Studies toward a conjugate vaccine for anthrax. Synthesis and characterization of anthrose [4,6-dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-O- methyl-D-glucopyranose] and its methyl glycosides

Article abstract of DOI:10.1016/j.carres.2005.04.010

The key step in the first chemical synthesis of anthrose (16) and its methyl α- (6) and β-glycoside (22) was inversion of configuration at C-2 in triflates 10, 2, and 18, respectively, obtained from the common intermediate, methyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (1). To prepare methyl α-anthroside (6), methylation at O-2 of the gluco product 3, obtained from 2, was followed by hydrogenation/hydrogenolysis of the formed 2-methyl ether 4, to simultaneously remove the protecting benzyl group and reduce the azido function. Subsequent N-acylation of the formed amine 5 with 3-hydroxy-3-methylbutyric acid gave the target methyl α-glycoside 6. Synthesis of methyl β-anthroside (22) comprised the same sequence of reactions, starting from the known methyl 4-azido-3-O-benzyl-4,6-dideoxy-β- D-mannopyranoside (17), which was prepared from 1. In the synthesis of anthrose (16), 1-thio-β-glucoside 11, obtained from 1 through 10, was methylated at O-2, and the azido function in the resulting benzylated 1-thioglycoside 12 was selectively reduced to give amine 13. After N-acylation with 3-hydroxy-3-methylbutyric acid, 1-thioglycoside 14 was hydrolyzed to give the corresponding reducing sugar, aldol 15, which was debenzylated to afford anthrose.

Full text of DOI:10.1016/j.carres.2005.04.010

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 1591–1600
Studies toward a conjugate vaccine for anthrax.
Synthesis and characterization of anthrose
[4,6-dideoxy-4-(3-hydroxy-3-methylbutanamido)-
2-O-methyl-D-glucopyranose] and its methyl glycosides
´ˇ*
Rina Saksena, Roberto Adamo and Pavol Kovac
NIDDK, LMC, National Institutes of Health, Bethesda, MD 20892-0815, USA
Received 15 March 2005; accepted 19 April 2005
Abstract—The key step in the first chemical synthesis of anthrose (16) and its methyl a- (6) and b-glycoside (22) was inversion of
configuration at C-2 in triflates 10, 2, and 18, respectively, obtained from the common intermediate, methyl 4-azido-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranoside (1). To prepare methyl a-anthroside (6), methylation at O-2 of the gluco product 3, obtained from 2,
was followed by hydrogenation/hydrogenolysis of the formed 2-methyl ether 4, to simultaneously remove the protecting benzyl
group and reduce the azido function. Subsequent N-acylation of the formed amine 5 with 3-hydroxy-3-methylbutyric acid gave
the target methyl a-glycoside 6. Synthesis of methyl b-anthroside (22) comprised the same sequence of reactions, starting from
the known methyl 4-azido-3-O-benzyl-4,6-dideoxy-b-D-mannopyranoside (17), which was prepared from 1. In the synthesis of anth-
rose (16), 1-thio-b-glucoside 11, obtained from 1 through 10, was methylated at O-2, and the azido function in the resulting
benzylated 1-thioglycoside 12 was selectively reduced to give amine 13. After N-acylation with 3-hydroxy-3-methylbutyric acid,
1-thioglycoside 14 was hydrolyzed to give the corresponding reducing sugar, aldol 15, which was debenzylated to afford anthrose.
Published by Elsevier Ltd.
Keywords: Methyl a-anthroside; Methyl b-anthroside; Thioglycoside hydrolysis; Perosamine
1. Introduction
tory distress occurs with shock and death following
shortly thereafter. Almost all cases of inhalational
anthrax in which treatment was begun after patients
have exhibited symptoms result in death, regardless of
post-exposure treatment. New concerns regarding
anthrax have recently emerged in connection with the
use of some form of Bacillus anthracis, which is the
etiological agent of anthrax, as a biological weapon.
As a result, a potent vaccine for anthrax has become a
pressing target worldwide.
B. anthracis is a Gram-positive spore-forming bacte-
rium.1 The spore surface provides the first interaction
with the host. It would be beneficial, therefore, to be
able to selectively inactivate B. anthracis spores. Killing
dispersed spores effectively has proven difficult and at
present requires the use of agents, which have very
broad toxicity, such as chlorine dioxide gas. The exospo-
rium is the outermost integument of the mature spore.
Anthrax is a serious disease, predominantly of domesti-
cated and wild animals. Humans become infected inci-
dentally when brought into contact with diseased
animals or contaminated soil. The most common form
of the disease in humans is cutaneous anthrax, which
is usually acquired via injured skin or mucous mem-
branes. Untreated contact or cutaneous anthrax has a
fatality rate of 5–20%. Inhalational anthrax is much
more deadly. Initial symptoms are generally nonspecific:
low-grade fever, a dry hacking cough, and weakness.
The person may briefly improve after 2–4 days; how-
ever, within 24 h after this brief improvement, respira-
*
Corresponding author. Tel.: +1 301 496 3569; fax: +1 301 402
0589; e-mail: kpn@helix.nih.gov
0008-6215/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.carres.2005.04.010

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R. Saksena et al. / Carbohydrate Research 340 (2005) 1591–1600
OH
H3C
O
HO
HO
O
H3C
O
HO
O
OH
H3C
O
O
OMe
O
CH3
HO
O
CH3
O
C
C
NH
HO
HO
NH
HO
O
OH
OH
H3C
HO
CH3
OMe
OH
β–Antp–(1→3)–α–
L
–Rhap–(1→3)–α–
L
–Rhap–(1→2)–L–Rhap–
A
B
Figure 1. Structure of the terminal tetrasaccharide of the surface glycoprotein of Bacillus anthracis (A) and of the terminal monosaccharide of the
OPS of Vibrio cholerae O:1, serotype Ogawa (B).
Thus, a vaccine for anthrax could be based on identify-
ing detailed structure of components unique to the
spores, and attacking them with specific, neutralizing
antibodies.
our involvement in the development of a conjugate vac-
cine for cholera from synthetic carbohydrate antigens,
this laboratory has considerable expertise in the chemis-
try of perosamine.9–11 Thus, while other routes could be
envisioned, it occurred to us that derivatives of anthrose
could be accessible through a suitable precursor of per-
osamine, and here we report the first synthesis of anth-
rose and its methyl glycosides from the known methyl
4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyranoside
(1).
