8 display low or no binding towards Con A as expected. 1-
thio- -mannose 1 displayed an EC50 value of 8.8 mM, almost
same as mannose. Methyl 3-thio- -mannoside 4 towards Con A
displayed an EC50 value of 4.1 mM, almost one-third as high in-
hibition as the methyl- -mannoside 14. Methyl-6-thio-
mannoside 3 and methyl-4-thio- -mannoside 9 displayed no
a-D
Scheme 4. Synthesis of methyl 2-thio- and 2,4-dithio-a/b-D-mannopyranosides; (a)
a-D
TBAOAc, Ac2O, MeCN, rt ꢁ40 ꢀC, 12h, 86%; (b) Tf2O, pyridine, CH2Cl2, ꢁ30 to ꢁ10 ꢀC, 4h;
(c) KSAc, DMF, rt, 24h, over 2 steps (25, 63%, 31, 81%), over 3 steps (27, 70%, 32, 78%);
(d) KOAc, DMF, rt, 1h; (e) i: NaOH, MeOH, rt, 4h, then with Hþ exchange resin; ii: DTT,
H2O, rt, 12h(5, 85%; 6, 81%; 7, 84%; 8, 78%).
a-
D
a-D-
a-D

4026
B. Wu et al. / Tetrahedron 71 (2015) 4023e4030
1-thio-β-mannose 2
1.0
0.5
0.0
methyl-2-thio-β-mannoside 7
1-thio-α-mannose 1
D
-mannose
methyl-3-thio-α-mannoside 4
methyl-α-mannoside 14
methyl-2-thio-α-mannoside 5
-2
0
2
log[man]/mM
Fig. 2. Competition binding plots of seven different mannosides towards Con A. (Competition plots of methyl-6-thio-
a-mannoside 3, methyl-2,4-di-thio-
a-mannoside 6, methyl-
2,4-di-thio-b-mannoside 8 and methyl-4-thio-a-mannoside 9 are not shown in this figure due to their extremely low binding affinity toward Con A).
Table 1
mannoside derivatives may lead to better inhibitor toward
mannoside-complexing proteins.
Estimation of EC50 values (50% inhibition of Concanavalin A binding) for tested
carbohydrates
Carbohydrates
EC50（mM）
R2
3. Conclusion
a1-S-
1-S-
a
-
D
D
-mannose 1
-mannose 2
-mannoside 3
8.8 (>5.0)30
>>30
>>30 (>>20)30
4.1
0.42
>>30
＞10
>>30
>>30
1.6
0.9951
d
b
-
A range of thio-a/b-D-mannose derivatives has been successfully
aMethyl 6-S-
a
-
D
d
synthesized using the strategies of protection/deprotection pat-
terns and inversions, especially using the double parallel or double
serial inversion for the synthesis of 2-thio- and 2,4-di-thio-man-
Methyl 3-S-
a
-
-
D
-mannoside 4
-mannoside 5
0.9975
0.9946
d
Methyl 2-S-
a
D
Methyl 2,4-S-
Methyl 2-S-
Methyl 2,4-S-
Methyl 4-S-
Methyl -mannoside 14
-mannose
a
-
D
-mannoside 6
-mannoside 7
-mannoside 8
-mannoside 9
b
-
D
d
nosides. The yields of 1-thio-
b-D-mannose 1 and methyl-6-thio-a-
b
-D
d
D-mannoside 3 were improved. The 2-, 3-, 4-, and 2,4-thio man-
a
-
D
d
nosides 4e9 were firstly synthesized. NaOH instead of NaOMe as
a catalyst catalyzing deacylation of acylated sulfur-containing gly-
cosides proved efficient. The binding affinity of these synthesized
positional thiol analogs of mannose towards Con A were evaluated,
a
-
D
0.9930
0.9942
a
/b
-D
8.6
a
The values in brackets were reported in reference.
and their EC50-values were then recorded. The thio-
and the thio- -mannosides where the 4- and 6-positions are dis-
placed with sulfur displayed no binding affinities towards Con A.
