Synthesis of alkyl and cycloalkyl α-D-mannopyranosides and derivatives thereof and their evaluation in the mycobacterial mannosyltransferase assay

Article abstract of DOI:10.1016/j.carres.2010.03.011

The synthesis of a series of alkyl (having from C6 to C20 aglycones), cyclohexyl, and cyclohexylalkyl α-Dmannopyranosides, 6-deoxygenated analogs, thioglycosides, and sulfones derived thereof, is reported. Here, under the in vitro assay conditions used, n

Full text of DOI:10.1016/j.carres.2010.03.011

Carbohydrate Research 345 (2010) 1339–1347
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of alkyl and cycloalkyl
a
-D-mannopyranosides and derivatives
thereof and their evaluation in the mycobacterial mannosyltransferase assay
b
a
b
Monika Poláková a, , Martina Belánová , Ladislav Petruš , Katarína Mikušová
*
ˇ
a Institute of Chemistry, Center for Glycomics, GLYCOMED, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38 Bratislava, Slovakia
b Department of Biochemistry, Comenius University, Faculty of Natural Sciences, Mlynská dolina, CH1, SK-842 15 Bratislava, Slovakia
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a series of alkyl (having from C6 to C20 aglycones), cyclohexyl, and cyclohexylalkyl a-D-
Received 14 January 2010
Received in revised form 5 March 2010
Accepted 9 March 2010
mannopyranosides, 6-deoxygenated analogs, thioglycosides, and sulfones derived thereof, is reported.
Here, under the in vitro assay conditions used, none of the 15 tested compounds acted as an inhibitor
of the mannose transfer catalyzed by the enzymes present in mycobacterial membrane and cell wall frac-
tions. Mannopyranosides comprising shorter aliphatic, up to 8 carbon atoms long linear, or cyclic agly-
cone served as the acceptor substrates in the mycobacterial mannosyltransferase reaction. The
thioglycosides exhibited similar behavior, in contrast to the sulfones, which were essentially not recog-
nized by the mycobacterial enzymes. 6-Deoxygenated glycosides were not processed by the enzymes,
suggesting that the mannose transfer occurs at position 6 of the acceptors.
Available online 15 March 2010
Keywords:
Synthetic
a-D-mannopyranosides
Mycobacteria
Mannosyltransferase
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
placed by a hydrogen, fluorine, amino, or methoxy group. All
these modified disaccharides, except 20-methyl ether, acted as sub-
Mycobacterial mannosyltransferases (ManTs) represent the
intriguing group of enzymes that take part in the biosynthesis of
the phosphatidylinositolmannosides (PIMs), lipomannan (LM),
and lipoarabinomannan (LAM), (Fig. 1), which are an important
structural and immunomodulatory set of molecules of the serious
human pathogen Mycobacterium tuberculosis.1 Activities of the en-
zymes catalyzing the transfer of mannose from GDP-Man to the
endogenous lipid acceptors in the mycobacterial membrane frac-
tions have been recognized for a long time.2–5 During the last 10
years, several ManTs were characterized including the enzymes
strates for mycobacterial ManTs.9 Further deoxygenation was car-
ried out at position C-60. Some of the disaccharides with altered C-
20 and C-60 substituents acted as the enzyme inhibitors.10 Recently,
using a simple iterative strategy developed by Williams and
Watt,11 the synthesis of all seven monomethoxy and seven mono-
deoxy analogs of octyl
a-(1?6)-mannodisaccharide has been
accomplished.12 Their screening has shown that the most tolerated
substitutions with regard to retaining their ability to serve as
acceptor substrates for the mycobacterial ManTs are deoxygen-
ation at 4- or 20-, and the attachment of methoxy group at the 3-,
4-, or 30-position.13
catalyzing the formation of
a-(1?2), as well as a-(1?6) glycosidic
bonds.6
Synthetic acceptors have also served as useful tools for the char-
acterization of mycobacterial mannosyltransferases. This is exem-
The very first reported examination of mycobacterial ManT
activities in the presence of exogenous acceptor substrates pro-
plified by studies where octyl
a-D-Manp-(1?6)-a-D-Manp has
vided proof that methyl
sides obtained by degradation of yeast mannan act as
substrates for
-(1?6)-ManTs from Mycobacterium smegmatis.7 La-
ter, syntheses of alkyl, thioalkyl, and alkenyl -(1?6)-di- and tri-
saccharides with 8 to 10 carbon atoms long aglycone have been
reported. These oligomannosides served as acceptor substrates of
a
-
D
-mannopyranoside and oligomanno-
been used for the investigation of the functional compartmentali-
a
a
a
OH
HO
O
HO
HO
polyprenolphosphomannose-dependent
a-(1?6)-ManT (Fig. 2)
O
from M. smegmatis present in a mycobacterial cell-free system.8
O
O
HO
HO
Lowary and co-workers described the synthesis of modified oc-
OH
O
O
tyl
a
-(1?6)-mannodisaccharides in which the 20-OH group is re-
HO
HO
O
X
* Corresponding author. Tel.: +421 259410272; fax: +421 259410222.
E-mail address: Monika.Polakova@savba.sk (M. Poláková).
Figure 1. Common mannan core structure of mycobacterial LAM and LM.
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.03.011

1340
M. Poláková et al. / Carbohydrate Research 345 (2010) 1339–1347
(A)
O
P
PPM
OH
HO
HO
synthase
O
GDP-Man
HO
O R
O
O
OH
O
HO
HO
HO
OH
OH
O
ManT
O
HO
HO
O
O
HO
HO
HO
OH
O
OH
O
O
O
HO
HO
O
P
HO
HO
O
O
O R
O
(B)
O
P
X =
Y =
O
N
OH
O
OH
HO
HO
HO
HO
HO
HO
O
O
P
O
P
N
N
O R
O
NH
NH2
8
O
O
O
O
O
R = X
R = Y
O
O
HO OH
GDP-mannose
4
structure of polyprenolphosphomannose derivatives (PPM)
Figure 2. (A) Reaction catalyzed by the polyprenolphosphomannose-dependent
a-(1?6)-ManT. (B) Structure of polyprenolphosphomannose derivatives (PPM) and GDP-
mannose.
zation of mannosyltransferases in M. smegmatis,14 or for the confir-
mation of -(1?6)-ManT activity of the MptA and MptB enzymes
OAc
O
OAc
O
OH
O
AcO
AcO
AcO
AcO
AcO
HO
HO
HO
a
b
AcO
a
OAc
involved in LAM/LM biosynthesis in M. tuberculosis and Corynebac-
OR
R= C6H13
3 R= C8H17
OR
R= C6H13
terium glutamicum, respectively.15,16
1
2
10
11 R= C8H17
The present study is focused on the synthesis and evaluation of
4
R= C12H25
12
13
R= C12H25
R= C16H33
a
5 R= C16H33
6 R= C20H41
a spectrum of aliphatic a-D-mannopyranosides varying in aglycone
a-D a-D-
-Manp7 and octyl
14 R= C20 41
H
length and shape. To date, methyl
7
R= Cyh
15
R= Cyh
16 R= CH2Cyh
17
Manp13 are the only monosaccharides evaluated as synthetic
8 R= CH2Cyh
9
R= CH2CH2Cyh
OH
R= CH2CH2Cyh
Cyh =
acceptors of mycobacterial ManTs. Concerning the aglycone part
a
-(1?6)-mannose oligomers, only octyl,8–10,13 octyl-1-thio,
OAc
O
of
AcO
AcO
AcO
HO
b
O
and dec-9-en-1-yl8 model compounds have been tested. The aim
of this study is to evaluate the tolerance of mycobacterial ManTs
toward the variable aglycone structure, initially for simple mono-
saccharide model compounds. The variable aglycone nature of
the synthetic mannoside acceptors may offer new opportunities
for examination of the function and mechanism of action of cur-
rently known mycobacterial ManTs, as well as of those yet to be
discovered.
