Synthesis of methyl 2-acetamido-2,6-dideoxy-α- and β-d-xylo-hexopyranosid-4-ulose, a keto sugar which misled the analytical chemists

Article abstract of DOI:10.1016/j.carres.2008.02.001

To understand the contradictory results on the structure of the lipopolysaccharide isolated from a Yersinia enterocolitica O:3, both anomers of methyl 2-acetamido-2,6-dideoxy-d-xylo-hexopyranosid-4-ulose were prepared. The key steps of the synthetic pathw

Full text of DOI:10.1016/j.carres.2008.02.001

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1004–1011
Synthesis of methyl 2-acetamido-2,6-dideoxy-a- and
b-D-xylo-hexopyranosid-4-ulose, a keto sugar which
misled the analytical chemists
Sabine Borowski,a Dirk Michalik,a,b Helmut Reinke,a Christian Vogel,a,*
Anna Hanuszkiewicz,c Katarzyna A. Dudac,d and Otto Holstc
aUniversity of Rostock, Institute of Chemistry, Albert-Einstein-Strasse 3a, D-18059 Rostock, Germany
bLeibniz Institute for Catalysis at the University of Rostock, Albert-Einstein-Strasse 29a, D-18059 Rostock, Germany
cResearch Centre Borstel, Leibniz-Centre for Medicine and Biosciences, Division of Structural Biochemistry,
Parkallee 1-40, D-23845 Borstel, Germany
dDepartment of Microbiology, University of Silesia, Jagiellon´ska 28, PL-40-032 Katowice, Poland
Received 6 December 2007; received in revised form 29 January 2008; accepted 1 February 2008
Available online 9 February 2008
Abstract—To understand the contradictory results on the structure of the lipopolysaccharide isolated from a Yersinia enterocolitica
O:3, both anomers of methyl 2-acetamido-2,6-dideoxy-D-xylo-hexopyranosid-4-ulose were prepared. The key steps of the synthetic
pathway were the selective acetylation of the methyl 2-acetamido-2,6-dideoxy-a,b-D-glucopyranosides, the oxidation of the 4-posi-
tion to form the keto-sugars, and deacetylation to provide the target compound. Surprisingly, the last step was accompanied by a
disproportionation to give methyl 2-acetamido-2,6-dideoxy-a- and b-D-glucopyranosides and N-(5-hydroxy-6-methyl-4-oxo-4H-
pyran-3-yl)acetamide as side-products.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: D-Glucosamine; Reduction; Oxidation; Anomerization; Keto-sugar; Selective acetylation
1. Introduction
reduction during various analytical protocols. The ratio
of the 4-epimeric sugars depended on the reducing
agents and the experimental conditions.3 To study this
phenomenon by using a model compound and, addi-
tionally, to obtain information on the influence of the
structure at the anomeric centre on the ratio of the 4-epi-
mers, both anomers of methyl 2-acetamido-2,6-dideoxy-
D-xylo-hexopyranosid-4-ulose (9a,b) were synthesized.
The lipopolysaccharides (LPS) of the wild type strain
Yersinia enterocolitica (Ye) 75S consist of an O-specific
polysaccharide, an outer and an inner core oligosaccha-
ride, and lipid A, whereas the LPS of the rough (R)
mutant YeO3-R1 bears only the core oligosaccharide
and lipid A.1 In contrast to earlier structural analyses
in which an N-acetyl-D-fucosamine (FucpNAc)2 or an
N-acetyl-D-quinovosamine (QuipNAc) unit was believed
to be the link between outer and inner core oligosaccha-
rides, improved NMR spectroscopic and mass spectro-
metric investigations [K. A. Duda et al. unpublished
results] indicated that a 4-keto sugar takes on this role
and FucpNAc and QuipNAc are artefacts formed by
2. Results and discussion
The a- and b-isomers of 4-ulose 9 were synthesized via a
synthetic approach shown in Scheme 1 using methyl 2-
acetamido-2-deoxy-a/b-D-glucopyranoside (1a,b) as
starting material. Applying conventional Fischer glycos-
ylation reaction conditions, N-acetyl-D-glucosamine in
methanol was converted in the presence of IR120 (H+)
*
Corresponding author. Tel.: +49 381 498 6430; fax: +49 381 498
6412; e-mail: christian.vogel@uni-rostock.de
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.02.001

S. Borowski et al. / Carbohydrate Research 343 (2008) 1004–1011
1005
R1
OH
O
O
O
OR2
OR
OH
OCH3
OCH3
OCH3
R2O
RO
HO
AcHN
1α,β
AcHN
AcHN
2α,β: R1 = OTs, R2 = H
3α,β: R1 = OTs, R2 = Ac
4α,β: R1 = I, R2 = Ac
5α,β: R = Ac
6α,β: R = H
R1
O
R1
O
OR3
O
OAc
R2
HO
R2
AcHN
AcHN
8α: R1 = H, R2 = OCH3, R3 = Ac
8β: R1 = OCH3, R2 = H, R3 = Ac
9α: R1 = H, R2 = OCH3, R3 = H
9β: R1 = OCH3, R2 = H, R3 = H
7α: R1 = H, R2 = OCH3
7β: R1 = OCH3, R2 = H
Scheme 1. Synthetic pathway for the preparation of both anomers of methyl 2-acetamido-2,6-dideoxy-D-xylo-hexopyranosid-4-ulose (9a and 9b)
starting from N-acetyl-D-glucosamine.