The 3-thio- -mannoside displayed almost one-third as high in-
hibition as methyl -mannoside, indicating that the O-atoms at
the C-3 positions is less important in binding site. It was surpris-
ingly found that the methyl 2-thio- -mannoside displayed four
times as high inhibition as methyl -mannoside, indicating that
modification of 2-OH of -mannoside derivatives may lead to
b-mannosides
a
binding towards Con A. These results support that the O-atoms at
the C-1, C-3, C-4 and C-6 positions are involved in binding,36,40 but
the O-atoms at the C-3 positions is less important for the binding
site. The S-atom is both larger and considerably poorer hydrogen-
bond donor than the O-atom. Thus the substitutions of S-atom
with O-atom lead to decreased binding affinities. Since the H-atom
at the 4-OH and 6-OH are also involved in binding,39 the sub-
stitutions of SH with OH at C-4 and C-6 positions led to dramatically
decreased binding affinities. However, a surprising result is that
a-D
a-D
a-D
a-D
a-D
better inhibitor towards mannoside-complexing proteins.
4. Experimental section
methyl-2-thio-
value of 0.42 mM, almost four times as high inhibition as the
methyl- -mannoside 14. Methyl-2,4-di-thio- -mannoside 6
a-D-mannoside 5 towards Con A displayed an EC50
4.1. General method for multiple regioselective acylation via
organotin51
a
-D
a-D
displayed no binding, indicating the importance of 4-OH in binding
site. According to a previous report,40 binding affinity sequence of
Methyl D-glycoside and dibutyltin oxide (2.2e3.3 equiv) were
their related tested sugars is
<2-deoxy-2-fluoro- -mannose. Since a fluorine atom is isosteric
with a hydroxyl group and is known to be a better hydrogen-bond
D-mannosez2-O-methyl-mannose
refluxed in methanol for two hours. After complete removal of
methanol via coevaporation with toluene, the residue was
dissolved in solvent (DMF, MeCN) and followed by an addition
dropwise of a solution of acetic anhydride (2.2e3.3 equiv) in dry
solvent at 0 ꢀC. The reaction proceeded at room temperature for
6e12 h. After removal of the solvent, the mixture was directly
purified by flash chromatography, affording the selectively
protected derivatives.
D
donor, it was suggested that the O-atom at the C-2 position of D-
mannose is involved in non-covalent binding to con A.40 If this is
the case, the substitutions of S-atom with O-atom should lead to
a decreased binding affinity, which is against our result. The study
of a complex of Con A with methyl a-D-mannoside shows that the
2-OH is not hydrogen-bonded to the binding site but linked
through a water bridge to an adjacent subunit.36 The results cannot
explain why methyl-2-thio-
binding affinity. Interestingly, it was reported recently that Me
Man(S2-3)Glc and Me Man(S2-2)Man Me also displayed 3e6
times as high binding constant as mannoside 14 towards Con A.62
These results indicated that modification of 2-OH of
a
-
D
-mannoside
5
displays higher
4.2. General method for multiple regioselective acylation via
acetate54
a
a
a
Methyl
D-glycoside (100 mg) were allowed to react with acetic
a-D-
anhydride (2.2 equiv) in dry acetonitrile (1 mL) at 40 ꢀC for 8e12 h

B. Wu et al. / Tetrahedron 71 (2015) 4023e4030
4027
in the presence of tetrabutylammonium acetate (0.6 equiv). The
reaction mixture was directly purified by flash column chroma-
tography (hexanes/EtOAc¼2:1 to 1:1), affording the pure selec-
tively protected derivatives.
system, they were subjected to three injections of bovine serum
albumin (BSA, 10 mg/mL in PBS, pH 7.4), two injections of low pH
buffer (pH 1.5) and finally two additional injections of BSA (10 mg/
mL) to fully block any nonfunctionalized surfaces. Binding to the
surfaces was monitored by frequency logging with Attester 1.1
(Attana), and adsorption/desorption to the surface recorded as the
resulting frequency shifts. Solutions of the lectins were then
injected into the system, where the resulting shifts in frequency
correspond to binding to the surfaces. Bound lectins were released
from the surfaces between measurements by two successive in-
jections of low pH buffer (PBS 10 mM, pH 1.5). The procedure was
then repeated at least two times for each carbohydrate inhibitor
concentration to give an average value and determine the surface
stability over time. The observed reduction in measured frequency,
compared to injections with pure Con A, was taken as a measure of
the competitive effect of the tested thio-mannosides. The quanti-
tative binding was then subjected to no-linear regression analysis
using Eq. 1.
4.3. General synthesis of triflate derivatives55
To a solution of the suitably O-protected methyl D-glycoside,
carrying unprotected OH groups at C-2 and C-4 (140 mg, 0.5 mmol),
in DCM (5 mL) was added pyridine (0.53 mL) at ꢁ30 ꢀC. Tri-
fluoromethanesulfonic anhydride (705 mg, 2.5 mmol) in DCM
(2 mL) was added dropwise, and the mixture was warmed to
ꢁ10 ꢀC and stirred for 4 h. The resulting mixture was subsequently
diluted with DCM and washed with 1M HCl, aqueous NaHCO3,
water, and brine. The organic phase was dried with MgSO4 and
concentrated in vacuum at low temperature. The residue was used
directly in the next step without further purification.