HO
HO
SR
SR
20
R= C6H13
18
R= C6H13
21 R= C8H17
19 R= C8H17
c
OAc
O
OH
HO
AcO
AcO
AcO
b
O
HO
HO
SO2R
R= C6H13
R= C8H17
SO2R
R= C6H13
25 R= C8H17
24
22
23
2. Results and discussion
2.1. Synthesis
Scheme 1. Reagents and conditions: (a) 1 M SnCl4 in CH2Cl2, alcohol, CH2Cl2, 16 h,
rt, or BF3ꢀOEt2, alcohol, CH2Cl2, 16 h, rt; (b) MeONa, MeOH/CH2Cl2, 16 h, rt; (c)
mCPBA, CH2Cl2, 90 min, rt.
The synthetic strategy is outlined in Schemes 1 and 2. Rapid ac-
cess to the target compounds was achieved by glycosylation of
acceptors with mannose pentaacetate 1 as a donor using Lewis acid
catalysis, which usually proceeded with high selectivity and in
good yields. Alcohols of varying alkyl chain length from C6 up to
C20, namely, hexanol, octanol, dodecanol, hexadecanol (cetyl alco-
hol), eicosanol, and thioalcohols such as hexanethiol and octane-
thiol, were chosen as acceptors. Furthermore, alcohols containing
a cyclohexyl ring, that is, cyclohexanol, cyclohexylmethanol, and
2-cyclohexylethanol were also employed as acceptors in the syn-
thesis of other model compounds. The alkyl/cycloalkyl portions
of the resulting glycosides are presumed to anchor the acceptor
substrates to the mycobacterial membrane where the ManT reac-
tion takes place.
OH
O
OBz
O
OBz
O
OH
O
HO
HO
HO
TsO
BzO
BzO
a
b
c
BzO
BzO
HO
HO
OR
OR
R= C8H17
OR
26 R= C8H17
OR
30 R= C8H17
11
17
R= C8H17
28
R= CH2CH2Cyh 29 R= CH2CH2Cyh
27
31
R= CH2CH2Cyh
R= CH2CH2Cyh
Scheme 2. Reagents and conditions: (a) (1) TsCl/CH2Cl2, py, 16 h, rt; (2) BzCl, py,
16 h, rt; (b) NaBH4, DMF, 6 h, 65 °C; (c) MeONa, MeOH–CH2Cl2, 16 h, rt.
(octanol, hexanethiol, and 2-cyclohexylethanol) to find a more effi-
cient strategy. The desired products were obtained in moderate
yields, which were not significantly dependent on the Lewis acid
used. While the SnCl4-catalyzed reaction provided products in
yields over 50%, slightly lower ones ranging from 42% to 53% were
Two alternative Lewis acid-catalyzed glycosylation procedures,
promoted under tin(IV) chloride (SnCl4) or boron trifluoride–
diethyl ether complex (BF3ꢀOEt2), were initially examined in the
reaction of donor 1 with representative acceptors of all three types

M. Poláková et al. / Carbohydrate Research 345 (2010) 1339–1347
1341
obtained using BF3ꢀOEt2 as the catalyst. Coupling of cyclohexanol
with donor 1 catalyzed by BF3ꢀOEt2 gave the required product 7
in only a 5% yield.17 In this specific case, using SnCl4 led to a signif-
icant improvement in the yield (64%), thus confirming the earlier
observation that it is a more efficient catalyst.18 Therefore in the
present paper, the SnCl4-catalyzed glycosylation procedure19 was
employed for coupling donor
1 with all tested acceptors
(Scheme 1). Its advantage was mainly the requirement for consid-
erably lower quantities of alcohol and catalyst (1.2 equiv alcohol/
1 equiv SnCl4; 5 equiv alcohol/10 equiv BF3ꢀOEt2). The SnCl4-cata-
lyzed reactions proceeded smoothly, although the yields decreased
slightly with elongation of the acceptor alkyl chain, due to their re-
duced reactivity.
As expected, the O-glycosylation was stereospecific with the
exclusive formation of the a-anomer. The anomeric proton of each
Figure 3. TLC analysis of the reaction products of the ManT reaction with synthetic
acceptors 10–17, 20, 21, 24, 25, and 30–32. The profile is a representation of three
separate experiments carried out at 4 mM concentration of the tested compounds.
Radioactivity (dpm) incorporated into the major band of the products formed is
shown at the bottom of the figure.
glycoside resonated in the range of d 4.80–4.77, whereas anomeric
carbon signals appeared at d 97.9–97.8. In contrast, S-glycosylation
afforded an anomeric mixture of products. Being in the range of
7:2–8:2, the
the strength of the Lewis acid used. Column chromatography of
the anomeric mixture of thioglycosides afforded the required -ano-
a
/b stereoselectivity20,21 was weakly influenced by
thioglycosides 18 and 19, while the BF3ꢀOEt2 method failed.22 This
is, however, in contradiction with previous reports20,21 and the
present paper, where the aforementioned compounds were ob-
tained in moderate yields.
a
mer, which was identified on the basis of a typical chemical shift for
H-1 at d 5.26, while the H-1 signal of b-anomer appeared at d 4.75.
These resonances are characteristic for thiomannosides 18 and 19.22
The thioglycosides can be further modified. Accordingly, 18 and
19 were easily converted to the sulfones 22 and 23 as the sole reac-
tion products, by oxidation with an excess of mCPBA (3 equiv) in
almost quantitative yields (96–98%). The transformation was evi-
dent from the 1H and 13C spectra of the product, that is, the H-1
chemical shift of sulfones (d 4.83) is shifted more upfield than that
of starting S-glycosides (d 5.26), C-1 of the products resonated at d
87.9 while that of starting compounds at d 82.8. Finally, the pro-
tecting groups were removed by treatment with MeONa in
MeOH–CH2Cl2 providing the desired compounds 10,23 11,11,20 12,
13,24 14, 15,17 16, 17,23 20,22 21,22 24, and 25.
The preparation of 6-deoxygenated compounds was accom-
plished over four steps (Scheme 2) through a selective tosylation
of the primary hydroxyl group of 3 and 9, followed by acylation
of secondary hydroxyl groups with BzCl. The fully protected glyco-
sides 26 and 27 were subsequently converted in moderate yields
(41–47%) into the corresponding 6-deoxy derivatives by reduction
with sodium borohydride. The deoxygenation was clearly evident
from the 13C NMR spectra. The characteristic chemical shift for C-
6 of 29 appeared at d 17.9, while the C-6 shift of the starting com-
pound 27 was significantly downfielded (d 68.5). Removal of ben-
zoyl protective groups under Zemplén conditions gave compounds
30 and 31. In comparison with a recent paper, wherein the use of
tin reagent resulted in a laborious purification of 30 achieved by
a repeated chromatography,11 our synthetic sequence afforded
the target compounds after one-step purification.
Octyl a-D-Manp-(1?6)-a-D-Manp 32, previously found to serve
as the best synthetic acceptor in the mycobacterial ManT assay,8,13
was used here as a reference synthetic substrate for the evaluation
of the potential of the other synthesized glycosides to be recog-
nized as the enzyme acceptors. For the synthesis of 32,13 which
is outlined in Scheme 3 2,3,4,6-tetra-O-acetyl-
trichloroacetimidate17 was chosen as the donor and octyl 2,3,4-tri-
O-benzyl-
-mannopyranoside9 as the acceptor.
a-D-mannopyranose
a
-D
2.2. Biological evaluation of the synthesized compounds
The synthetic compounds 10–17, 20, 21, 24, 25, and 30–32 (15
compounds in total) were evaluated at 4 mM concentration in the
in vitro assay monitoring ManT activities of the mycobacterial
membrane and cell wall fractions. Comparison of the TLC profiles
of the radiolabelled reaction products proved that none of the
tested compounds inhibited in vitro production of natural mann-
olipids: PIMs and polyprenylphosphoryl mannoses, which were
produced in comparable quantity in all reaction mixtures, includ-
ing the control without the acceptor (data not shown).