Amberlite resin into the corresponding methyl glycoside.
In contrast to the work of Bornaghi and Poulsen,4 we
did not use anhydrous methanol and after 8 h at 70 °C
we reproducibly observed a/b ratios from 2:1 through
5:1. The total yield of isolated methyl glycosides 1a,b
was 74%. Although it was possible by flash-chromato-
graphy to separate the a,b-isomers formed during each
synthetic step, further reactions were carried out with
the anomeric mixtures until the oxidation step, since
(i) both isomers were needed for reduction experiments
and (ii) each synthetic step with a pure b-isomer was
always accompanied by anomerization. Consequently,
it made sense under these conditions to work on a
multi-step pathway with anomeric mixtures as long as
possible.
The synthesis of the 6-deoxy-sugars 6a,b was carried
out by using a procedure developed by Kajihara et al.5
for the b-anomer (Scheme 1). The sequence comprised
selective tosylation (2a,b), acetylation (3a,b), exchange
of tosyl function by iodine (4a,b), reduction (5a,b) and
deacetylation (6a,b). It was of advantage to conduct
the synthesis of 3a,b from 1a,b as an one-pot reaction.
Otherwise, the total yield was significantly lower. As
mentioned before, each anomer was separated for ana-
lytical characterization and since the Japanese authors
had not published the 13C NMR data for the b-anomers,
the complete set of analytical data for both a- and b-iso-
mers (2–6) are given in the experimental part.
7a and 7b in nearly 40% yields. As side-products only
the completely acetylated compounds 5a and 5b were
isolated by flash chromatography, respectively, which
could be used again in the previous reaction steps. The
1H NMR data proved the acetylation of the O-3 posi-
tion of 7a and 7b. The multiplets at d 3.51–3.34 (2H,
H-3, H-5) and d 3.26–3.11 (2H, H-3, H-5) of 6a and
6b, respectively, were splitted up in the spectra of 7a
and 7b. As expected, the acetyl function in the O-3 posi-
tion caused a considerable downfield shift of about
1.5 ppm of the H-3 signals of both anomers to d 4.99.
Furthermore, the O-3 position of the acetyl group was
also confirmed by the HMBC spectrum recorded for
7a. In this spectrum, a correlation between the proton
H-3 and the carbonyl carbon atom of the acetyl group
was observed involving interaction through three bonds.
Additionally, X-ray diffraction studies were per-
formed to establish the structures of 7a and 7b (Figs. 1
It had to be proved that an acetyl group could be
introduced regioselectively at O-3 position of compound
6. Dropwise addition of acetyl chloride diluted by tolu-
ene to a solution of both compounds 6a and 6b in pyr-
idine at ꢀ40 °C gave exclusively the desired derivatives
Figure 1. An ORTEP plot of 7a with 50% probability for the thermic
ellipsoids.

1006
S. Borowski et al. / Carbohydrate Research 343 (2008) 1004–1011
Figure 3. An ORTEP plot of 10 with 50% probability for the thermic
ellipsoids. In compound 10 the molecules form hydrogen bonded
Figure 2. An ORTEP plot of 7b with 50% probability for the thermic
dimers around a centre of inversion.
ellipsoids.
and 2). Both structures showed a number of hydrogen
bond interactions. In the tetragonal crystal of 7a (Hall
space group: P4abw 2nw), there are two donating groups
and two acceptor sites per molecule. The starting mole-
cule is linked to its translational equivalent molecule
parallel to a-axis via pairs of again translational equiva-
lent molecules with symmetry codes 0.5 ꢀ x, 0.5 + y,
0.25 ꢀ z and 0.5 ꢀ x, 1.5 + y, 0.25 ꢀ z, thus forming a
planar network of hydrogen bonded molecules perpen-
dicular to the c-axis. In the monoclinic 7b (Hall space
group: P2yb) there is also a planar network of hydrogen
bonded molecules perpendicular to the c-axis. However,
this plane consists of two symmetry related chains of
molecules (symmetry code: ꢀx, 0.5 + y, ꢀz). The mole-
cules in each chain are connected to each other via
hydrogen bonds between the OH group at C-4 and the
carbonyl oxygen of the N-acetyl group in the next mole-
cule. The symmetry related chains are then connected
via a link between the NH-group in one chain to the
oxygen at 4-position of the neighbouring chain.