Daubenspeck et al.2 have recently established the
structure of the tetrasaccharide (Fig. 1A) of the exospo-
rium glycoprotein Bc1A. Since multiple copies of the tet-
rasaccharide were found to be linked to Bc1A, the
tetrasaccharide or its constituents should be suitable tar-
gets for specific, homologous antibodies. Accessibility of
synthetic derivatives of anthrose is crucial for making its
glycosyl donors, to be used for synthesis of anthrose-
containing oligosaccharides and conjugate immunogens
from them. Within our work toward a carbohydrate-
based, conjugate immunogens for anti-anthrax exospo-
rium antibodies, herein, we report the synthesis of anth-
rose (16), the terminal determinant of the B. anthracis
exosporium glycoprotein tetrasaccharide and methyl a-
(6) and b-anthroside (22). While the syntheses described
here, starting from 1, are not the shortest that could be
designed as synthetic routes to anthrose and its deriva-
tives, they are viable alternatives, since the large-scale
preparation of starting material 1 is well established.3–5
The key step in syntheses of the three title substances
from 1 is inversion of configuration at C-2. Literature
precedents show that nucleophilic displacements at posi-
tion 2 in b-manno and b-gluco derivatives usually give
better yields of displacement products12–17 than in their
a-counterparts.12,13 With some nucleophiles, formation
of byproducts, resulting from elimination, fragmenta-
tion, ring-contraction and neighboring group participa-
tion, can be minimized by subjecting 3-hydroxy-2-O-
triflates to SN2 displacement reactions.18 Since our tar-
get compounds were to carry a methoxyl group at C-
2, the latter approach was not readily applicable. Albert
et al.19 described direct conversion of carbohydrate tri-
flates into epi-hydroxy compounds with the nitrite ion
as a nucleophile. Accordingly, we have treated the triflyl
derivative of 1, mannopyranoside 2, with NaNO2 or
Bu4NNO2 in DMF and obtained crystalline methyl 4-
azido-3-O-benzyl-4,6-dideoxy-a-D-glucopyranoside (3)
in 50–60% yield after chromatography (Scheme 1). The
use of acetonitrile19 as solvent, with either NaNO2 or
Bu4NNO2, resulted in much lower yield of 3. While
the yield of 3 was better than those observed for conven-
tional SN2 displacement in some similar systems,12,13 the
reaction was accompanied by side reactions (TLC). The
major byproduct formed in the conversion of 2 was
identified (MS, NMR) as the 2,3-olefin, methyl 4-azi-
do-3-O-benzyl-2,4,6-trideoxy-a-D-erythro-hex-2-enopyr-
anoside. Extensive formation of products of elimination
2. Results and discussion
The similarity of the terminal determinant of the B.
anthracis exosporium glycoprotein tetrasaccharide
[anthrose,2 4,6-dideoxy-4-(3-hydroxy-3-methylbutanam-
ido)-2-O-methyl-D-glucose] to the terminal determinant
of the O–PS of Vibrio cholerae O:1, serotype Ogawa
and co-workers6–8 [Fig. 1B, 4-(3-deoxy-L-glycero-tetro-
namido)-4,6-dideoxy-2-O-methyl-D-mannose (perosa-
mine)] is remarkable. The two constituent saccharides
are epimeric 4,6-dideoxy-4-aminohexoses of the D-series,
they are methylated at O-2, and their amino groups are
acylated with derivatives of butyric acid. As a result of

R. Saksena et al. / Carbohydrate Research 340 (2005) 1591–1600
1593
OH
O
Me
OTf
O
Me
Me
Me
(a)
OMe
(b)
(c)
(d)
O
O
N3
BnO
N3
BnO
N3
BnO
N3
BnO
OMe
HO OMe
MeO OMe
1
2
3
4
O
C
Me
Me
O
O
(e)
H2N
NH
HO
HO
Me
Me
HO
OMe
OMe
MeO
MeO
5
6
Scheme 1. Reagents and conditions: (a) Tf2O, Py; (b) NaNO2, DMF, 80 °C; (c) MeI, Ag2O, Me2S, DME; (d) H2, Pd/C, MeOH; (e) 3-hydroxy-3-
methylbutyric acid, CH2Cl2, HATU, DIPEA.
(53–82%) was observed by Haradahira et al.13 during
displacements of 2-triflates in a-gluco- and a-mannopyr-
anosides. Other byproducts formed in our situation,
some unstable, were likely analogous to those whose
formation was observed in similar systems when inver-
sion of configuration of secondary alcohols was effected
hydrolysis of the glycoside, or by acetolysis followed
by deacetylation. Treatment of 6 with dilute mineral
or trifluoroacetic acid, under conditions which normally
cleaves alkyl glycosides,22,23 or acetolysis under condi-
tions that yielded the desired product from a related
methyl 4-acylamido hexopyranoside24 did not afford
the expected products. Therefore, we chose to prepare
anthrose from a thioglycoside, instead from a methyl
glycoside. 1-Thioglycosides can be readily converted to
reducing sugars (hemiacetals) under mild conditions.25–27
The use of 1-thio-b-glycoside 9 as the starting material
(Scheme 2) for preparation of anthrose, and methyl gly-
coside 17 as the starting material for preparation of 22
(Scheme 3), offered also an opportunity to compare
the effect of anomeric configuration and the nature of
b y S 2 displacement reaction using nitrite salts.20
N
Conversion of 3 to methyl a-anthroside 6 by succes-
sive methylation21 at O-2 (!4), hydrogenation/hydro-
genolysis (!5) and N-acylation with 3-hydroxy-3-
methylbutyric acid was uneventful, and afforded the
target product in crystalline state.
Methyl a-anthroside (6) can be made from 1 in only
five steps (Scheme 1). Therefore, our original plan was
to make anthrose by further conversion of 6, either by
OAc
OAc
OH
O
Me
Me
Me
O
(d)
O
(a)
(b)
(c)
N3
BnO
N3
BnO
N3
BnO
SEt
1
SEt
OAc
7
8
9
OTf
O
Me
Me
Me
O
O
(e)
(f)
(g)
N3
BnO
N3
BnO
N3
BnO
SEt
SEt
SEt
NH
OH
11
OMe
12
10
O
C
Me
Me
O
O
H2N
BnO
(h)
(i)
SEt
SEt
Me
BnO
Me
OH
Me
OMe
OMe
13
14
O
C
O
C
Me
O
O
NH
BnO
(j)
NH
HO
OH
OH
Me
Me
Me
OH
Me
OMe
OH
OMe
16
15
Scheme 2. Reagents: (a) Ac2O, H2SO4, AcOH; (b) EtSH, BF3ÆEt2O, CH2Cl2; (c) NaOMe, MeOH; (d) Tf2O, CH2Cl2/Py; (e) NaNO2, DMF, 50 °C; (f)
MeI, Ag2O, Me2S, THF; (g) H2S, H2O–Py; (h) 3-hydroxy-3-methylbutyric acid, HATU, DIPEA, CH2Cl2; (i) NIS, H2O, acetone; (j) H2, Pd/C,
MeOH.

1594
R. Saksena et al. / Carbohydrate Research 340 (2005) 1591–1600
OH
O
OTf
O
Me
Me
Me
O
3 steps
Ref. 29
(a)
(b)
(c)
N3
BnO
N3
BnO
N3
BnO
1
OMe
OMe
OMe
OH
17
18
19
Me
O
Me
Me
O
O
O
(d)
(e)
N3
BnO
H2N
HO
C
NH
HO
OMe
OMe
OMe
Me
Me
OMe
21
OMe
20
OMe
22
OH
Scheme 3. Reagents and conditions: (a) Tf2O, CH2Cl2–Py; (b) NaNO2, DMF, rt; (c) MeI, Ag2O, Me2S, DME; (d) H2, Pd/C, MeOH; (e) 3-hydroxy-
3-methylbutyric acid, HATU, DIPEA, CH2Cl2.