Eight of the compounds served as substrates in the ManT reac-
tion (Fig. 3). While the efficacies of linear six- and eight-carbon al-
kyl
a-D-mannopyranosides 10 and 11 to serve as acceptor
substrates were comparable under our experimental conditions,
those with 12 and more carbon atoms (compounds 12–14) were
not used by the ManTs. Nor did the reduction of concentration of
compounds 12–14 to 2 mM led to any transfer product detection,
presumably due to their limited solubility. It is noteworthy that
mannopyranosides 15–17 comprising cyclohexyl and cyclo-
hexylalkyl aglycones were also recognized as the substrates. Sulfur
counterparts of 10 and 11, thioglycosides 20, and 21 also acted as
the substrates, although the latter compound with lower effi-
Concerning intermediates, there are several reports on the syn-
thesis of per-O-acetylated octyl glycoside 311,20,25 and hexadecyl
glycoside 16.26 The latter was prepared as in this paper, but using
a different Lewis acid (FeCl3) as the catalyst. The same catalyst
(FeCl3) has been successfully employed also in the synthesis of
OH
HO
O
HO
HO
OBn
OH
O
OAc
O
HO
BnO
BnO
O
a
O
AcO
AcO
AcO
+
HO
HO
O
CCl3
NH
O(CH2)7CH3
O(CH2)7CH3
32
Scheme 3. Building blocks in the synthesis of octyl a-D-Manp-(1?6)-a-D-Manp 32. Reagents and conditions: (a) (1) BF3ꢀOEt2, CH2Cl2, 30 min, rt; (2) MeONa, MeOH–CH2Cl2,
16 h, rt; (3) H2, Pd/C, MeOH, 3 h, rt.

1342
M. Poláková et al. / Carbohydrate Research 345 (2010) 1339–1347
ciency. However, transformation of these thioglycosides to sulf-
ones 24 and 25 led to almost complete loss of their ability to func-
tion as the enzyme acceptors; and in the latter case, the products
were seen only after prolonged exposure of the TLC plate to radio-
graphic film (data not shown).
While compounds 10, 11, 15–17, 20, and 21 clearly served as
substrates for mycobacterial ManTs, the efficiency of the most ac-
tive compounds was about 20 times lower than that of the disac-
charide reference compound 32 (Fig. 3). This is based on the
quantification of radioactivity of the major individual bands corre-
sponding to the reaction products. For the representative TLC plate
shown in Figure 3 the value obtained for compound 32 was 5546
dpm, while the incorporation of radioactivity into the products de-
rived from compounds 11, 16, 17, and 20 ranged from 240 to 268
dpm. This observation agrees with previous reports in which
monosaccharides bearing methyl7 and octyl13 aglycones acted as
poor acceptors for the ManT reaction.
TOF Premier instrument (Waters, Millford, MA, USA) with ESI
(electrospray ionization) operated in positive mode. 1H NMR and
13C NMR spectra were recorded at 25 °C with either Bruker AM
300, VNMRS 400 MHz Varian or INOVA AV 600 MHz spectrome-
ters. Chemical shifts are referenced to either TMS (d 0.00, CDCl3,
DMSO-d6, pyridine-d5 for 1H) or HOD (d 4.87, CD3OD for 1H), and
to CDCl3 (d 77.23), DMSO-d6 (d 39.51), pyridine-d5 (d 123.87) or
CD3OD (d 49.15) for 13C, as internal standards. In the glycosylation
step, the a-stereochemistry of the glycosidic linkage was also con-
1
firmed by the one-bond JC-1,H-1 heteronuclear coupling constant
with a typical value of approximately 170 Hz.18,28 TLC was per-
formed on aluminum sheets precoated with Silica Gel 60 F254
(Merck). Flash chromatography was carried out on Silica Gel 60
(0.040–0.060 mm, Merck) with distilled solvents (hexane, ethyl-
acetate, CH2Cl2, and MeOH). Reaction solvents were dried and dis-
tilled before use. All reactions containing sensitive reagents were
carried out under argon atmosphere.
Due to the low efficiency of glycosyl transfer to the mannose
monosaccharides, we did not attempt to isolate and characterize
the products of the enzyme reactions. However, their structures
may be anticipated from previous studies using similar assay con-
4.2. General procedures for glycosylation
4.2.1. Procedure A using BF3ꢀOEt2 as promoter
ditions,8,13 in which particularly
a-(1?6)-ManT activity of the en-
To a stirred solution containing 1,2,3,4,6-penta-O-acetyl-D-
zyme was observed. This conclusion is further confirmed by the
fact that 6-deoxygenated derivatives 30 and 31 (counterparts of
11 and 17) did not serve as substrates in the ManT reaction.
Despite the low efficacy of the enzymatic mannose transfer to the
monosaccharidesyntheticsubstratesundertheexperimentalcondi-
tions used, we identified several aglycones, including cyclic struc-
tures, which are well-tolerated by mycobacterial ManTs. Indeed,
the nature of the aglycone should not be underestimated in the de-
sign of the appropriate acceptors for in vitro investigation of the gly-
cosyltransferases. This is well documented by the work of May
et al.27 who tested a series of synthetic acceptors with mycobacterial
galactofuranosyltransferase GlfT2. The study showed that only the
compound with appropriate aglycone chain length and structural
features enabled efficient polymerization of the galactofuran on
the synthetic acceptor. This observation led to the proposal of a teth-
ering mechanism for the galactan chain length elongation.
mannopyranose (1) (1 g, 2.56 mmol, 1 equiv) in CH2Cl2 (20 mL)
the corresponding alcohol (5 equiv) was added. The reaction mix-
ture was stirred for 20 min, cooled down on an ice bath, and
BF3ꢀOEt2 (3.64 g, 3.16 mL, 25.6 mmol, 10 equiv) was added drop-
wise. The resulting mixture was stirred for 15 min, brought to rt,
and stirring was continued for 24 h. The reaction mixture was di-
luted with CH2Cl2 (100 mL) and poured into ice cold water
(200 mL) under stirring. The organic phase was separated, washed
with satd NaHCO3 (3 ꢁ 100 mL), water (100 mL), dried with anhy-
drous Na2SO4, filtered, and concentrated. The crude product was
purified by flash chromatography (hexane–EtOAc, 5:1?2.5:1).
4.2.1.1.
(3).
Octyl
2,3,4,6-tetra-O-acetyl-a-D-mannopyranoside
Yield 0.57 g, 49%, yellowish oil.
4.2.1.2. 2-Cyclohexylethyl 2,3,4,6-tetra-O-acetyl-a-D-mannopy-
The investigation of monosaccharide synthetic substrates
serves as a pilot study, which will be further extended toward a
preparation and biological evaluation of disaccharide homologs
of the tested monosaccharides in order to obtain a deeper insight
into the structural requirements of the catalyst on the acceptor.
ranoside (9). Yield 0.62 g, 53%, yellowish oil.
4.2.1.3. Hexyl 2,3,4,6-tetra-O-acetyl-1-thio-a-D-mannopyrano-
side (18). Yield 0.48 g, 42%, yellowish oil.
4.2.2. Procedure B using SnCl4 as promoter
A stirred solution containing 1,2,3,4,6-penta-O-acetyl-
D-manno-
3. Conclusion
pyranose (1) (1 g, 2.56 mmol, 1 equiv) in CH2Cl2 (20 mL) was
cooled down on an ice bath, and 1 M SnCl4 in CH2Cl2 (2.56 mL,
2.56 mmol, 1 equiv) was added. The reaction mixture was stirred
for 10 min and then the corresponding alcohol (1.2 equiv) was
added dropwise. The resulting mixture was stirred for 15 min,
brought to rt, and stirring was continued for 16 h. The reaction
mixture was diluted with CH2Cl2 (80 mL) and poured into ice cold
satd NaHCO3 (100 mL) under stirring. The organic phase was sep-
arated, washed with water (100 mL), dried with anhydrous
Na2SO4, filtered, and concentrated. The crude product was purified
by flash chromatography (hexane–EtOAc, 5:1?2.5:1).
A rapid access to aliphatic a-D-mannopyranosides bearing vari-
ous aglycone, the corresponding thioglycosides, and sulfones is de-
scribed. The synthesis was accomplished by Lewis acid-catalyzed
glycosylation of hydrophobic acceptors with mannose pentaace-
tate 1 as the donor. 6-Deoxygenated analogs were obtained
through modification of the primary alcohol group of selected
mannopyranosides by tosylation followed by reduction of the tosyl
function. It was demonstrated that alkyl and cycloalkyl
a-D-man-
nopyranosides consisting of up to an eight carbon atom long agly-
cone, and their thio analogs, can act as substrates for mycobacterial
a
-(1?6)-Man reaction.