The yields (5–10%) such as the reaction rate were pretty
low. Switching to pyridinium dichromate (PDC)8 8a and
8b were obtained in 75% and 50% yields, respectively,
but the reaction time of 3 weeks was still unacceptable.
A significant improvement of the oxidation with PDC
was achieved by the addition of molecular sieves9 and
a catalytic amount of glacial acetic acid10 to the reaction
mixture. Now, 4-uloses 8a and 8b were obtained after
12 h in 91% and 60% yield, respectively. Parallely, the
reaction conditions of Swern-oxidation11 were applied
to 7a and 7b. The observed yields for 8a (93%) and 8b
(57%) were comparable with the improved PDC oxida-
tion. However, Swern-oxidation necessitated purifica-
tion by column chromatography while PDC oxidation
provided analytically pure products. The structure of
the 4-uloses 8a and 8b was confirmed by the analytical
and by the NMR data. Thus, in the 13C NMR spectra
of the 4-uloses the oxidation of the 4-position of 7a
and 7b resulted in a characteristic downfield shifts d
74.2 and d 72.0 to d 197.9 and d 198.3, respectively.
Surprisingly, the final step of the synthesis of the
model compounds was quite cumbersome and acco-
mpanied by an unexpected side reaction. Deacetyla-
The crucial point of the synthetic route was the oxida-
tion step to gain 4-uloses 8a and 8b. The first experi-
ments using pyridinium chlorochromate (PCC)6 or the
conditions of Jones oxidation7 were not very auspicious.
´
tion under Zemplen conditions furnished the target
O
O
O
OAc
2
O
OCH3
OH
O
OCH3
OCH3
+
HO
O
NHAc
NHAc
NHAc
8α,β
6α,β
O
HO
O
NHAc
10
Scheme 2. Suggested mechanism of the side-reaction occurring during deacetylation of compounds 8a and 8b.

S. Borowski et al. / Carbohydrate Research 343 (2008) 1004–1011
1007
compounds 9a and 9b in yields of 72% and 40%, respec-
tively. The application of methanolic 0.28 M hydrochloric
acid12 gave only 44% and 12%, respectively.
hydrogen atoms were put into theoretical positions
and refined using the riding model. Crystallographic
data for the structural analysis has been deposited with
the Cambridge Crystallographic Data Centre, CCDC
No. 669295–CCDC 669297 for compounds 7a, 7b and
10. Copies of this information may be obtained free of
charge from: The Director, CCDC, 12 Union Road,
Cambridge, CB2 1EZ UK, Fax. (int code)
+44(1223)336-033 or via Email: deposit@ccdc.cam.
ac.uk or www:http://www.ccdc.cam.ac.uk. All washing
solutions were cooled to ꢁ5 °C. The NaHCO3 soln
was saturated. Reactions were monitored by thin-layer
chromatography (TLC, Silica Gel 60 F254, Merck
KGaA). The followings solvents systems (v/v) were
used: (A1) 4:1, (A2) 9:1 chloroform–ethanol; (B1) 3:1,
(B2) 6:1, (B3) 10:1 chloroform–methanol. The spots were
made visible by dipping the TLC plates into a methano-
lic 10% H2SO4 soln and charring with a heat gun for
3–5 min. Preparative flash chromatography was per-
formed by elution from columns of slurry-packed Silica
Gel 60 (Merck, 63–200 lm). All solvents and reagents
were purified and dried according to standard proce-
dures.13 After classical workup of the reaction mixtures,
the organic layer was dried over MgSO4, and then con-
centrated under reduced pressure (rotary evaporator).
Both reactions yielded an unknown side-product (10)
together with the reduced derivative 6a originating from
8a or the anomeric mixture 6a,b starting from 8b. The
structural determination of the side-product 10 was
finally achieved by X-ray diffraction studies (Fig. 3).
All other analytical data were in good agreement with
the proposed structure of 10. Thus, in the 1H,13C
HMBC spectrum recorded for 10 the following correla-
tions have been found: H-2 with C-3,4 and 6; methyl
group with C-5 and C-6; CH3CO with CH3CO; and
NH with C-2 and CH3CO. The formation of 10 together
with compounds 6a,b suggested that a disproportion-
ation took place during deacetylation either under base
or acid catalysis. The diketo structure shown in Scheme
2 was not stable and, consequently, by the elimination of
methanol the side-product 10 was formed.