4.4. General double parallel inversion
D
fmax ꢁ
D
fmin
D
f ¼
D
f min þ
(1)
1 þ 10ðlog cꢁlog EC50Þ
During the binding studies, when high inhibitor concentrations
KSAc (5 equiv) was added to a solution of the protected triflate
residue (100 mg) in dry DMF (1.0 mL). After stirring at room tem-
perature for 24e36 h under nitrogen atmosphere, the mixture was
diluted by ethyl acetate, and washed with brine. The organic phase
was dried with MgSO4 and concentrated in vacuum. Purification of
the residue by flash column chromatography (ethyl acetate/petrol,
1:4e1:7) afforded the inversion products.
were injected, an unexpected binding behavior was observed
(Fig. S2a). Control injections in the absence of Con A showed the
same behavior (Fig. S2b), indicating that the resulting frequency
change emanated from an instrumental artifact due to the change
of buffer composition. The corrected binding was obtained by the
subtraction of the Con A response curves with the corresponding
inhibitor response curves (Fig. S2c).
4.5. General double serial inversion
KOAc (1.1 equiv) was added to a solution of the protected triflate
residue (100 mg) in dry DMF (1.0 mL). After stirring at room tem-
perature for 1 h, KSAc (3.0 equiv) was added to the mixture and
kept at room temperature for 24e36 h. The mixture was diluted by
ethyl acetate, and washed with brine. The organic phase was dried
with MgSO4 and concentrated in vacuum. Purification of the resi-
due by flash column chromatography (ethyl acetate/petro, l:4e1:7)
afforded the inversion products.
4.8. 2,3,4,6-Tetra-O-acetyl-1-S-acetyl-b-D
-mannopyranose 1346
To a solution of penta-O-acetyl-
2.56 mmol) in DCM (10 mL) was added HBr-AcOH (33%, 15 mL) at
ꢀC. After the reaction proceeded for two hours, the mixture was
extracted and concentrated, giving 2,3,4,6-tetra-O-acetyl-a-D-
D-mannopyranose 11 (1.00 g,
0
mannopyranosyl bromide 12 (946 mg, 90%), which could be used in
the next step without further purification. To a solution of 12
(700 mg, 2.6 mmol) in toluene (10 mL) was added TBASAc (1.65 g,
5.2 mmol). The reaction proceeded for 5 h at room temperature.
After extracted and concentrated, the resulting residue was purified
by flash chromatography (ethyl acetate/petrol; 1:2) to afford
4.6. General deacylation of thio-containing carbohydrate
derivatives
Sodium hydroxide (1.1 equiv for each of thioacetyl groups) was
added in the solution of acylated thio-containing methyl D-glyco-
compound 13 (589 mg, 85%). 1H NMR (400 MHz, CDCl3)
d¼5.50 (m,
side derivative in methanol. The reaction mixture was stirred at
room temperature for 4e8 h under nitrogen protection and mon-
itored with TLC. Then the mixture was neutralized with Amberlite
IR-120 (Hþ) ion exchange resin and filtered. DTT (3.0e5.0 equiv)
was added to the filtration. The mixture was stirred at room tem-
perature for 12 h. The solvent was removed under vacuum. Puri-
fication of the residue by flash column chromatography (CHCl3/
MeOH 10:1e20:1) afforded the deprotected products 1e9.
2H, H-1, H-2), 5.27 (t, J¼10.0 Hz,1H, H-4), 5.16 (dd, J¼3.3 Hz,10.1 Hz,
1H, H-3), 4.28 (dd, J¼5.3 Hz, 12.4 Hz, 1H, H-6), 4.13 (dd, J¼2.3 Hz,
12.4 Hz, 1H, H-60), 3.83 (ddd, J¼2.3 Hz, 5.3 Hz, 9.9 Hz, 1H, H-5), 2.38
(s, 3H, SAc), 2.20 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.06 (s, 3H, OAc),1.99
(s, 3H, OAc).