4.2.2.1.
(2).
Hexyl
2,3,4,6-tetra-O-acetyl-a-D-mannopyranoside
Yield 0.61 g, 55%, colorless oil. [a] +38.0 (c 1.0, CHCl3).
D
HRMS: m/z: calcd for C20H32O10Na [M+Na]+: 455.1893; found:
4. Experimental
455.1899.
4.1. General methods
4.2.2.2.
(3).
HRMS: m/z: calcd for
Octyl
2,3,4,6-tetra-O-acetyl-
a
-
D
-mannopyranoside
Optical rotations were measured on a Perkin–Elmer 241 polar-
imeter at 20 °C. Melting points were determined on a Kofler hot
stage and are uncorrected. HRMS spectra were acquired on a Q-
Yield 0.67 g, 57%, yellowish oil. [
a
]
+38.2 (c 1.0, CHCl3).
D
C
22H37O10 [M+H]+: 461.2387; found:
461.2383.

M. Poláková et al. / Carbohydrate Research 345 (2010) 1339–1347
1343
4.2.2.3. Dodecyl 2,3,4,6-tetra-O-acetyl-
(4). Yield 0.71 g, 54%, oil. [ +41.0 (c 1.0, CHCl3). HRMS: m/
z: calcd for C26H44O10Na [M+Na]+: 539.2832; found: 539.2833.
a
-
D
-mannopyranoside
CHCl3), lit.20
C
½
a 2D5
+93.0 (c 1.1, CHCl3). HRMS: m/z: calcd for
ꢂ
a
]
D
20H32O9SNa [M+Na]+: 485.1820; found: 485.1843.
4.3. General procedure for oxidation of alkyl 2,3,4,6-tetra-O-
acetyl-1-thio- -mannopyranosides
4.2.2.4. Hexadecyl 2,3,4,6-tetra-O-acetyl-
(5). Yield 0.76 g, 52%, oil. [ D +33.0 (c 1.0, CHCl3). HRMS: m/z:
calcd for C30H52O10Na [M+Na]+: 595.3458; found: 595.3481.
a-D-mannopyranoside
a-D
a
]
To a stirred and 0 °C precooled solution containing compound
18 (0.3 g, 0.67 mmol, 1 equiv) or 19 (0.3 g, 0.63 mmol, 1 equiv) in
CH2Cl2 (10 mL) mCPBA (3 equiv) was added. The reaction mix-
ture was stirred at rt for 90 min, diluted with CH2Cl2 (20 mL),
and washed with satd NaHCO3 (2 ꢁ 20 mL) and water (20 mL).
The organic phase was dried with anhydrous Na2SO4, filtered,
and concentrated. The crude product was purified by flash chro-
matography (hexane–EtOAc, 3:1?1:1).
4.2.2.5. Eicosyl 2,3,4,6-tetra-O-acetyl-
(6). Yield 0.80 g, 50%, oil. [ +24.0 (c 0.5, CHCl3). HRMS: m/
z: calcd for C34H60O10Na [M+Na]+: 651.4085; found: 651.4098.
a-D-mannopyranoside
a]
D
4.2.2.6. Cyclohexylmethyl 2,3,4,6-tetra-O-acetyl-
a-D
-mannopy-
ranoside (8).
Yield 0.83 g, 73%, colorless oil. [a]D +48.0 (c 1.0,
CHCl3). HRMS: m/z: calcd for C21H32O10Na [M+Na]+: 467.1893;
found: 467.1899.
4.3.1. Hexyl 2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl sulfone
(22)
4.2.2.7. 2-Cyclohexylethyl 2,3,4,6-tetra-O-acetyl-
a-D
-mannopy-
Yield 0.31 g, 98%, white solid, mp = 98–99 °C. [a] +44.0 (c 1.0,
D
ranoside (9).
Yield 0.66 g, 56%, yellowish oil. [a]D +44.0 (c 1.0,
CHCl3). HRMS: m/z: calcd for C22H36O11SNa [M+Na]+: 531.1876;
CHCl3). HRMS: m/z: calcd for C22H34O10Na [M+Na]+: 481.2050;
found: 531.1886.
found: 481.2059.
4.3.2. Octyl 2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl sulfone
(23)
4.2.2.8. Hexyl 2,3,4,6-tetra-O-acetyl-1-thio-a-D-mannopyrano-
side (18).
Yield 0.60 g, 52%, yellowish oil. [a] +85.0 (c 1.0,
D
Yield 0.31 g, 96%, white solid, mp = 99–101 °C. [a]D +44.1 (c 1.0,
CHCl3). HRMS: m/z: calcd for C20H32O9SNa [M+Na]+: 471.1665;
CHCl3). HRMS: m/z: calcd for C14H29O7S [M+H]+: 341.1634; found:
found: 471.1682.
341.1658.
1H and 13C NMR data for compounds 2–6, 8, 9, 18, 19, 22, and
23 are in Tables 1 and 2.
4.2.2.9. Octyl 2,3,4,6-tetra-O-acetyl-1-thio-
side (19).
a
-
D
-mannopyrano-
Yield 0.62 g, 51%, yellowish oil. [a] +84.0 (c 1.0,
D
Table 1
1H NMR chemical shifts (in ppm) and 1H–1H coupling constants of 2–6, 8, 9, 18, 19, 22, and 23a
H-1
(J1,2
H-2
(J2,3
H-3
H-4
H-5
H-6
H-60
Aglycone (JH,H
)
Acetyl groups
0
0
)
)
(J3,4
)
(J4,5
)
(J5,6
)
(J6,6
)
(J5,6
)
2
3
4
5
6
8
9
4.80 d 5.23 dd
(1.5) (3.4)
5.36 dd
(9.9)
5.28 dd
(9.8)
3.98
ddd
(2.3)
3.97
ddd
(2.4)
3.98
ddd
(2.4)
3.99
ddd
(2.4)
3.99
ddd
(2.4)
3.97
ddd
(2.3)
3.98
ddd
(2.4)
4.39
dd
(2.0)
4.39
dd
(2.0)
4.67
ddd
(2.4)
4.67
ddd
(2.4)
4.11 dd
(ꢃ12.3)
4.28
dd
(5.3)
4.27
dd
(5.3)
4.28
dd
(5.3)
4.28
dd
(5.3)
4.28
dd
(5.3)
4.27
dd
(5.3)
4.28
dd
(5.4)
4.09
dd
3.68 dt (9.5, 6.7), 1H, OCH2, 3.45 dt (9.5, 6.6), 1H, OCH2, 1.62–0.86 2.15, 2.09, 2.04,
m, 11H, 4 ꢁ CH2, 1 ꢁ CH3 1.99 each s
4.78 d 5.24 dd
(1.4) (3.3)
5.34 dd
(10.0)
5.28 dd
(9.9)
4.11 dd
(ꢃ12.2)
3.68 dd (9.3, 6.8), 1H, OCH2, 3.45 dd (9.3, 6.2), 1H, OCH2, 1.75–0.89 2.16, 2.11, 2.05,
m, 15H, 6 ꢁ CH2, 1 ꢁ CH3 1.99 each s
4.80 d 5.23 dd
(1.5) (3.4)
5.36 dd
(10.0)
5.28 dd
(9.8)
4.10 dd
(ꢃ12.2)
3.67 dt (9.5, 6.8), 1H, OCH2, 3.45 dt (9.6, 6.6), 1H, OCH2, 1.58–0.86 2.15, 2.10, 2.04,
m, 23H, 10 ꢁ CH2, 1 ꢁ CH3 1.99 each s
4.80 d 5.23 dd
(1.5) (3.3)