Table 1. 1H and 13C chemical shiftsa (ppm) of the b-anthrosyl residue in 22 and in the tetrasaccharide side chain of the Bacillus anthracis exosporium
Residue
H-1
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6
C-6
b-Anthrosyl in 22
b-Anthrosyl in Ab
4.108
103.33
2.747
84.00
3.302
72.84
3.397
56.45
3.284
70.33
1.074
17.91
4.582
103.4
2.850
84.3
3.301
72.7
3.401
56.4
3.278
70.3
1.082
18.1
NH
H-20
C-20
2CH3
2CH3
CH3-2
CH3-2
CO
C-30
b-Anthrosyl in 22
b-Anthrosyl in Ab
7.683
7.754
2.210
48.61
1.160; 1.148
29.47; 29.36
3.424
59.83
171.38
68.61
2.211
48.6
1.160; 1.148
29.4; 29.5
3.526
59.9
Not reported
171.4
a Spectra taken in Me2SO-d6 at 22 °C, at 600 MHz for 1H and 150 MHz for 13C.
b Data taken from Ref. 2.
the aglycon upon the outcome of the SN2 displacement
reaction at C-2 in similarly protected glycosides. Thus,
b-thioglycoside 8, obtained along with the a-anomer
from 1 through 7,28 was deacetylated, and the crystalline
alcohol 9 was converted to its epimer, the gluco analog
11, through the same sequence of reactions as in the con-
version 1!3 (Scheme 1). As with 3, the formation of 11
from triflate 10 was accompanied by side reactions, and
the yields of the desired products in the two SN2 dis-
placement reactions described above were comparable.
After methylation21 (!12) and reduction of the azido
group, amidation of the formed amine 13 with 3-hydr-
oxy-3-methylbutyric acid gave ethyl 3-O-benzyl-1-thio-b-
anthroside (14). Hydrolysis of the latter, effected with
N-iodosuccinimide, gave crystalline hemiacetal 15 as a
mixture of anomers. Hydrogenolytic cleavage of the
benzyl protecting group then gave anthrose as an ano-
meric mixture.
Synthesis of methyl b-anthroside (22) from the
known29 b-mannopyranoside 17 was readily carried
out through the same reaction sequence as for the prep-
aration of a-substance 6 from 1. The yield of the gluco
product 19 of the nucleophilic displacement of the tri-
flate group in 18 with NO2 anion was higher than those
of displacement products from triflates 2 and 10. Meth-
ylation21 at O-2 (!20) and catalytic hydrogenation/
hydrogenolysis gave the crystalline amine 21. Condensa-
tion of the latter with 3-hydroxy-3-methylbutyric acid
gave methyl b-anthroside 22, whose NMR data were
similar to those for the terminal b-anthrosyl residue in
the spectrum of the tetrasaccharide of the exosporium
glycoprotein of B. anthracis (Table 1). The difference
in the chemical shift for H-1 in 22 and the b-anthrosyl
residue in A (Fig. 1) is attributable to the different sub-
stituents at C-1, namely the O-methyl group in 22, ver-
sus the rhamnose trisaccharide in A.
3. Experimental
3.1. General methods
Unless stated otherwise, optical rotations were mea-
sured at ambient temperature for solutions in chloro-
form, with a Perkin–Elmer automatic polarimeter,
Model 341. All reactions were monitored by thin-layer
chromatography (TLC) on Silica Gel 60 coated glass
slides (E. Merck). Column chromatography was per-
formed by gradient elution from columns of silica gel
with the High-Performance Rapid Flash Chromatogra-
phy System (RT Scientific) or with the CombiFlash
Companion Chromatograph (Isco, Inc.). Solvent mix-
tures less polar than those used for TLC were used at
the onset of separation. Nuclear magnetic resonance

R. Saksena et al. / Carbohydrate Research 340 (2005) 1591–1600
1595
(NMR) spectra were measured with a Varian Gemini or
Varian Mercury instrument at 300 MHz (1H) and
75 MHz (13C), or with Bruker Avance 600 spectrome-
ters. Assignments of NMR signals were made by homo-
nuclear and heteronuclear two-dimensional correlation
spectroscopy, run with the software supplied with the
spectrometers. When reporting assignment of NMR sig-
nals, nuclei associated with the N-amido side chain are
denoted with a prime. Liquid chromatography–electron
spray-ionization mass spectrometry (ESIMS) was per-
formed with a Hewlett–Packard 1100 MSD spectrome-
ter. Matrix assisted laser desorption-ionization time-of-
flight mass spectrometry (MALDI-TOFMS) was per-
formed with a Shimadzu AXIMA/CFR spectrometer.
Attempts have been made to obtain correct analytical
data for all new compounds. However, some com-
pounds tenaciously retained traces of solvents, despite
exhaustive drying, and analytical figures for carbon
could not be obtain within 0.4%. Structures of these
compounds follow unequivocally from the mode of syn-
thesis, NMR data, and m/z values found in their mass
spectra, and their purity was verified by TLC and
NMR spectroscopy. Pd/C catalyst (5%, ESCAT 103)
was a product of Engelhard Industries. HATU {N-
[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]-pyridin-1-yl-
methylene]-N-methylmethanaminium hexafluorophos-
phate N-oxide} was purchased from Applied Biosys-
tems. Solutions in organic solvents were dried with
anhydrous Na2SO4, and concentrated at 40 °C/2 kPa.
and that one major and several minor byproducts were
formed. After concentration, chromatography gave first
a mixture of byproducts, which were difficult to resolve.
The main component in the mixture, later eluted pure,
was the material whose NMR and mass spectra showed
that it was methyl 4-azido-3-O-benzyl-2,4,6-trideoxy-a-
D-erythro-hex-2-enopyranoside, [a]D +249 (c 0.5). 1H
NMR (300 MHz, CDCl3): d 4.98 (br d, distorted by long
range coupling, 1H, H-2), 4.91 (dd, 1H, J1,2 3.5, J1,3
1.1 Hz, H-1), 4.85, 4.75 (2d, 1H each, 2J 11.5 Hz,
CH2Ph), 3.90–3.79 (m, 1H, H-5), 3.72, 3.69 (2t, 1H,
J4,5 10.5, J2,4 1 Hz, H-4), 3.37 (s, 3H, OCH3), 1.34 (d,
3H, J5,6 6.0 Hz, H-6); 13C NMR (75 MHz, CDCl3): d
154.90 (C-3), 96.63 (C-1), 96.37 (C-2), 69.53 (CH2Ph),
65.99 (C-5), 61.62 (C-4), 55.22 (OCH3), 18.61 (C-6);
CIMS: (on addition of LiCl): m/z 282 ([M+Li]+). Anal.
Calcd for C14H17N3O3: C, 61.08; H, 6.22; N, 15.26.
Found: C, 61.40; H, 6.28; N, 15.29.