4.9. Methyl 6-S-acetyl-a-D-mannopyranside 17
4.7. Binding affinity study
To a solution of 15 (200 mg) in dry DMF (2 mL) was added KSAc
(100 mg). After the mixture was stirred at 35 ꢀC under nitrogen
atmosphere for 8 h, the mixture was added KNO2 (15 mg) and
stirred at 35 ꢀC for 2 h. After removal of the solvent, the residue was
purified directly by flash chromatography with solvent system
DCM/MeOH 25:1 giving compound 17 (101 mg, 70%). 1H NMR
QCM analyses were performed using a flow-through Attana
A100 C-Fast QCM system. The gold plated 10 MHz QCM crystals
were obtained from Attana AB and further coated with poly-
styrene. The mannose-functionalized surface was subjected to
injections of solutions containing Con A (200 mg/mL) and either of
the synthesized thio-mannosides in various concentrations. A
continuous flow of running buffer (PBS 10 mM, pH 7.4, 25 L/min)
was used throughout the experiments, and the sample of Con A
was prepared in the same buffer. The crystals were washed/
equilibrated with buffer solution prior to manipulations/mea-
surements. After equilibration of the crystals in the flowthrough
(CDCl3, 400 MHz):
d
4.69 (d,1H, J¼0.8 Hz,1-H), 3.95e3.96 (m,1H, 2-
H), 3.84 (dd, 1H, J¼3.2 Hz, 9.2 Hz, 3-H), 3.73e3.77 (m, 1H, 5-H), 3.53
(t, 1H, J¼9.2 Hz, 4-H), 3.43 (dd, 1H, J¼4.4 Hz, 12 Hz, 6-H), 3.36 (s, 3H,
OMe), 3.16 (dd, 1H, J¼3.2 Hz, 12.0 Hz, 6-H0), 2.49 (s, 3H, SAc). 13C
NMR (CDCl3, 100 MHz):
d 199.3, 100.8, 70.9, 70.2, 70.0, 69.2, 55.1,
30.9, 30.5. HRMS (ESI-TOF) m/z: [MþNa]þ Calcd for C9H16O6SNa
275.0565; found 275.0560.

4028
B. Wu et al. / Tetrahedron 71 (2015) 4023e4030
4.10. Methyl 3-thio-
a
-
D
-mannopyranoside 4
(57 mg, 78%). 1H NMR (400 MHz, CDCl3)
d
¼8.06e7.31 (m, 15H,
Phx3), 5.74 (t, J¼10.6 Hz 1H, H-4), 5.31 (dd, J¼1.6 Hz, 2.9 Hz, 1H, H-
2), 4.89 (d, J¼1.5 Hz, 1H, H-1), 4.66 (dd, J¼3.0 Hz, 11.3 Hz, 1H, H-3),
4.63e4.56 (m, 1H, H-6), 4.47e4.35 (m, 2H, H-5, H-60), 3.51 (s, 3H,
Prepared from methyl 2,4,6-tri-O-benzoyl-3-S-acetyl-a-D-man-
nopyranoside 21 in light of the general deacetylation of thio-
containing carbohydrate derivatives procedure, 14% total yield. 1H
OMe), 2.13 (s, 3H, SAc). 13C NMR (100 MHz, CDCl3)
d
¼193.4, 166.2,
NMR (400 MHz, D2O)
d
¼4.83 (d, J¼1.2 Hz, 1H, H-1), 3.95 (m, 2H, H-
165.7, 165.5, 133.7, 133.6, 133.1, 130.1, 130.0, 129.8, 129.3, 129.2,
128.8, 128.7, 128.5, 97.7, 73.3, 69.7, 67.3, 63.6, 55.6, 44.2, 30.6. HRMS
(ESI-TOF) m/z: [MþNa]þ Calcd for C30H28O9SNa 587.1352; found
587.1348.
6,H-2), 3.81 (dd, J¼6.1 Hz, 12.1 Hz, 1H, H-60), 3.70 (m, 1H, H-5), 3.56
(t, J¼10.2 Hz, 1H, H-4), 3.48 (s, 3H, OMe), 3.19 (dd, J¼3.8 Hz, 7.6 Hz,
1H, H-3). 13C NMR (100 MHz, D2O)
d¼100.0, 73.6, 71.3, 67.8, 61.2,
54.6, 43.6. HRMS (ESI-TOF) m/z: [MþNa]þ Calcd for C7H14O5SNa
233.0460; found 233.0478.