5.36 dd
(9.9)
5.29 dd
(9.8)
4.11 dd
(ꢃ12.2)
3.68 dt (9.5, 6.8), 1H, OCH2, 3.45 dt (9.6, 6.6), 1H, OCH2, 1.60–0.88 2.15, 2.10, 2.04,
m, 31H, 14 ꢁ CH2, 1 ꢁ CH3 1.99 each s
4.80 d 5.23 dd
(1.4) (3.4)
5.36 dd
(9.9)
5.29 dd
(9.8)
4.10 dd
(ꢃ12.2)
3.67 dt (9.5, 6.7), 1H, OCH2, 3.45 dt (9.6, 6.6), 1H, OCH2, 1.62–0.85 2.16, 2.10, 2.04,
m, 39H, 18 ꢁ CH2, 1 ꢁ CH3 1.99 each s
4.77 d 5.24 dd
(1.4) (3.3)
5.34 dd
(9.9)
5.28 dd
(9.8)
4.11 dd
(ꢃ12.2)
3.48 dd (9.3, 6.8), 1H, OCH2, 3.25 dd (9.3, 6.2), 1H, OCH2, 1.75–0.89 2.16, 2.11, 2.05,
m, 11H, 5 ꢁ CH2, 1 ꢁ CH 1.99 each s
4.77 d 5.23 dd
(1.5) (3.4)
5.35 dd
(9.9)
5.28 dd
(10.0)
4.11 dd
(ꢃ12.2)
3.73 dd (9.7, 6.9), 1H, OCH2, 3.49 dd (9.7, 6.8), 1H, OCH2, 1.73–0.88 2.16, 2.10, 2.05,
m, 13H, 6 ꢁ CH2, 1 ꢁ CH
1.99 each s
18 5.26 d 5.35–
(1.1) 5.28 m
5.35–
5.28 m
5.35–
5.28 m
4.32 dd
(ꢃ12.0)
2.61 m, 2H, SCH2, 1.67–0.86 m, 11H, 4 ꢁ CH2, 1 ꢁ CH3
2.16, 2.10, 2.05,
1.99 each s
(5.3)
4.09
dd
(5.3)
4.15
dd
(5.6)
4.15
dd
19 5.26 d 5.35–
(1.1) 5.28 m
5.35–
5.28 m
5.35–
5.28 m
4.32 dd
(ꢃ12.0)
2.62 m, 2H, SCH2, 1.71–0.85 m, 15H, 6 ꢁ CH2, 1 ꢁ CH3
3.12 m, 2H, SCH2, 1.87–0.86 m, 11H, 4 ꢁ CH2, 1 ꢁ CH3
3.12 m, 2H, SCH2, 1.91–0.86 m, 15H, 6 ꢁ CH2, 1 ꢁ CH3
2.17, 2.10, 2.05,
1.99 each s
22 4.83 d 5.95 dd
(2.2) (3.6)
5.59 dd
(9.1)
5.30 t
(9.5)
4.28 dd
(ꢃ12.5)
2.17, 2.10, 2.06,
2.02 each s
23 4.83 d 5.95 dd
(2.2) (3.6)
5.59 dd
(9.1)
5.30 t
(9.5)
4.28 dd
(ꢃ12.5)
2.17, 2.11, 2.07,
2.02 each s
(5.6)
a
300 MHz in chloroform-d.

1344
M. Poláková et al. / Carbohydrate Research 345 (2010) 1339–1347
Table 2
13C NMR chemical shifts (in ppm) of 2–6, 8, 9, 18, 19, 22, and 23a
C-1
C-2
C-3
C-4
C-5
C-6
Aglycone
Acetyl groups
2
3
4
5
6
8
9
97.8 70.0 69.4 66.5 68.6 62.8 68.8 OCH2, 31.7, 29.4, 25.9, 22.7 CH2, 14.2 CH3
170.9, 170.3, 170.1, 170.0 CH3CO, 21.1, 20.9 (3ꢁ)
CH3CO
97.8 70.0 69.4 66.6 68.6 62.8 68.8 OCH2, 32.2, 29.5 (2ꢁ), 29.4, 26.3, 22.8 CH2, 14.3 CH3
170.9, 170.3, 170.1, 170.0 CH3CO, 21.1, 20.9 (3ꢁ)
CH3CO
97.8 70.0 69.4 66.6 68.6 62.8 68.8 OCH2, 32.1, 29.8 (2ꢁ), 29.7, 29.6 (2ꢁ), 29.5 (2ꢁ), 26.3, 22.9
CH2, 14.3 CH3
170.9, 170.3, 170.1, 170.0 CH3CO, 21.1, 20.9 (3ꢁ)
CH3CO
97.8 70.0 69.4 66.6 68.6 62.8 68.8 OCH2, 32.1, 29.9 (4ꢁ), 29.8 (3ꢁ), 29.7, 29.6 (2ꢁ), 29.5, 26.3,
170.8, 170.3, 170.1, 170.0 CH3CO, 21.1, 20.9 (3ꢁ)
22.9 CH2, 14.3 CH3
CH3CO
97.8 70.0 69.4 66.6 68.6 62.8 68.8 OCH2, 32.1, 29.9 (9ꢁ), 29.8 (2ꢁ), 29.6 (2ꢁ), 29.5 (2ꢁ), 26.3,
22.9 CH2, 14.3 CH3
97.9 69.9 69.4 66.5 68.6 62.8 74.2 OCH2, 37.8 CH,30.2, 30.1, 26.7, 26.0, 25.9 CH2
170.9, 170.3, 170.1, 170.0 CH3CO, 21.1, 20.9 (3ꢁ)
CH3CO
170.8, 170.3, 170.1, 170.0 CH3CO, 21.1, 20.9 (3ꢁ)
CH3CO
97.8 70.0 69.4 66.5 68.7 62.8 66.7 OCH2, 34.7 CH, 36.9, 33.5, 33.4, 26.7, 26.4 (2ꢁ) CH2
170.8, 170.3, 170.1, 170.0 CH3CO, 21.1, 20.9 (3ꢁ)
CH3CO
18 82.8 71.4 69.7 66.6 69.1 62.7 31.6, 31.5, 29.6, 28.7, 22.7 CH2, 14.2 CH3
170.8, 170.2, 170.0, 169.9 CH3CO, 21.1, 20.9 (2ꢁ),
20.8 CH3CO
19 82.8 71.4 69.7 66.6 69.1 62.7 32.0, 31.6, 29.6, 29.4, 29.3, 29.0, 22.8 CH2, 14.3 CH3
22 87.9 65.0 69.0 65.6 73.6 62.6 51.1 SO2CH2, 31.3, 28.4, 22.5, 21.7 CH2, 14.1 CH3
23 87.9 65.0 69.0 65.6 73.6 62.6 51.1 SO2CH2, 31.9, 29.1 (2ꢁ), 28.7, 22.8, 21.8 CH2, 14.2 CH3
170.8, 170.2, 170.0, 169.9 CH3CO, 21.1, 20.9 (2ꢁ),
20.8 CH3CO
170.5, 169.8, 169.5, 169.4 CH3CO, 20.9 (2ꢁ), 20.8,
20.7 CH3CO
170.6, 169.9, 169.5, 169.4 CH3CO, 20.9 (2ꢁ), 20.8,
20.7 CH3CO
a
75 MHz in chloroform-d.
4.4. General procedure for removal of acetyl groups
4.4.8. Hexyl 1-thio-
a-
D-mannopyranoside (20)
Yield 0.12 g, 97%, pale yellow oil. [
a]D +186.0 (c 1.0, MeOH). HRMS:
Acetylated mannopyranoside (0.2 g) was dissolved in MeOH–
CH2Cl2 (7.5:2 mL), and 1 M MeONa (0.5 mL) was added. The
reaction mixture was stirred for 24 h at rt, neutralized with
Dowex 50 H+-form, filtered, and concentrated. The crude product
was purified by flash chromatography (CH2Cl2–MeOH,
10:1?5:1).
m/z: calcd for C12H24O5SNa [M+Na]+: 303.1242; found: 303.1260.
4.4.9. Octyl 1-thio-
Yield 0.12 g, 95%, pale yellow oil. [
+142.6 (c 0.4, Me2SO). HRMS: m/z: calcd for C14H28O5SNa
[M+Na]+: 331.1555; found: 331.1583.
a
-
D
-mannopyranoside (21)
a]
D +164.0 (c 1.0, MeOH), lit.20
½ ꢂ
a 2D5
4.4.10. a-D-Mannopyranosyl hexyl sulfone (24)
4.4.1. Hexyl
Yield 0.12 g, 96%, colorless oil. [
m/z: calcd for C12H24O6Na [M+Na]+: 287.1471; found: 287.1480.
a
-
D
-mannopyranoside (10)
Yield 0.12 g, 93%, mp = 105–106 °C. [a] +80.0 (c 1.0, MeOH).