Eluted next was the title glucopyranoside (3, 2.3 g,
54.7%), mp 97.5–98 °C (from i-Pr2O), [a]D +242 (c
1
0.5). H NMR (300 MHz, CDCl3 + D2O): d 4.93, 4.84
(2d, 1H each, 2J 11.1 Hz, CH2Ph), 4.69 (d, 1H, J1,2
3.7 Hz, H-1), 3.68–3.48 (m, 3H, H-2,3,5), 3.41 (s, 3H,
OCH3), 3.07 (t, 1H, J 9.7 Hz, H-4), 1.30 (d, 3H, J5,6
6.2 Hz, C-6); 13C NMR (CDCl3 + D2O): d 99.29 (C-1),
81.10 (C-3), 75.19 (CH2Ph), 72.97 (C-2), 67.53 (C-4),
66.14 (C-5), 55.32 (OCH3), 18.30 (C-6); ESIMSꢁ: m/z:
293.15 ([M]ꢁ), 279 ([MꢁN]ꢁ), 265 ([Mꢁ2Nꢁ]). Anal.
Calcd for C14H19N3O3: C, 57.33; H, 6.53; N, 14.33.
Found: C, 57.32; H, 6.66; N, 14.29.
3.2. Methyl 4-azido-3-O-benzyl-4,6-dideoxy-a-D-gluco-
pyranoside (3)
A comparable yield of 3 was obtained when the reac-
tion was carried out in DMF as solvent and Bu4NNO2
as the source of nucleophile, but diminished yields were
observed when CH3CN was the solvent.
Triflic anhydride (2.7 mL, 16.3 mmol) was added at 0 °C
to a stirred solution of methyl 4-azido-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranoside (1)3–5 (4 g, 13.6 mmol)
in pyridine (7 mL). The cooling bath was removed,
and stirring was continued until TLC (10:1 toluene–ace-
tone) showed that the reaction was complete (ꢀ1 h).
After concentration, the residue was chromatographed
to give methyl 4-azido-3-O-benzyl-4,6-dideoxy-2-O-tri-
fluoromethanesulfonyl-a-D-mannopyranoside (2, 7 g,
ꢀ100%). 1H NMR (CDCl3): d 5.05 (br t, 1H, H-2),
4.80, 4.57 (2d, partially overlapped, J 11.5 Hz, 2H,
2CH2Ph), 4.80 (d, partially overlapped, 1H, J1,2
1.6 Hz, H-1), 3.80 (dd, 1H, J2,3 3.0, J3,4 9.8 Hz, H-3),
3.57–3.45 (m, 1H, H-5), 3.39 (t, partially overlapped, J
9.8 Hz, H-4), 3.37 (s, partially overlapped, OCH3),
1.35 (d, 3H, J5,6 6.2 Hz, H-6); 13C NMR (CDCl3): d
97.63 (C-1), 80.68 (C-2), 74.84 (C-3), 72.53 (CH2Ph),
67.18 (C-5), 63.57 (C-4), 55.36 (OCH3), 18.22 (C-6);
ESIMS: m/z 448 ([M+Na]+).
3.3. Methyl 4-azido-3-O-benzyl-4,6-dideoxy-2-O-methyl-
a-D-glucopyranoside (4)
MeI (3 mL), followed by Ag2O (1.2 g) and a catalytic
amount of Me2S, was added to a solution of alcohol 3
(600 mg, 2 mmol) in 1,2-dimethoxyethane (5 mL), and
the mixture was stirred in the dark, overnight at room
temperature. TLC (3:1 hexane–EtOAc) showed the reac-
tion was complete, and that one product was formed.
The mixture was filtered through a Celite pad, the fil-
trate was concentrated, and the residue was chromato-
1
graphed to give 4 (540 mg, 87%), [a]D +247 (c 0.4). H
NMR (CDCl3): d 4.89, ꢀ4.78 (2d, partially overlapped,
2J 10.7 Hz, CH2Ph), ꢀ4.79 (d, partially overlapped, J1,2
3.5 Hz, read from H-2 signal, H-1), 3.75 (t, 1H, J 9.5 Hz,
H-3), 3.57–3.51 (m, 4H, H-5, incl. s, 3.52, OCH3-2), 3.41
(s, 3H, OCH3-1), 3.32 (dd, 1H, J2,3 9.5 Hz, H-2), 3.08 (t,
1H, J 9.7 Hz, H-4), 1.29 (d, 3H, J5,6 6.3 Hz, H-6); 13C
NMR (CDCl3): d 97.29 (C-1), 82.31 (C-2), 79.73 (C-3),
75.47 (CH2Ph), 67.85 (C-4), 65.82 (C-5), 58.95 (OCH3-
2), 55.21 (OCH3-1), 18.36 (C-6); ESIMS: m/z 330
NaNO2 (2 g, 29.6 mmol) was added to a solution of 2
(6.3 g, 14.8 mmol) in DMF (50 mL), and the mixture
was stirred at 80 °C for 8 h. TLC (3:1 hexane–EtOAc)
then showed that all starting material was consumed,

1596
R. Saksena et al. / Carbohydrate Research 340 (2005) 1591–1600
([M+Na]+). Anal. Calcd for C15H21N3O4: C, 58.62; H,
6.89; N, 13.57. Found: C, 59.07; H, 7.07; N, 13.50.
mannopyranose (7) following the protocol28 aimed at
the preparation of ethyl 2-O-acetyl-4-azido-3-O-benzyl-
4,6-dideoxy-1-thio-a-D-mannopyranoside from 1 (6.4
g). Chromatography of the crude product gave first
the a-anomer (4.72 g, 74%), whose NMR data agreed
with those published.28
3.4. Methyl 4-amino-4,6-dideoxy-2-O-methyl-a-D-gluco-
pyranoside (5)
A solution of the foregoing azide 4 (540 mg) in MeOH
(15 mL) was treated overnight at room temperature with
hydrogen in the presence of 5% Pd/C catalyst (0.5 g).
(Caution! Extreme fire hazard.) After filtration, the res-
idue was chromatographed, to give 5 (250 mg, 75%), mp
146–147 °C (from EtOH; the compound sublimes when
Continued elution gave the title b-anomer 8 (0.44 g,
6.9%), which solidified on standing, mp 103–104 °C
(EtOH), [a]D ꢁ28 (c 0.5); lit.28 [a]D +107.7 (c 0.5) for
amorphous material reported as a byproduct of this
reaction, whose reported NMR data28 do not support
structure 8. 1H NMR (CDCl3): d 5.62 (dd, 1H, J1,2
1
2
heated over 70 °C/133 Pa), [a]D +157 (c 0.5). H NMR
1.0 Hz, J2,3 3.3 Hz, H-2), 4.76, 4.49 (2d, 1H each, J
(CDCl3): d 4.84 (d, 1H, J1,2 3.6 Hz, H-1), 3.58 (t, 1H,
J 9.5 Hz, H-3), 3.56–3.51 (m, 1H, H-5), 3.49 (s, 3H,
OCH3-2), 3.40 (s, 3H, OCH3-1), 3.17 (dd, 1H, J2,3
9.4 Hz, H-2), 2.43 (t, 1H, J 9.6 Hz, H-4), 1.26 (d, 3H,
J5,6 6.3 Hz, H-6); 13C NMR (CDCl3): d 96.86 (C-1),
81.68 (C-2), 71.97 (C-3), 67.93 (C-5), 58.77 (C-4),
57.87 (OCH3-2), 54.72 (OCH3-1), 17.70 (C-6); ESIMS:
m/z 214 ([M+Na]+). Anal. Calcd for C8H7NO4: C,
50.25; H, 8.96; N, 7.32. Found: C, 50.26; H, 8.97; N,
7.24.