An investigation of the reduction of the model com-
pounds 9a and 9b under conditions which are typical
for analytical procedures of bacterial polysaccharides
is presently underway.
3. Experimental
3.2. Selective tosylation of methyl 2-acetamido-2-deoxy-
a,b-D-glucopyranoside (1a,b)
3.1. General methods
Melting points were determined with a Boetius micro-
heating plate BHMK 05 (Rapido, Dresden) and were
not corrected. Optical rotation was measured for
solutions in a 2-cm cell with an automatic polarimeter
GYROMAT (Dr. Kernchen Co.). 1H NMR spectra
(250.13 MHz, 300.13 MHz, and 500.13 MHz) and
13C NMR spectra (62.89 MHz; 75.47 MHz; and
125.76 MHz) were recorded on Bruker instruments AC
250, ARX 300, and AVANCE 500, respectively, with
CDCl3, CD3OD or Me2SO-d6 as solvents. The calibra-
tion of spectra was carried out on solvent signals
A soln of p-tolysulfonyl chloride (930 mg, 4.90 mmol) in
dry pyridine (3 mL) was added dropwise to a soln of
anomeric mixture 1a,b (1.0 g, 4.25 mmol)4 in dry pyri-
dine (10 mL) at ꢀ10 °C. The mixture was stirred at that
temperature for 3 h, then at 5 °C for 12 h, and finally at
ambient temperature for 24 h. Ice-water (5 mL) was
added, and after 30 min the aqueous phase was
extracted with chloroform (4 ꢂ 70 mL). The combined
extracts were successively washed with ice-water
(3 ꢂ 30 mL), aq 1 M H2SO4 (30 mL), ice-water
(30 mL), aq NaHCO3 (30 mL), ice-water (30 mL), dried
and concentrated. Traces of pyridine were removed by
evaporation with repeated addition of toluene. The res-
idue was then subjected to column chromatography
(solvent A2) to provide 2a and 2b.
(CDCl3: d H 7.25, d 13C 77.0; CD3OD: d H 4.78, d
13C 49.0; Me2SO-d6: d 1H 2.50, d 13C 39.7). 1H and
13C NMR signals were assigned by DEPT and two-
dimensional 1H,1H COSY, and 1H,13C correlation spec-
tra (HMBC and HSQC). Mass spectra were recorded on
an AMD 402/3 spectrometer (AMD Intectra GmbH).
Elemental analysis was performed on a CHNS-Flash-
EA-1112 instrument (Thermoquest). For the X-ray
structural determination of compounds 7a, 7b and 10
an X8Apex system with CCD area detector was used
1
1
3.3. Methyl 2-acetamido-2-deoxy-6-O-p-tolysulfonyl-a-D-
glucopyranoside (2a)
Colourless crystals (744 mg, 45%); mp 69–72 °C; lit.14
24
23
70–73 °C; ½aꢃD +55.6 (c 1.0, CHCl3), lit.14 ½aꢃD +68.0
(c 2.0, CHCl3); Rf 0.40 (solvent A1); 1H NMR
(300.13 MHz, CDCl3): d 7.78 (m, 2H, o-C6H4), 7.32
˚
(k = 0.71073 A, graphite monochromator). The struc-
tures were solved by direct methods (Bruker-SHELXTL).
The refinement calculations were done by the full-matrix
least-squares method of Bruker SHELXTL, Vers.5.10,
Copyright 1997, Bruker Analytical X-ray Systems. All
non-hydrogen atoms were refined anisotropically. The
3
(m, 2H, m-C6H4), 6.22 (d, 1H, JNH,2 8.8 Hz, NH),
4.59 (d, 1H, J1,2 3.6 Hz, H-1), 4.34–4.21 (m, 2H,
H-6), 3.98 (ddd, 1H, J2,3 10.2 Hz, H-2), 3.77–3.66 (m,
1H, H-5), 3.61 (dd, 1H, J3,4 9.2 Hz, H-3), 3.43 (‘t’,
3
3
3

1008
S. Borowski et al. / Carbohydrate Research 343 (2008) 1004–1011
3
1H, J4,5 9.2 Hz, H-4), 3.31 (s, 3H, OCH3), 2.42 (s, 3H,
CH3C6H4), 2.00 (s, 3H, COCH3); 13C NMR
(2 ꢂ COCH3); EIMS (m/z, %) 473 (5) [M+H]+. Anal.