D
a
]D +50.0 (c 1.0, MeOH). HRMS:
HRMS: m/z: calcd for C12H24O7SNa [M+Na]+: 335.1141; found:
335.1156.
4.4.2. Octyl
Yield 0.12 g, 95%, pale yellow oil. [
+57.0 (c 0.82, MeOH); lit.29
+56.0 (c 0.85, H2O). HRMS: m/
z: calcd for C14H28O6Na [M+Na]+: 315.1784; found: 315.1796.
a
-
D
-mannopyranoside (11)
4.4.11.
a
-
D
-Mannopyranosyl octyl sulfone (25)
+74.0 (c 1.0, MeOH).
a]
D +59.0 (c 1.0, MeOH), lit.11
Yield 0.13 g, 95%, mp = 103–105 °C. [
HRMS: m/z: calcd for
341.1678.
a
]
D
½
a 2D4
ꢂ
½ ꢂ
a 2D5
C
14H29O7S [M+H]+: 341.1634; found:
1H and 13C NMR data for compounds 10–14, 16, 17, 20, 21, 24,
and 25 are in Tables 3 and 4.
4.4.3. Dodecyl
Yield 0.12 g, 90%, white solid. [
m/z: calcd for C18H36O6Na [M+Na]+: 371.2410; found: 371.2476.
a
-
D
-mannopyranoside (12)
a]
+60.0 (c 0.5, CHCl3). HRMS:
D
4.5. General procedure for the synthesis of alkyl 2,3,4-tri-O-
benzoyl-6-O-tosyl-a-D-mannopyranosides
4.4.4. Hexadecyl
Yield 0.12 g, 85%, mp = 76–78 °C. [
+31.0 (c 1.0, MeOH). HRMS: m/z: calcd for C22H45O6 [M+H]+:
a
-
D
-mannopyranoside (13)
a]
D
+46.0 (c 0.5, CHCl3), lit.24
[a]
D
A stirred solution containing 11 (0.20 g, 0.68 mmol) or 17
(0.20 g, 0.69 mmol) in pyridine (5 mL) was cooled down on an
ice bath and tosyl chloride (1.3 equiv) in CH2Cl2 (2 mL) was added
dropwise. The resulting mixture was brought to rt and stirring was
continued for 16 h. The reaction mixture was diluted with CH2Cl2
(20 mL), washed with 1 M HCl (2 ꢁ 10 mL), satd NaHCO3
(2 ꢁ 10 mL), water (20 mL), dried with anhydrous Na2SO4, filtered,
and concentrated. The crude product was dissolved in pyridine
(4 mL) and cooled down on an ice bath. Benzoyl chloride
(4.5 equiv) was added dropwise. The resulting mixture was
brought to rt and stirring was continued for 16 h. The reaction mix-
ture was diluted with water (10 ml) and CH2Cl2 (30 mL). The or-
ganic phase was separated, washed with 1 M HCl (3 ꢁ 15 mL),
satd NaHCO3 (3 ꢁ 10 mL), dried with anhydrous Na2SO4, filtered,
and concentrated. The crude product was purified by flash chroma-
tography (hexane–EtOAc, 5:1?1.5:1).
405.3216; found: 405.3249.
4.4.5. Eicosyl
Yield 0.12 g, 81%, mp = 89–91 °C. [
HRMS: m/z: calcd for C26H53O6 [M+H]+: 461.3842; found: 461.3874.
a
-D
-mannopyranoside (14)
a
]
+29.0 (c 0.45, pyridine).
D
4.4.6. Cyclohexylmethyl a-D-mannopyranoside (16)
Yield 0.12 g, 96%, mp = 114–117 °C. [a] +68.0 (c 1.0, MeOH).
D
HRMS: m/z: calcd for
C
13H25O6 [M+H]+: 277.1651; found:
277.1678.
4.4.7. 2-Cyclohexylethyl
a-
D
-mannopyranoside (17)
Yield 0.12 g, 95%, mp = 101–103 °C. [a] +64.0 (c 1.0, MeOH).
D
HRMS: m/z: calcd for C14H26O6Na [M+Na]+: 313.1627; found:
313.1656.

M. Poláková et al. / Carbohydrate Research 345 (2010) 1339–1347
1345
Table 3
1H NMR chemical shifts (in ppm) and 1H–1H coupling constants of 10–14, 16, 17, 20, 21, 24, and 25
H-1
(J1,2
H-2
(J2,3
H-3
H-4
H-5
H-6
H-60
Aglycone (JH,H)
0
0
)
)
(J3,4
)
(J4,5
)
(J5,6
)
(J6,6
)
(J5,6
)
10a 4.75 3.80 dd
3.77–
3.69 m
(9.3)
3.78–
3.69 m
(9.3)
3.63 t
(9.3)
3.56
ddd
(2.4)
3.55
ddd
3.85 dd
(ꢃ11.7)
3.77–
3.69 m
(5.6)
3.78–
3.69 m
(5.6)
3.77–3.69 m, 1H, OCH2, 3.44 dt (9.6, 6.3), 1H, OCH2, 1.61 m, 2H, OCH2CH2, 1.46–1.31
m, 6H, 3 ꢁ CH2, 0.94 t (6.7), 3H, CH3
d
(3.2)
(1.4)
11a 4.76 3.81 dd
3.64 t
(9.3)
3.85 dd
(ꢃ11.7)
3.78–3.69 m, 1H, OCH2, 3.44 dt (9.6, 6.3), 1H, OCH2, 1.61 m, 2H, OCH2CH2, 1.45–1.30
m, 10H, 5 ꢁ CH2, 0.94 t (6.7), 3H, CH3
d
(3.1)
(1.4)
(2.4)
3.50 d
12b 4.81 3.99–
3.85 dd
(9.6)
3.98–
3.90 m
(9.7)
3.76 d
(ꢃ11.3)
3.99–
3.92 m
3.60 dt (9.4, 6.6), 1H, OCH2, 3.36 dt (9.5, 6.6), 1H, OCH2, 1.52 m, 2H, OCH2CH2, 1.30–
1.21 m, 18H, 9 ꢁ CH2, 0.88 t (7.0), 3H, CH3
s
3.92 m
(2.1)
13c 4.57 3.60–
3.45–
3.35–
3.35–
3.27 m
(2.0)
3.63 ddd 3.45–
3.60–3.55 m, 1H, OCH2, 3.35–3.27 m, 1H, OCH2, 1.49 m, 2H, OCH2CH2, 1.30–1.19 m,
26H, 13 ꢁ CH2, 0.85 t (7.0), 3H, CH3
d
3.55 m
3.41 m
(4.52 d,
3–OH,
3.27 m
(4.69 d,
4-OH,
(ꢃ11.5)
(4.40 t,
6-OH,
3.41 m
(6.0)
(1.4) (4.66 d,
2–OH,
J2,OH 4.4) J3,OH 6.1) J4,OH 5.4)
J6,OH 6.0)
4.60–
4.55 m
(ꢃ11.4)
3.85 dd
(ꢃ11.7)
14d 5.36 4.60–
4.55 m
4.60–
4.55 m
(8.8)
3.76–
3.69 m
(9.3)
3.78–
3.69 m
(9.3)
3.95–
3.90 m
4.67 dd
(9.7)
4.33 m
4.41 dd 3.98 dt (15.8, 6.8), 1H, OCH2, 3.50 dt (15.7, 6.5), 1H, OCH2, 1.63 m, 2H, OCH2CH2,
s
(5.9)
1.36–1.19 m, 34H, 17 ꢁ CH2, 0.87 t (6.4), 3H, CH3
16a 4.75 3.81 dd
3.63 t
(9.3)
3.57–
3.52 m
(2.3)
3.56
ddd
3.76–
3.69 m
3.57–3.52 m, 1H, OCH2, 3.24 dd (9.4, 6.0), 1H, OCH2, 1.84–0.99 m, 11H, 5 ꢁ CH2,
1 ꢁ CH
d
(1.4)
(3.1)
17a 4.76 3.81 dd
3.64 t
(9.3)
3.87 dd
(ꢃ11.7)
3.78–
3.69 m
(5.6)
3.78–3.69 m, 1H, OCH2, 3.48 dt (9.8, 6.4), 1H, OCH2, 1.82–0.91 m, 13H, 6 ꢁ CH2,
1 ꢁ CH
d
(1.4)
(3.1)
(2.4)
20a 5.24 3.95–
3.70–
3.67 m
3.70–
3.67 m
(2.4)
3.70-
3.67 m
(2.4)
3.85 dd
(ꢃ11.8)
3.77 dd 2.66 m, 2H, SCH2, 1.67–1.32 m, 8H, 4 ꢁ CH2, 0.94 t (6.8), 3H, CH3
d
(1.2)
3.90 m
(5.6)
21a 5.24 3.95–
3.95–
3.70-
3.85 dd
(ꢃ11.8)
3.77 dd 2.67 m, 2H, SCH2, 1.72–1.30 m, 12H, 6 ꢁ CH2, 0.93 t (6.6), 3H, CH3
d
(1.2)
3.90 m
3.90 m
3.67 m
(5.6)
24a 4.95 4.53 dd
4.00 dd
(9.2)
3.74–
3.66 m
4.18 m
(2.0)
3.92 dd
(ꢃ12.1)
3.74–
3.66 m
3.25 m, 2H, SCH2, 1.90–1.80 m, 2H, SCH2CH2, 1.56–1.34 m, 6H, 3 ꢁ CH2, 0.96 t (6.8),
d
(1.2)
(3.6)
3H, CH3
25a 4.95 4.53 dd
4.00 dd
(9.2)
3.74–
3.66 m
4.18
ddd
(2.0)
3.92 dd
(ꢃ12.2)
3.74–
3.66 m
3.25 m, 2H, SCH2, 1.90–1.80 m, 2H, SCH2CH2, 1.56–1.31 m, 10H, 5 ꢁ CH2, 0.94 t (6.9),
d
(1.2)
(3.6)