11.1 Hz, CH2Ph), 4.61 (d, 1H, H-1), 3.52 (dd, 1H, J3,4
9.8 Hz, H-3), 3.41 (t, 1H, H-4), 3.26–3.17 (m, 1H, H-
5), 2.77–2.66 (m, 2H, CH2CH3), 2.17 (s, 3H, COCH3),
1.39 (d, 3H, J5,6 6.1 Hz, H-6), 1.28 (t, 3H, J 7.4 Hz,
CH2CH3); 13C NMR (CDCl3):
d 81.80 (JC-1,H-1
151.0 Hz, C-1), 79.24 (C-3), 75.26 (C-5), 71.44 (CH2Ph),
68.42 (C-2), 63.55 (C-4), 25.38 (CH2CH3), 20.69
(COCH3), 18.67 (C-6), 14.81 (CH2CH3). FABMS: m/z
366 ([M+H]+), 388 ([M+Na]+). Anal. Calcd for
C17H23N3O4S: C, 55.87; H, 6.34; N, 11.50; S, 8.77.
Found: C, 55.69; H, 6.22; N, 11.42; S, 8.67.
An intermediate, mixed fraction (0.52 g) was also
obtained.
3.5. Methyl 4,6-dideoxy-4-(3-hydroxy-3-methylbutanam-
ido)-2-O-methyl-a-D-glucopyranoside (6)
The structure of the compound previously described28
as the b-anomer 8 is unknown.
To a stirred solution of amine 5 (200 mg, 1.04
mmol) and 3-hydroxy-3-methylbutyric acid (184 mg,
1.56 mmol) in CH2Cl2 (2 mL) was slowly added HATU
(530 mg, 1.56 mmol) followed by N,N-diisopropylethyl-
amine (0.2 mL, 1.56 mmol), and the stirring was contin-
ued at room temperature. After 0.5 h, TLC (10:1
CH2Cl2–MeOH) showed that the reaction was com-
plete. The mixture was concentrated, and the residue
was chromatographed to afford the coupling product 6
(236 mg, 79%), mp 128–129 °C (from EtOAc),
[a]D +134 (c 0.5). 1H NMR (CDCl3): d 6.05 (d, 1H,
J4,NH 8.0 Hz, NH), 4.86 (d, 1H, J1,2 3.5 Hz, H-1),
3.82–3.76 (m, 2H, H-3,4), 3.71–3.63 (m, 2H, H-5, OH-
30), 3.52 (s, 3H, OCH3-2), 3.42 (s, 3H, OCH3-1), 3.25
(dd, 1H, J2,3 9.1 Hz, H-2), 3.00 (br s, 1H, OH-3), 2.41,
3.7. Ethyl 4-azido-3-O-benzyl-4,6-dideoxy-1-thio-b-D-
mannopyranoside (9)
A solution of thioglycoside 8 (1.6 g, 4.38 mmol) in
MeOH (40 mL) was made strongly alkaline by addition
of 0.1 M NaOMe, and stirred overnight at room temper-
ature. TLC (3:1 hexane–EtOAc) showed the reaction
was complete, and dry ice was added to neutralize the
base. The mixture was concentrated, and chromatogra-
phy gave the deacetylated product 9 (1.35 g, 94%), mp
1
90–92 °C; [a]D ꢁ235 (c 0.8, CHCl3). H NMR (CDCl3):
d 4.73, 4.68 (2d, 2H, 2J 11.7 Hz, CH2Ph), 4.52 (br d, 1H,
J1,2 ꢀ 0.9 Hz, H-1), 4.09 (br d, 1H, H-2), 3.48–3.44 (m,
2H, H-3,4), 3.20–3.16 (m, 1H, H-5), 2.74–2.67 (m, 2H,
CH2CH3), 2.43 (s, 1H, OH), 1.37 (d, 3H, J5,6 6.3 Hz,
H-6), 1.29 (t, 3H, J 7.5 Hz, CH2CH3); 13C NMR
(CDCl3): d 82.97 (C-1), 80.83 (C-3), 75.07 (C-5), 71.48
(CH2Ph), 68.40 (C-2), 63.53 (C-4), 25.30 (CH2CH3),
18.58 (C-6), 14.88 (CH2CH3); CIMS: m/z 346
([M+Na]+). Anal. Calcd for C15H21N3O3S: C, 55.71;
H, 6.54; N, 12.99. Found: C, 55.77; H, 6.57; N, 13.11.
2
2.37 (2d, 2H, J 14.4 Hz, H-20), 1.31, 1.30 (2s, 3H each,
2CH3 ), 1.25 (d, 3H, J5,6 6.3 Hz, H-6); 13C NMR
0
(CDCl3): d 172.93 (CO), 96.89 (C-1), 82.02 (C-2),
71.34 (C-3), 69.80 (C-30), 66.37 (C-5), 58.50 (OCH3-2),
56.67 (C-4), 55.28 (OCH3-1), 48.68 (C-20), 29.77, 29.16
+
(2CH3 ), 17.93 (C-6); ESIMS: m/z 292 ([M+H] ). Anal.
0
Calcd for C13H25NO6: C, 53.59; H, 8.65; N, 4.81.
Found: C, 53.30; H, 8.56; N, 4.74.
3.6. Ethyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-1-
thio-b-D-mannopyranoside (8)
3.8. Ethyl 4-azido-3-O-benzyl-4,6-dideoxy-2-O-methyl-1-
thio-b-D-glucopyranoside (12)
This compound was formed in a small amount from 1,2-
di-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-1-thio-a-D-
To a stirred solution of the 2-hydroxy compound 9
(1.3 g, 4.02 mmol) in 9:1 CH2Cl2–pyridine (40 mL) was

R. Saksena et al. / Carbohydrate Research 340 (2005) 1591–1600
1597
added triflic anhydride (0.87 mL, 5.2 mmol). The
mixture was stirred at room temperature for 0.5 h, when
TLC (4:1 hexane–acetone) showed that the reaction was
complete. The mixture was partitioned between CH2Cl2
and aq NaHCO3, the organic layer was concen-
trated, and chromatography of the residue gave ethyl
4-azido-3-O-benzyl-4,6-dideoxy-1-thio-2-O-triflyl-b-D-
[M+Li]+. Anal. Calcd for C16H23N3O3S: C, 56.95; H,
6.87; N, 12.45. Found: C, 57.25; H, 7.00; N, 12.30.
3.9. Ethyl 3-O-benzyl-4,6-dideoxy-4-(3-hydroxy-3-meth-
ylbutanamido)-2-O-methyl-1-thio-b-D-glucopyranoside
(14)
1
mannopyranoside (10, 1.57 g, 86%). H NMR (CDCl3):
At room temperature, H2S was passed, for 30 min,
through a stirred solution of azido sugar 12 (230 mg,
0.68 mmol) in a 2:1 mixture H2O–pyridine (12 mL).