4.14. Methyl 2-thio-
a-D-mannopyranoside 5
4.11. Methyl 2,3,6-tri-O-benzoyl-
a
-D
-altropyranoside 19 and
prepared from 3,4,6-tri-O-acetyl-2-S-acetyl-a-D-mannopyrano-
methyl 2,4,6-tri-O-benzoyl- -altropyranoside 20
a
-
D
side 27 according to the general deacetylation of thio-containing
carbohydrate derivatives procedure, 51% total yield. 1H NMR
Starting from methyl-
2 mmol), methyl 3-O-benzyl-
was obtained in light of the literature.58 Benzylation with BzCl
(903 L) and pyridine (4 mL) afforded methyl 2,4,6-tri-O-benzoyl-
3-O-benzyl- -mannopyranoside (736 mg, 95%). After removal of
a
-
a
D
-mannopyranoside 14 (304 mg,
(400 MHz, D2O)
d
¼4.94 (s, 1H, H-1), 4.04 (dd, J¼4.9 Hz, 8.8 Hz, 1H,
-
D
-mannopyranoside (369 mg, 83%)
H-3), 3.88 (dd, J¼2.0 Hz, 12.3 Hz, 1H, H-6), 3.78 (dd, J¼5.3 Hz,
12.3 Hz, 1H, H-60), 3.73e3.60 (m, 2H, H-4, H-5), 3.45 (d, J¼5.0 Hz,
m
1H, H-2), 3.41 (s, 3H, OMe). 13C NMR (100 MHz, D2O)
d
¼102.4, 72.9,
a-
D
69.0, 66.5, 60.7, 54.8, 44.4. HRMS (ESI-TOF) m/z: [MþNa]þ Calcd for
benzyl group by catalytic hydrogenation, compound 18 (612 mg,
98%) was given. To a solution of compound 18 (200 mg) in DCM
C7H14O5SNa 233.0460; found 233.0460.
(5 mL) was added pyridine (210
fluoromethanesulfonic anhydride (200
m
L) at ꢁ30 ꢀC. Tri-
4.15. Methyl 2-S-acetyl-3,4,6-tri-O-acetyl-a-D-mannopyrano-
side 27
mL) in DCM (2 mL) was
added dropwise, and the mixture was warmed to ꢁ10 ꢀC and
stirred for 4 h. The resulting mixture was subsequently extracted
and concentrated. The residue was dissolved in DMF with the ad-
dition of KNO2 (101 mg, 3.0 equiv). The mixture was stirred at 50 ꢀC
for 6 h. After extracted and concentrated, the crude product was
purified by flash chromatography with solvent system petrol/ethyl
acetate 7:1 giving compound 19 (110 mg, 55%) and compound 20
(66 mg, 33%). For compound 19: 1H NMR (400 MHz, CDCl3)
Prepared from methyl 3,6-di-O-acetyl-a-D-galactoside 23
according to the general synthesis of triflate derivatives and general
double serial inversion procedure. The yield over three steps is 70%.
Compound 23 was obtained in 86% yield starting from methyl
a-D-
galactoside 22 in light of the literature.54 1H NMR (400 MHz, CDCl3)
d
¼5.54 (dd, J¼4.8 Hz, 9.9 Hz, 1H, H-3), 5.05 (t, J¼10.0, 1H, H-4), 4.73
(s, 1H, H-1), 4.24 (dd, J¼1.1 Hz, 4.7 Hz, 1H, H-2), 4.18 (dd, J¼4.9 Hz,
12.2 Hz, 1H, H-6), 4.07 (dd, J¼2.4 Hz, 12.2 Hz, 1H, H-60), 3.93 (ddd,
J¼2.4 Hz, 4.8 Hz, 9.9 Hz,1H, H-5), 3.37 (s, 3H, OMe), 2.34 (s, 3H, SAc),
2.08 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.93 (s, 3H, OAc). 13C NMR
d
¼8.13e7.32 (m, 15H, Phx3), 5.57 (t, J¼3.3 Hz, 1H, H-3), 5.36 (d,
J¼3.3 Hz, 1H), 4.84 (s, 1H, H-1), 4.77 (dd, J¼4.7 Hz, 12.0 Hz, 1H, H-6),
4.68 (dd, J¼2.1 Hz, 12.0 Hz, 1H, H-60), 4.41 (ddd, J¼2.1 Hz, 4.5 Hz,
9.8 Hz, 1H, H-5), 4.27 (dd, J¼3.5 Hz, 9.9 Hz, 1H, H-4), 3.48 (s, 3H,
(100 MHz, CDCl3)
d
¼193.5, 170.7, 169.8, 169.7, 101.0, 68.7, 68.6, 66.9,
OMe), 2.84 (s, 1H, OH). 13C NMR (100 MHz, CDCl3)
d¼166.9, 166.4,
62.4, 55.5, 47.2, 30.6, 20.8, 20.7. HRMS (ESI-TOF) m/z: [MþNa]þ
164.9, 133.5, 133.3, 130.2, 130.1, 130.0, 129.9, 129.8, 129.7, 129.3,
128.6,128.5,128.4, 98.5, 70.0, 69.8, 67.2, 64.4, 64.2, 55.7. HRMS (ESI-
TOF) m/z: [MþNa]þ Calcd for C28H26O9Na 529.1475; found
529.1475.