3H, CH3
a
b
c
300 MHz in methanol-d4.
600 MHz in chloroform-d.
600 MHz in dimethylsulfoxide-d6.
400 MHz in pyridine-d5.
d
Table 4
13C NMR chemical shifts (in ppm) of 10–14, 16, 17, 20, 21, 24, and 25
C-1
C-2
C-3
C-4
C-5
C-6
Aglycone
10a
11a
12b
13c
14d
16a
17a
20a
21a
24a
25a
101.7
101.7
100.0
99.7
101.9
101.8
101.7
86.6
72.4
72.4
71.1
70.4
72.6/73.5
72.4
72.5
73.4
73.4
72.8
72.8
71.7
71.0
68.8
68.8
66.2
67.0
69.6
68.7
68.8
69.0
69.0
67.8
67.8
74.7
74.7
72.1
73.9
75.7
74.7
74.8
75.0
75.0
80.0
80.0
63.1
63.1
61.0
61.3
63.6
63.1
63.1
62.9
62.9
63.3
63.3
68.7 OCH2, 33.0, 30.7, 27.2, 23.8 CH2, 14.5 CH3
68.7 OCH2, 33.1, 30.8, 30.7, 30.6, 27.5, 23.9 CH2, 14.6 CH3
68.0 OCH2, 32.2, 29.7 (3ꢁ), 29.6, 29.5, 29.4 (2ꢁ), 26.1, 22.7 CH2, 14.1 CH3
66.2 OCH2, 31.3, 29.04–29.00 (9ꢁ), 28.9, 28.7, 25.8, 22.1 CH2, 14.0 CH3
67.9 OCH2, 32.5, 30.4–30.3 (13ꢁ), 30.1, 30.0, 27.0, 23.3, CH2, 14.6 CH3
74.3 OCH2, 39.4 CH, 31.4, 31.2, 27.8, 27.2, 27.1 CH2
66.7 OCH2, 36.1 CH, 38.3, 34.7, 34.5, 27.8, 27.6, 27.5 CH2
32.7, 32.0, 30.8, 29.7, 23.8 CH2, 14.5 CH3
33.1, 32.0, 30.9, 30.5, 30.4, 30.0, 23.8 CH2, 14.6 CH3
51.3 SCH2, 32.5, 29.4, 23.6, 22.6 CH2, 14.4 CH3
72.9
72.8
73.9
73.9
72.9
72.9
86.6
93.0
93.0
67.0
67.0
51.2 SCH2, 33.1, 30.3 (2ꢁ), 29.7, 23.8, 22.6 CH2, 14.5 CH3
a
b
c
75 MHz in methanol-d4.
150 MHz in chloroform-d.
150 MHz in dimethylsulfoxide-d6.
100 MHz in pyridine-d5.
d
4.5.1. Octyl 2,3,4-tri-O-benzoyl-6-O-tosyl-
a-
D-mannopyranoside
4.5.2. 2-Cyclohexylethyl 2,3,4-tri-O-benzoyl-6-O-tosyl-a-D-
(26)
mannopyranoside (27)
Yield 0.32 g, 61% over two steps, oil. [
a]
ꢃ60.0 (c 1.0, CHCl3).
Yield 0.23 g, 44% over two steps, oil. [
a
]
D
ꢃ41.0 (c 1.0, CHCl3).
D
HRMS: m/z: calcd for C42H46O11SNa [M+Na]+: 781.2658; found:
HRMS: m/z: calcd for C42H44O11SNa [M+Na]+: 779.2502; found:
781.2667.
779.2538.

1346
M. Poláková et al. / Carbohydrate Research 345 (2010) 1339–1347
4.6. General procedure for the synthesis of alkyl 2,3,4-tri-O-
benzoyl-6-deoxy- -mannopyranosides
4.6.2. 2-Cyclohexylethyl 2,3,4-tri-O-benzoyl-6-deoxy-a-D-
mannopyranoside (29)
a
-D
Yield 83.7 mg, 47%, pale yellow oil. [
a]
D
ꢃ98.0 (c 1.0, CHCl3).
Compound 26 (0.32 g, 4.2 mmol, 1 equiv) or 27 (0.23 g,
3.0 mmol, 1 equiv) was dissolved in DMF (4 mL), and sodium boro-
hydride (5 equiv) was added slowly. The resulting mixture was
stirred at 65 °C for 6 h. The reaction mixture was cooled to rt and
neutralized with glacial AcOH and diluted with CH2Cl2 (30 mL).
The organic phase was separated, washed with satd NaHCO3
(3 ꢁ 20 mL), water (2 ꢁ 20 mL), dried with anhydrous Na2SO4, fil-
tered, and concentrated. The crude product was purified by flash
chromatography (hexane–EtOAc, 7:1?4:1).
HRMS: m/z: calcd for C35H38O8Na [M+Na]+: 609.2465; found:
609.2488.
4.7. General procedure for removal of benzoyl groups
Benzoylated compound 28 (0.10 g, 0.17 mmol) or 29 (0.08 g,
0.14 mmol) was dissolved in MeOH–CH2Cl2 4:1 (5 mL), and 1 M
MeONa (0.25 mL) was added. The reaction mixture was stirred
for 24 h at rt, neutralized with Dowex 50 H+-form, filtered, and
concentrated. The crude product was purified by column chroma-
tography (CH2Cl2–MeOH, 10:1?5:1).
4.6.1. Octyl 2,3,4-tri-O-benzoyl-6-deoxy-a-D-mannopyranoside
(28)
4.7.1. Octyl 6-deoxy-
Yield 42.2 mg, 90%, colorless oil. [
+52.0 (c 0.5, MeOH). HRMS: m/z: calcd for C14H28O5Na
[M+Na]+: 299.1834; found: 299.1851.
a
-
D
-mannopyranoside (30)
D ꢃ103.0 (c 1.0, CHCl3), lit.11
a]
D +56.0 (c 0.55, MeOH), lit.11
Yield 0.10 g, 41%, pale yellow oil. [
a]
a 2D5
ꢂ
ꢃ115.0 (c 0.8, CHCl3). HRMS: m/z: calcd for C35H41O8 [M+H]+:
½ ꢂ
a 2D5
½
589.2802; found: 589.2842.