The solution was stirred at 40 °C for 3 h, when TLC
(95:5 CH2Cl2–MeOH) showed the reaction to be com-
plete. The mixture was concentrated, and the residue
was chromatographed to give ethyl 4-amino-3-O-benz-
yl-4,6-dideoxy-2-O-methyl-1-thio-b-D-glucopyranoside
d 5.22 (br d, 1H, J2,3 ꢀ 2.8 Hz, H-2), 4.90, 4.58 (2d, 2H,
2J 11.7 Hz, CH2Ph), 4.62 (s, 1H, H-1), 3.51 (dd, 1H, J3,4
9.8 Hz, H-3), 3.40 (t, 1H, J 9.4 Hz, H-4), 3.28–3.18 (m,
1H, H-5), 2.78–2.70 (m, 2H, CH2CH3), 1.38 (d, 3H,
J5,6 6.0 Hz, H-6), 1.29 (t, 3H, J 7.5 Hz, CH2CH3); 13C
NMR (CDCl3): d 118.22 (q, CF3SO2), 83.39 (C-2),
80.65 (C-1), 78.32 (C-3), 75.87 (C-5), 72.64 (CH2Ph),
63.36 (C-4), 25.84 (CH2CH3), 18.56 (C-6), 14.74
(CH2CH3); CIMS: m/z 456 ([M+H]+).
1
(13, 205 mg, 95%). H NMR (CDCl3): d 4.98, 4.69 (2d,
2H, J 11.4 Hz, CH2Ph), 4.38 (d, 1H, J1,2 9.6 Hz, H-1),
2
NaNO2 (455 mg, 6.6 mmol) was added to a solution
of 10 (1 g, 2.2 mmol) in DMF (10 mL), and the
mixture was stirred for 1 h at 50 °C. TLC (5:1 tolu-
ene–EtOAc) showed that the starting material was
consumed and that several products were formed. After
concentration, chromatography gave the major product,
ethyl 4-azido-3-O-benzyl-4,6-dideoxy-1-thio-b-D-gluco-
pyranoside (11, 350 mg, 50%) as an oil. 1H NMR
(CDCl3): d 4.96, 4.86 (2d, 2H, 2J 10.9 Hz, CH2Ph),
4.26 (d, 1H, J1,2 9.8 Hz, H-1), 3.54 (ddd, 1H, J2,3 8.7,
J2,OH 1.6 Hz, H-2), 3.43 (t, 1H, J 9.5 Hz, H-3), 3.29–
3.24 (m, 1H, H-5), 3.16 (t, 1H, H-4), 2.75–2.68 (m,
2H, CH2CH3), 2.42 (d, 1H, OH), 1.35 (d, 3H, J5,6
6.1 Hz, H-6), 1.31 (t, 3H, J 7.5 Hz, CH2CH3); 13C
NMR (CDCl3): d 86.03 (C-1), 83.84 (C-3), 75.10 (C-2),
75.03 (CH2Ph), 73.46 (C-5), 67.26 (C-4), 24.36
(CH2CH3), 18.76 (C-6), 15.74 (CH2CH3); CIMS: m/z
346 ([M+Na]+).
3.63 (s, 3H, OCH3), 3.25–3.09 (m, 3H, H-3,5,2, in that
order), 2.83–2.67 (m, 2H, CH2CH3), 2.46 (t, 1H, J
9.3 Hz, H-4), 1.31 (t, 3H, J 7.4 Hz, CH2CH3) 1.26 (d,
3H, J5,6 6.1 Hz, H-6); 13C NMR (CDCl3): d 85.89
(C-2), 84.71 (C-1), 84.22 (C-3), 76.83 (C-5), 75.07
(CH2Ph), 60.60 (OCH3), 57.95 (C-4), 24.74 (CH2CH3),
18.11 (C-6), 14.85 (CH2CH3); CIMS: m/z 312 ([M+1]+).
To a solution of amine 13 (180 mg, 0.18 mmol) in
CH2Cl2 (2 mL) was slowly added HATU (102.6 mg,
0.27 mmol) followed by N,N-diisopropylethylamine
(0.054 mL, 0.27 mmol), and the mixture was stirred at
room temperature for 1 h. TLC (25:1 CH2Cl2–MeOH)
showed that the reaction was complete. The mixture
was concentrated, and the residue was chromatographed
to afford coupling product 14 in virtually theoretical
yield, mp 112–114 °C (from hexane containing a few
1
drops of i-Pr2O), [a]D ꢁ80.3 (c 0.8). H NMR (CDCl3):
2
d 5.53 (d, 1H, JNH,4 8.5 Hz, NH), 4.89, 4.65 (2d, 2H, J
MeI (2.5 mL, 40 mmol), followed by Ag2O (1 g,
15 mmol) and a catalytic amount of Me2S, was added
to a solution of alcohol 11 (330 mg, 1.02 mmol) in
THF (2.5 mL, 40 mmol), and the mixture was stirred
overnight, protected from light, at room temperature.
TLC (6:1 hexane–EtOAc) showed that the reaction
was complete, and that one product was formed. The
mixture was filtered through a Celite pad, the filtrate
was concentrated, and the residue was chromato-
graphed, to give pure 12 (265 mg, 87%) as a crystalline
solid, mp 36–38 °C, which could not be recrystallized
11.7 Hz, CH2Ph), 4.36 (d, 1H, J1,2 9.8 Hz, H-1), 3.65–
3.58 (m, 4H, H-4, incl. s, 3.64, OCH3), 3.54–3.43 (m,
2H, H-3,5), 3.17 (dd, 1H, J2,3 H-2), 2.80–2.68 (m, 2H,
2
CH2CH3), 2.20, 2.10 (2d, 2H, J 15.1 Hz, H-20), 1.31
(t, 3H, J 7.2 Hz, CH2CH3), 1.24, 1.23 (2s, 3H each,
2CH3 ), 1.21 (d, 3H, J5,6 6.1 Hz, H-6); 13C NMR
0
(CDCl3): d 84.52 (C-1), 84.06 (C-2), 81.99 (C-3), 74.99
(C-5), 74.20 (CH2Ph), 0 69.52 (C-30), 60.78 (OCH3),
0
56.09 (C-4), 47.67 (C-2 ), 29.31, 29.22 (2CH3 ), 24.67
(CH2CH3), 18.25 (C-6), 14.85 (CH2CH3); CIMS: m/z
412 ([M+1]+). Anal. Calcd for C21H33NO5S: C, 61.29;
H, 8.08; N, 3.40. Found: C, 61.03; H, 8.00; N, 3.34.