Calcd for C15H22O9SNa 401.0882; found 401.0879.
4.16. Methyl 2,4-di-thio-
a-D-mannopyranoside 6
Prepared from 3,6-di-O-acetyl-2,4-di-S-acetyl-
a-D-mannopyr-
4.12. For compound 20
anoside 25 according to the general deacetylation of thio-
containing carbohydrate derivatives procedure procedure, 44% to-
1H NMR (400 MHz, CDCl3)
d
¼8.17e7.32 (m, 15H, Phx3), 5.59 (dd,
tal yield. 1H NMR (400 MHz, CDCl3)
d
¼4.86 (s, 1H, H-1), 3.93e3.79
J¼3.2 Hz, 10.3 Hz, 1H, H-4), 5.30 (dd, J¼1.1 Hz, 3.6 Hz, 1H, H-2), 4.96
(s, 1H, H-1), 4.72 (dd, J¼2.3 Hz, 11.8 Hz, 1H, H-6), 4.59 (ddd,
J¼2.3 Hz, 4.9 Hz,10.3 Hz,1H, H-5), 4.52 (dd, J¼5.0 Hz,11.8 Hz,1H, H-
(m, 3H, H-3, H-6, H-60), 3.60e3.51 (m, 1H, H-5), 3.35 (ddd, J¼1.2 Hz,
4.5 Hz, 9.7 Hz, 1H, H-2), 3.31 (s, 3H, OMe), 3.03 (td, J¼8.3 Hz,
10.5 Hz, 1H, H-4), 2.78 (s, 2H, OH), 1.75 (d, J¼9.7 Hz, 1H, SH), 1.58 (d,
6), 4.45 (t, J¼3.3 Hz, 1H, H-3), 3.53 (s, 3H, OMe), 3.42 (s, 1H, OH). 13
C
J¼8.3 Hz, 1H, SH). 13C NMR (100 MHz, CDCl3)
¼102.5, 74.4, 70.3,
d
NMR (100 MHz, CDCl3)
d
¼166.4, 165.7, 165.3, 133.9, 133.8, 133.7,
62.8, 55.4, 45.6, 39.9. HRMS (ESI-TOF) m/z: [MþNa]þ Calcd for
133.3, 130.4, 130.1, 130.0, 129.9, 129.6, 129.5, 129.3, 128.8, 128.7,
128.6, 99.1, 70.5, 68.1, 67.5, 64.5, 63.8, 56.2. HRMS (ESI-TOF) m/z:
[MþNa]þ Calcd for C28H26O9Na 529.1475; found 529.1470.
C7H14O4S2Na 249.0231; found 249.0228.
4.17. Methyl 2,4-di-S-acetyl-3,6-di-O-acetyl-a-D-mannopyr-
anoside 25
4.13. Methyl 2,4,6-tri-O-benzoyl-3-S-acetyl-a-D-mannopyr-
anoside 21
Prepared from compound 23 according to the general synthesis
of triflate derivatives and general double parallel inversion pro-
cedure. The yield over two steps is 63%. 1H NMR (400 MHz, CDCl3)
To a solution of compound 20 (66 mg) in DCM (2 mL) was added
pyridine (68
(66
m
L) at ꢁ30 ꢀC. Trifluoromethanesulfonic anhydride
d
¼5.52 (dd, J¼4.4 Hz, 11.5 Hz, 1H), 4.83 (d, J¼1.3 Hz, 1H, H-1), 4.34
m
L) in DCM (0.5 mL) was added dropwise, and the mixture was
(dd, J¼5.2 Hz, 12.1 Hz, 1H, H-6), 4.25 (dd, J¼1.6 Hz, 4.4 Hz, 1H, H-2),
4.18 (dd, J¼2.1 Hz, 12.1 Hz, 1H, H-60), 3.99 (ddd, J¼2.0 Hz, 5.2 Hz,
11.2 Hz, 1H, H-5), 3.81 (t, J¼11.4 Hz, 1H, H-4), 3.40 (s, 3H, OMe), 2.39
warmed to ꢁ10 ꢀC and stirred for 4 h. The resulting mixture was
extracted and concentrated. The residue was dissolved in toluene
(1.5 mL) with the addition of TBASAc (20 equiv, 827 mg). After being
stirred at rt for 5 h, the mixture was extracted and concentrated.