Table 5
1H NMR chemical shifts (in ppm) and 1H–1H coupling constants of 26–31 (300 MHz)
H-1
(J1,2
H-2
(J2,3
H-3
(J3,4
H-4
H-5
H-6
H-60
Aglycone (JH,H
)
Other groups
0
0
)
)
)
(J4,5
)
(J5,6
)
(J6,6
)
(J5,6
)
26a 4.98 5.61 5.84
dd dd
5.74
dd
(9.9)
5.67
dd
4.32–
4.32–
4.32–
3.75 dt (9.6, 6.7), 1H, OCH2, 3.49 dt (9.6, 6.6), 1H, OCH2, 1.65 m, 2H,
8.08–7.14 m, 19H, Ar,
2.33 s, 3H, CH3
d
4.20 m 4.20 m 4.20 m OCH2CH2, 1.42–1.26 m, 10H, 5 ꢁ CH2, 0.90 t (5.8), 3H, CH3
(1.6) (3.2) (10.0)
28a 4.99 5.65 5.84
4.19 m 1.36 d
(6.3)
3.78 dt (9.6, 6.7), 1H, OCH2, 3.54 dt (9.6, 6.6), 1H, OCH2, 1.74–1.26 m,
12H, 6 ꢁ CH2, 0.90 t (6.2), 3H, CH3
8.12–7.22 m, 15H, Ar
d
dd
dd
(3H)
(1.3) (3.4) (10.1)
(10.0)
3.37
30b 4.68 3.82 3.66–
3.66–
1.28 d
3.71 dt (9.6, 6.7), 1H, OCH2, 3.41 dt (9.6, 6.6), 1H, OCH2, 1.60 m, 2H,
d
dd
3.55 m dd
3.55 m (3H)
(6.2)
OCH2CH2, 1.43–1.32 m, 10H, 5 ꢁ CH2, 0.94 t (6.5), 3H, CH3
(1.2) (3.3)
(9.3)
5.76
dd
27a 4.98 5.60 5.83
4.31–
4.31–
4.31–
3.79 dt (9.7, 6.9), 1H, OCH2, 3.53 dt (9.7, 6.8), 1H, OCH2, 1.76–0.86 m,
8.08–7.23 m, 19H, Ar,
2.33 s, 3H, CH3
d
dd
dd
4.22 m 4.22 m 4.22 m 13H, 6 ꢁ CH2, 1 ꢁ CH
(1.6) (3.2) (10.0)
(9.8)
5.67
dd
29a 4.99 5.65 5.84
4.18 m 1.36 d 3.83 dt (9.6, 6.9), 1H, OCH2, 3.58 dt (9.7, 6.7), 1H, OCH2, 1.79–0.86 m,
8.12–7.23 m, 15H, Ar
d
dd
dd
(6.3)
(3H)
13H, 6 ꢁ CH2, 1 ꢁ CH
(1.3) (3.4) (10.1)
(10.0)
3.40
31b 4.67 3.80 3.66–
3.66–
1.29 d
3.76 dt (9.6, 6.8), 1H, OCH2, 3.47 dt (9.6, 6.7), 1H, OCH2, 1.78–0.89 m,
d
dd
3.55 m dd
(9.5)
3.55 m (3H)
(6.2)
13H, 6 ꢁ CH2, 1 ꢁ CH
(1.2) (3.3)
a
In chloroform-d.
In methanol-d4.
b
Table 6
13C NMR chemical shifts (in ppm) of 26–31 (75 MHz)
C-1 C-2 C-3 C-4 C-5
C-6
Aglycone
Other groups
26a
28a
97.6 70.7
70.1 67.1
68.9 68.6 69.0 OCH2, 32.0, 29.6, 29.5, 26.3, 22.9, 21.8 CH2, 165.7, 165.6 (2ꢁ) PhCO, 144.9 Ar (Ts), 133.7–128.2 Ar,
14.3 CH3 21.8 CH3 (Ts)
66.8 17.9 68.7 OCH2, 32.1, 29.7, 29.6, 29.5, 26.4, 22.9 CH2, 166.0, 165.8, 165.7 PhCO, 133.6–128.5 Ar
97.8 71.3/
72.2
70.3 71.3/
72.2
14.3 CH3
30b 101.8 72.5/72.6
74.1
69.9 18.1 68.7 OCH2, 33.1, 30.8, 30.6, 30.5, 27.5, 23.8 CH2,
14.6 CH3
27a
29a
97.6 70.8
70.1 67.2
69.0 68.5 67.0 OCH2, 34.7 CH, 36.9, 33.5, 33.4, 26.7, 26.4
165.7, 165.6 (2ꢁ) PhCO, 144.9 Ar (Ts), 134.0–128.2 Ar,
21.8 CH3 (Ts)
166.0, 165.9, 165.7 PhCO, 133.6–128.5 Ar.
(2ꢁ) CH2
97.7 71.3/
72.2
70.3 71.3/
72.2
66.8 17.9 66.6 OCH2, 34.8 CH, 37.1, 33.6, 33.5, 26.8, 26.5
(2ꢁ) CH2
31b 101.8 72.5/72.6
74.1
69.9 18.1 66.6 OCH2, 36.0 CH, 38.3, 34.8, 34.4, 27.8, 27.6,
27.5 CH2
a
In chloroform-d.
In methanol-d4.
b

M. Poláková et al. / Carbohydrate Research 345 (2010) 1339–1347
1347
4.7.2. 2-Cyclohexylethyl 6-deoxy-a-D-mannopyranoside (31)
Acknowledgments
Yield 32.5 mg, 87%, colorless oil. [
a]
+51.0 (c 0.6, MeOH).
D
HRMS: m/z: calcd for C14H26O5Na [M+Na]+: 297.1678; found:
This work was supported by the APVV-51-046505 and
VEGA-2/0199/09 grants, the Slovak State Programme Project
No. 2003SP200280203, and by the European Commission under
contract LSHP-CT-2005-018923‘NM4TB‘. Drs. Jana Korduláková
and Vladimír Puchart are appreciated for helpful discussion.
297.1691.
1H and 13C NMR data for compounds 26–31 are in Tables 5 and
6.
ˇ
Dr. Iveta Uhliariková and Mrs. Anna Karovicová are acknowl-
4.8. Octyl
(32)
a-D-mannopyranosyl-(1?6)-a-D-mannopyranoside
edged for technichal assistance in NMR analyses. The authors
thank Dr. Ray Marshall for his kind help in the preparation of
this paper.
The disaccharide 32 was obtained after coupling reaction fol-
lowed by stepwise deprotection. Data are in agreement with those
previously reported.13 13C NMR (100 MHz, CD3OD): d 101.7, 101.5
(C-1, C-10), 74.5, 73.2, 73.0, 72.8 (C-3, C-30, C-5, C-50), 72.3, 72.2 (C-
2, C-20), 68.7 (3ꢁ) (OCH2 octyl, C-4, C-40), 67.5 (C-6), 63.0 (C-60),
33.2, 30.8, 30.7, 30.6, 27.6, 23.9 (6 ꢁ CH2 octyl), 14.6 (CH3 octyl).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2010.03.011.
References
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50
mixtures were incubated at 60 °C for 30 min. After neutralization
with 10 L of 0.2 M NaOH, the samples were dried under a stream
of nitrogen. They were then subjected to mild alkali hydrolysis per-
formed with 150 L CHCl3–MeOH (2:1, v/v) and 150 L 0.2 M
lL of 1-propanol, and 100 lL of 20 mM HCl was added. The
l
l
l
NaOH in MeOH for 20 min at 37 °C. After neutralization with a
drop of glacial acetic acid, the mixtures were dried and subjected
to n-butanol/water partitioning. Pooled butanol extracts contain-
ing the radiolabeled products originating from the synthetic glyco-
sides were dried under N2, resuspended in n-butanol, and analyzed
by TLC on aluminum-coated Silica 60 F254 plate (Merck). The TLC
plate was developed in CHCl3–MeOH–concd NH4OH–H2O
(65:25:0.5:4; by vol.) and exposed to radiographic film (Kodak
Bio Max MR). Incorporation of the radioactive label to the products
was quantified by scraping the silica from the corresponding bands
to scintillation vials and measuring the radioactivity using 5 mL of
EcoLite(+) liquid scintillation fluid (MP Biomedicals) in the scintil-
lation counter Tri-Carb 2900 TR (Perkin–Elmer).