1
from common organic solvents, [a]D +14.4 (c 0.5). H
NMR (CDCl3): d 4.90, 4.82 (2d, 2H, 2J 10.7 Hz,
CH2Ph), 4.31 (d, 1H, J1,2 9.9 Hz, H-1), 3.62 (s, 3H,
OCH3), 3.42 (t, 1H, J 8.8 Hz, H-3), 3.25–3.08 (m, 3H,
H-5,4,2, in that order), 2.81–2.64 (m, 2H, CH2CH3),
1.33 (d, 3H, J5,6 5.5 Hz, H-6), 1.30 (t, 3H, J 7.4 Hz,
CH2CH3); 13C NMR (CDCl3): d 84.64 (C-1), 84.45 (C-
3), 83.65 (C-2), 75.48 (CH2Ph), 74.55 (C-5), 67.50 (C-
4), 60.94 (CH3), 24.82 (CH2CH3), 18.57 (C-6), 14.84
(CH2CH3); FABMS (after addition of LiCl): m/z 344
3.10. 3-O-Benzyl-4,6-dideoxy-4-(3-hydroxy-3-methyl-
butanamido)-2-O-methyl-a,b-D-glucopyranose
(3-O-benzylanthrose, 15)
NIS (48 mg, 0.45 mmol), followed by a drop of water,
was added at 0 °C, to a stirred solution of thioglycoside
14 (90 mg, 0.21 mmol) in acetone (2 mL). After 1 h,
TLC (EtOAc) showed that the reaction was complete

1598
R. Saksena et al. / Carbohydrate Research 340 (2005) 1591–1600
0
and that one product, showing slower chromatographic
mobility than 14, was formed. After concentration,
chromatography of the residue gave 15 (83 mg, 91%),
which crystallized as a mixture of anomers (a:b ꢀ 3:1,
NMR), mp 168–170 °C (from i-Pr2O containing a few
drops of EtOAc), [a]D +5.2 (c 0.4). 1H NMR (a-anomer,
CDCl3): d 5.89 (d, JNH,4 9.0 Hz, NH), 5.33 (d, J1,2
3.2 Hz, H-1), 4.83, 4.62 (2d, partially overlapped, 2J
11.7 Hz, CH2Ph), 3.96–3.91 (m, H-5), 3.81 (q, J
9.4 Hz, H-4), 3.71 (t, partially overlapped, H-3), 3.51
(s, partially overlapped, OCH3), 3.39 (dd, J2,3 8.6 Hz,
2CH3 ), 1.17 (d, J5,6 6.3 Hz, H-6); 13C NMR (a-anomer,
D2O): d 176.76 (CO), 92.05 (C-1), 83.86 (C-2), 73.02 (C-
30), 72.41 (C-3), 69.00 (C-5), 60.37 (OCH3), 59.46 (C-4),
1
0
0
51.60 (C-2 ), 31.01, 30.86 (2CH3 ), 19.80 (C-6); H NMR
(b-anomer, D2O): d 4.63 (d, J1,2 7.9 Hz, H-1), 3.63 (par-
tially overlapped, H-4), 3.61 (s, CH3O), 3.58–3.51 (m,
H-5, incl. 3.53, t, J 9.3 Hz, H-3), 3.04 (dd, 1H, J2,3
9.3 Hz, H-2), 2.48–2.44 (m, H-20), 1.31, 1.30 (2s,
2CH3 ), 1.20 (d, 3H, J5,6 6.1 Hz, H-6); 13C NMR (b-ano-
0
mer, D2O): d 176.76 (CO), 98.29 (C-1), 87.10 (C-2),
75.90 (C-3), 73.64 (C-5), 73.02 (C-30), 60.37 (OCH3),
2
0
H-2), 2.15 (dd, J 14.8 Hz, H-20a,b), 1.23, 1.20 (2s,
59.29 (C-4), 51.64 (C-2 ), 31.04, 30.84 (2CH3 ), 19.80
(C-6); ESIMS: m/z 330 ([M+Na]+).
0
2CH3 ), 1.22 (d, J5,6 6.7 Hz, H-6); 13C NMR (a-anomer,
0
CDCl3): d 172.40 (CO), 90.23 (JC,H 168 Hz, C-1), 82.46
(C-2), 77.17 (C-3), 73.86 (CH2Ph), 69.55 (C-30), 67.24
(C-5), 58.62 (OCH3), 54.83 (C-4), 47.73 (C-20), 29.30,
3.12. Methyl 4-azido-3-O-benzyl-4,6-dideoxy-b-D-gluco-
pyranoside (19)
29.27 (2CH3 ), 18.06 (C-6); 1H NMR (b-anomer,
0
CDCl3): d 5.89 (d, JNH,4 9.0 Hz, NH), 4.85, 4.63 (2d,
partially overlapped, J 11.5 Hz, CH2Ph), 4.61 (d, par-
Triflic anhydride (0.4 mL, 2.5 mmol) was added at 0 °C
to a stirred solution of methyl 4-azido-3-O-benzyl-4,6-
dideoxy-b-D-mannopyranoside (17,29 500 mg, 1.7 mmol)
in CH2Cl2 (15 mL) containing pyridine (0.85 mL,
10 mmol). The cooling was removed and, when TLC
(4:1 hexane–EtOAc) showed that the reaction was com-
plete (ꢀ1 h) the mixture was concentrated. Chromato-
graphy gave pure methyl 4-azido-3-O-benzyl-4,6-dide-
oxy-2-O-trifluoromethanesulfonyl-b-D-glucopyranoside
2
tially overlapped, H-1), ꢀ3.71 (m, partially overlapped,
H-4), 3.62 (s, 3H, OCH3), 3.56–3.46 (m, partially over-
lapped, H-3,5), 3.15 (t, J 8.2 Hz, H-2), 2.15 (dd, 2J
14.8 Hz, H-20a,b), 1.25 (d, J5,6 6.3 Hz, H-6), 1.23, 1.20
(2s, 2CH3 ); 1H NMR (b-anomer, C6D6): d 5.02 (d,
0
2
JNH,4 8.9 Hz, NH), 4.79, 4.53 (2d, J 11.7 Hz, CH2Ph),
4.38 (d, J1,2 8.5 Hz, H-1), ꢀ3.76–3.71 (m, partially over-
lapped, H-5), 3.56 (q, J 9.8 Hz, H-4), 3.49 (s, OCH3),
3.31 (t, H-3), 2.99 (dd, J2,3 8.3 Hz, H-2), 1.98, 1.87
1
(18, 570 mg, ꢀ100%). H NMR (CDCl3): d 5.12 (br d,
2
1H, H-2), 4.85, 4.57 (2d, 2H, J 11.3 Hz, CH2Ph), 4.36
2
0
0
(2d, J 14.8 Hz, H-2 a,b), 1.13, 1.11 (2s, 2CH3 ), 1.07
(d, 1H, J1,2 ꢀ 0.6 Hz, H-1), 3.52 (s, 3H, OCH3), 3.48
(dd, 1H, J2,3 2.8, J3,4 9.9 Hz, H-3), 3.37 (t, 1H, J
9.9 Hz, H-4), 3.23–3.13 (m, 1H, H-5), 1.39 (d, 3H, J5,6
6.0 Hz, H-6); 13C NMR (CDCl3): d 97.87 (C-1), 80.76
(C-2), 77.00 (C-3), 72.18 (CH2Ph), 71.30 (C-5), 63.39
(C-4), 56.87 (OCH3), 18.16 (C-6); ESIMS: m/z 448
([M+Na]+).