The crude product was purified by flash chromatography with
solvent system petrol/ethyl acetate 8:1 giving compound 21
(s, 3H, SAc), 2.35 (s, 3H, SAc), 2.13 (s, 3H, OAc), 1.97 (s, 3H, OAc). 13
C
NMR (100 MHz, CDCl3)
63.6, 55.5, 47.8, 41.8, 30.8, 30.7, 20.9, 20.8. HRMS (ESI-TOF) m/z:
[MþNa]þ Calcd for C15H22O8S2Na 417.0654; found 417.0655.
d
¼193.7, 192.9, 170.9, 169.8, 101.1, 70.0, 67.2,

B. Wu et al. / Tetrahedron 71 (2015) 4023e4030
4029
4.18. Methyl 2-thio-
b-
D-mannopyranoside 7
4.23. Methyl 2,3,6-tri-O-acetyl-4-S-acetyl-
side 35
a-D-mannopyrano-
Prepared from methyl 2-S-acetyl-3,4,6-tri-O-acetyl-
b-D-man-
nopyranoside 32 according to the general deacetylation of thio-
Starting from methyl
a-D-mannopyranoside 14, methyl 2,3,6-tri-
containing carbohydrate derivatives procedure, 56% total yield. 1H
O-acetyl-a-D-mannopyranoside 33 was obtained in 85% yield in
NMR (400 MHz, D2O)
d
¼4.79 (d, J¼1.8 Hz, 1H, H-1), 3.97e3.85 (m,
light of the literature.51 Compound 33 (160 mg, 0.5 mmol) was
triflated in general procedure, following an inversion by TBANO2
(417 mg) in toluene (4 mL) at 50 ꢀC for 5 h. The crude product was
purified by flash chromatography with solvent system Hex: EtOAc
2H, H-3, H-6), 3.75 (dd, J¼6.1 Hz, 12.3 Hz, 1H, H-60), 3.65e3.57 (m,
2H, H-2, H-4), 3.55 (s, 3H, OMe), 3.45e3.37 (m, 1H, H-5). 13C NMR
(100 MHz, D2O)
d
¼100.3, 76.8, 71.9, 66.5, 60.9, 56.7, 46.3. HRMS
(ESI-TOF) m/z: [MþNa]þ Calcd for C7H14O5SNa 233.0460; found
2:1 giving 141 mg of a mixture of methyl 2,3,6-tri-O-acetyl-a-D-
233.0455.
talopyranoside 34 and its side products. The mixture was triflated
in general procedure, following an inversion by TBASAc (419 mg) in
toluene (2 mL) at rt for 6 h. The mixture was extracted and con-
centrated. Purification of the residue by flash column chromatog-
raphy (ethyl acetate/petrol: 1:5) afforded the desired product
4.19. Methyl 2-S-acetyl-3,4,6-tri-O-acetyl-b-D-mannopyrano-
side 32
compound 35 (62 mg, 33%). 1H NMR (400 MHz, CDCl3)
d
¼5.24 (dd,
Prepared from methyl 3,6-di-O-acetyl-b-D-galactoside 30
J¼3.0 Hz, 11.2 Hz, 1H, H-3), 5.13 (dd, J¼1.9 Hz, 3.2 Hz, 1H, H-2), 4.72
(d, J¼1.7 Hz, 1H, H-1), 4.33 (dd, J¼5.3 Hz, 12.1 Hz, 1H, H-6), 4.17 (dd,
J¼1.4 Hz, 12.1 Hz, 1H, H-60), 4.01e3.88 (m, 2H, H-5, H-4), 3.36 (s, 3H,
OMe), 2.30 (s, 3H, SAc), 2.13 (s, 3H, OAc), 2.06 (s, 3H, OAc), 1.94 (s,
according to the general synthesis of triflate derivatives and gen-
eral double serial inversion procedure. The yield over three steps is
78%. Compound 30 was obtained in 90% yield starting from methyl
b-D
-galactoside 29 in light of the literature.54 1H NMR (400 MHz,
3H, OAc). 13C NMR (100 MHz, CDCl3)
d¼193.1, 170.9, 170.3, 169.9,
CDCl3)
d
¼5.20 (dd, J¼4.3 Hz and 9.6 Hz, 1H, H-3), 5.09 (t, J¼9.5 Hz,
98.8, 69.9, 68.7, 67.6, 63.9, 55.4, 40.7, 30.9, 21.0, 21.0, 20.8. HRMS
(ESI-TOF) m/z: [MþNa]þ Calcd for C15H22O9SNa 401.0882; found
401.0879.