(d, J5,6 6.1 Hz, H-6); 13C NMR (b-anomer, CDCl3): d
172.53 (CO), 97.07 (JC,H 161 Hz, C-1), 85.45 (C-2),
80.14 (C-3), 73.77 (CH2Ph), 70.97 (C-5), 69.53 (C-30),
60.41 (OCH3), 55.70 (C-4), 47.83 (C-20), 29.33, 29.30
+
(2CH3 ), 18.17 (C-6); CIMS: m/z 390 ([M+Na] ). Anal.
0
Calcd for C19H29NO6: C, 62.11; H, 7.96; N, 3.81.
Found: C, 61.85; H, 7.87; N, 3.78.
NaNO2 (267 mg, 3.87 mmol) was added to a solution
of the above triflate (550 mg, 1.29 mmol) in DMF
(5 mL), and the solution was stirred at room tempera-
ture for 24 h. TLC (4:1 hexane–EtOAc) showed that
the reaction was complete and that three products,
two showing faster and one slower mobility than the
starting material, were formed. The mixture containing
some precipitate was concentrated, and the residue
was chromatographed to give the desired, title com-
pound 19 (276 mg, 73%), mp 68.5–69 °C (from i-Pr2O–
hexane), [a]D ꢁ9 (c 0.4). 1H NMR (CDCl3): d 4.95,
4.85 (2d, 2H, 2J 11 Hz, CH2Ph), 4.11 (d, 1H, J1,2
7.7 Hz, H-1), 3.54–3.51 (m, 4H, H-2, incl. 3.54, s,
OCH3), 3.44 (t, 1H, J 9.1 Hz, H-3), 3.27–3.23 (m, 1H,
H-5), 3.15 (t, 1H, J 9.5 Hz, H-4), 2.72 (d, 1H, J2,OH
2.6 Hz, OH), 1.34 (d, 3H, J5,6 6.1 Hz, H-6); 13C NMR
(CDCl3): d 103.38 (C-1), 82.35 (C-3), 74.95 (CH2Ph),
74.79 (C-2), 70.66 (C-5), 67.25 (C-4), 57.08 (OCH3),
18.35 (C-6); ESIMS: m/z 316 ([M+Na]+). Anal. Calcd
for C14H19N3O4: C, 57.33; H, 6.53; N, 14.33. Found:
C, 57.33; H, 6.58; N, 14.33.
3.11. 4,6-Dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-
O-methyl-a,b-D-glucopyranose (anthrose, 16)
A
solution of the above anthroside 15 (40 mg,
0.10 mmol) in 1:1 H2O–MeOH (5 mL) was stirred under
hydrogen for 48 h at 50 °C in the presence of 5% Pd/C
catalyst (40 mg). TLC (4:1 CH2Cl2–MeOH) showed that
the reaction was complete and that one product was
formed. After filtration and concentration of the filtrate,
the residue was chromatographed, mainly to remove
residual catalyst. Fractions containing anthrose were
concentrated, a solution of the residue in H2O was fil-
tered through a syringe filter (0.02 lm porosity) and
freeze dried, to give anthrose (16 anomeric mixture,
a:b ꢀ 4.5:5.5, NMR) as a white solid in virtually theo-
1
retical yield. H NMR (a-anomer, D2O): d 5.44 (d, J1,2
3.6 Hz, H-1), 3.99–3.94 (m, H-5), 3.73 (t, J 9.7 Hz, H-
3), 3.63 (m, partially overlapped, H-4), 3.48 (s, OCH3),
3.32 (dd, H-2), 2.48–2.44 (m, H-20), 1.31, 1.30 (2s,

R. Saksena et al. / Carbohydrate Research 340 (2005) 1591–1600
1599
3.13. Methyl 4-azido-3-O-benzyl-4,6-dideoxy-2-O-
methyl-b-D-glucopyranoside (20)
150 °C (from EtOAc), [a]D ꢁ33.5 (c, 0.5). 1H NMR
(CDCl3): d 6.60 (d, 1H, J4,NH 8.8 Hz, NH), 4.19 (d,
1H, J1,2 7.8 Hz, H-1), 4.12, 3.78 (2br s, 2H, 2OH),
3.66 (q, 1H, J 9.7 Hz, H-4), 3.60 (s, 3H, OCH3-2), 3.53
(s, partially overlapped, OCH3-1), 3.52 (t, partially over-
lapped, J 9.7 Hz, H-3), 3.49–3.45 (m, 1H, H-5), 3.00 (dd,
A mixture of alcohol 19 (740 mg), MeI (4 mL), Ag2O
(1.2 g), and a catalytic amount of Me2S in 1,2-dimeth-
oxyethane (10 mL) was stirred at room temperature
for 8 h, protected from light. A fresh portion of MeI
(4 mL) and Ag2O (1.2 g) were added, and stirring was
continued overnight. TLC (4:1 hexane–EtOAc) showed
that the reaction was complete and that a faster moving
product was formed. After filtration and concentration
of the filtrate, chromatography gave 20 (690 mg, 89%),
2
1H, J2,3 8.9 Hz, H-2), 2.41, 2.38 (2d, 2H, J 14.3 Hz, H-
0
0
2 a,b), 1.31, 1.30 (2s, 6H, 2CH3 ), 1.28 (d, 3H, J5,6
6.2 Hz, H-6); 13C NMR (CDCl3): d 173.02 (CO),
103.97 (C-1), 83.87 (C-2), 74.04 (C-3), 70.67 (C-5),
69.85 (C-30), 60.58 (OCH3-2), 57.02 (C-4), 56.77
0
0
(OCH3-1), 48.62 (C-2 ), 29.70, 29.21 (2CH3 ), 17.98 (C-
1
mp 67–68 °C (from EtOH), [a]D +48 (c 0.5). H NMR
6); ESIMS: m/z 314 ([M+Na]+). Anal. Calcd for
C13H25NO6: C, 53.59; H, 8.65; N, 4.81. Found: C,
53.34; H, 8.58; N, 4.82.
(CDCl3): d 4.89, 4.79 (2d, 2H, 2J 10.8 Hz, CH2Ph),
4.14 (d, 1H, J1,2 7.7 Hz, H-1), 3.58 (s, 3H, OCH3-2),
3.52 (s, 3H, OCH3-1), 3.39 (t, 1H, J 9.2 Hz, H-3),
3.21–3.16 (m, 1H, H-5), 3.12–3.09 (m, 2H, H-2,4), 1.33
(d, 3H, J5,6 5.9 Hz, H-6). 13C NMR (CDCl3): d 104.21
(C-1), 84.17 (C-2), 82.75 (C-3), 75.38 (CH2Ph), 70.32
(C-5), 67.45 (C-4), 60.52 (OCH3-2), 56.93 (OCH3-1),
18.37 (C-6); ESIMS: m/z 330 ([M+Na]+). Anal. Calcd
for C15H21N3O4: C, 58.62; H, 6.89; N, 13.67. Found:
C, 58.73; H, 6.89; N, 13.72.
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