1H, H-4), 4.68 (d, J¼1.7 Hz, 1H, H-1), 4.38 (dd, J¼1.7 Hz, 4.3 Hz, 1H,
H-2), 4.20 (dd, J¼4.9 Hz, 12.2 Hz, 1H, H-6), 4.13 (dd, J¼2.9 Hz,
12.2 Hz, 1H, H-60), 3.64 (ddd, J¼2.9 Hz, 4.9 Hz, 9.3 Hz, 1H, H-5), 3.50
(s, 3H, OMe), 2.36 (s, 3H, SAc), 2.07 (s, 3H, OAc), 2.02 (s, 3H, OAc),
1.94 (s, 3H, OAc).
Acknowledgements
This study was supported by the National Nature Science
Foundation of China (Nos. 21272083), and the Chutian Project-
Sponsored by Hubei Province. The authors are also grateful to the
staffs in the Analytical and Test Center of HUST for support with the
NMR instruments.
4.20. Methyl 2,4-thio-
b-D-mannopyranoside 8
Prepared from methyl 2,4-di-S-acetyl-3,6-di-O-acetyl-
mannopyranoside 31 according to the general deacetylation of
b-D-
thio-containing carbohydrate derivatives procedure, 54% total
yield. 1H NMR (400 MHz, CDCl3)
d
¼4.51 (s, 1H, H-1), 3.94 (dd,
J¼12.2, 2.1, 1H, H-6), 3.83 (dd, J¼4.6 Hz and 12.2 Hz, 1H, H-60), 3.57
(m, 2H, H-2, H-3), 3.48 (s, 3H, OMe), 3.35 (s, 1H, OH), 3.29e3.21 (m,
1H, H-5), 3.12e2.97 (m, 1H, H-4), 2.77 (s, 1H, OH), 1.72 (d, J¼6.3 Hz,
1H, SH), 1.59 (d, J¼8.2 Hz, 1H, SH). 13C NMR (100 MHz, CDCl3)
Supplementary data
General methods, Fig. S1 and S2, 1H and 13C NMR-spectra of
compounds 4, 5, 6, 7, 8, 9, 19, 20, 21, 25, 27, 31, and 35. Supple-
mentary data associated with this article can be found in the online
version, at http://dx.doi.org/10.1016/j.tet.2015.04.060.
d
¼100.8, 78.6, 74.0, 62.6, 57.1, 47.3, 39.4. HRMS (ESI-TOF) m/z:
[MþNa]þ Calcd for C7H14O4S2Na 249.0231; found 249.0226.
4.21. Methyl 2,4-di-S-acetyl-3,6-di-O-acetyl-b-D-mannopyr-
anoside 31
References and notes
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Prepared from compound 30 according to the general synthesis
of triflate derivatives and general double parallel inversion pro-
cedure. 1H NMR (400 MHz, CDCl3)
d
¼5.22 (dd, J¼4.1 Hz, 10.9 Hz, 1H,
H-3), 4.62 (d, J¼1.6 Hz, 1H, H-1), 4.36 (dd, J¼1.5 Hz, 4.1 Hz, 1H, H-2),
4.30 (dd, J¼12.1 Hz, 4.9 Hz, 1H, H-6), 4.21 (dd, J¼2.1 Hz, 12.1 Hz, 1H,
H-60), 3.76 (ddd, J¼2.2 Hz, 4.9 Hz, 10.7 Hz, 1H, H-5), 3.68 (t,
J¼10.8 Hz, 1H, H-4), 3.49 (s, 3H, OMe), 2.36 (s, 3H, SAc), 2.31 (s, 3H,
SAc), 2.08 (s, 3H, OAc), 1.94 (s, 3H, OAc). 13C NMR (100 MHz, CDCl3)
d
¼194.2, 192.9, 170.8, 169.9, 100.3, 73.8, 69.8, 63.4, 57.2, 49.4, 41.6,
30.8, 30.7, 20.8, 20.7. HRMS (ESI-TOF) m/z: [MþNa]þ Calcd for
C
15H22O8S2Na 417.0654; found 417.0658.
4.22. Methyl 4-thio-a-D-mannopyranoside 9
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