Calcd for C20H27NO10S (473.14): C, 50.73; H, 5.75;
N, 2.96; S, 6.77. Found: C, 50.54; H, 5.77; N, 2.77; S,
6.70.
(75.47 MHz, CDCl3):
d 172.08 (COCH3), 144.90
(p-C6H4), 132.82 (i-C6H4), 129.88 (m-C6H4), 127.98
(o-C6H4), 98.25 (C-1), 73.61 (C-3), 70.71 (C-4), 69.41
(C-5), 69.19 (C-6), 55.23 (OCH3), 53.45 (C-2), 23.21
(COCH3), 21.62 (CH3C6H4); CIMS (m/z, %) 390 (100)
[M+H]+. Anal. Calcd for C16H23NO8S (389.11): C,
49.35; H, 5.95; N, 3.60; S, 8.23. Found: C, 49.14; H,
5.97; N, 3.46; S, 8.18.
3.6. Methyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-6-O-p-
tolysulfonyl-b-D-glucopyranoside (3b)
Mp 179 °C (ethanol, dec), lit.5 mp 192 °C (dec; this melt-
ing point was not reproducible even after several crystal-
22
23
D
lisations); ½aꢃD +21.8 (c 1.0, CHCl3), lit.5 ½aꢃ +19.7 (c
3.4. Methyl 2-acetamido-2-deoxy-6-O-p-tolysulfonyl-b-D-
glucopyranoside (2b)
0.6, CH2Cl2); Rf 0.34 (ethyl acetate); 1H NMR
(250.13 MHz, CDCl3): d 7.77 (m, 2H, o-C6H4), 7.32
3
(m, 2H, m-C6H4), 5.52 (d, 1H, JNH,2 9.0 Hz, NH),
24
3
3
Colourless crystals (33 mg, 2%); mp 101–102 °C; ½aꢃD
5.22 (dd, 1H, J2,3 10.5 Hz, J3,4 9.3 Hz, H-3), 4.88 (‘t’,
1
3
3
ꢀ30.7 (c 1.0, CHCl3); Rf 0.27 (solvent A1); H NMR
1H, J4,5 10.0 Hz, H-4), 4.50 (d, 1H, J1,2 8.4 Hz, H-1),
4.14–4.02 (m, 2H, H-6), 3.85–3.70 (m, 2H, H-2, H-5),
3.41 (s, 3H, OCH3), 2.44 (s, 3H, CH3C6H4), 2.00, 1.99
(2s, 2 ꢂ 3H, 2 ꢂ COCH3), 1.92 (s, 3H, NHCOCH3);
13C NMR (75.47 MHz, CDCl3): d 170.86 (NHCOCH3),
170.22, 169.47 (2 ꢂ COCH3), 145.15, 132.41 (2 ꢂ
i-C6H4), 129.88, 128.05 (2 ꢂ o-, 2 ꢂ m-C6H4), 101.44
(C-1), 72.10 (C-3), 71.58 (C-5), 68.88 (C-4), 68.01
(C-6), 56.76 (OCH3), 54.41 (C-2), 23.32 (NHCOCH3),
21.65 (CH3C6H4), 20.62, 20.56 (2 ꢂ COCH3); CIMS
(m/z, %) 474 (76) [M+H]+. Anal. Calcd for C20H27-
NO10S (473.14): C, 50.73; H, 5.75; N, 2.96; S, 6.77.
Found: C, 51.04; H, 5.90; N, 2.85; S, 6.48.
For transformation of compounds 3a,b (10.0 g,
21.14 mmol) into methyl 3,4-di-O-acetyl-2-acetamido-
2,6-dideoxy-a,b-D-glucopyranosides (5a,b) via the corres-
ponding iodine compound 4a,b conditions of Kajihara
et al.5 were used again. Purification by column chroma-
tography (eluent ethyl acetate gradient 80?100% in
petrol ether) afforded 5a,b (5.33 g, 83%). Analytical
samples were obtained by column chromatography
using ethyl acetate as eluent.
3
(500.13 MHz, Me2SO-d6): d 7.67 (d, 1H, JNH,2 9.0 Hz,
NH), 7.79 (m, 2H, o-C6H4), 7.48 (m, 2H, m-C6H4),
4.25 (dd, 1H, J6a,6b 10.7 Hz, J5,6a 1.8 Hz, H-6a), 4.17
(d, 1H, J1,2 8.3 Hz, H-1), 4.07 (dd, 1H, J5,6b 6.6 Hz,
2
3
3
3
H-6b), 3.40–3.28 (m, 2H, H-2, H-5), 3.24 (dd, 1H,
3J2,3 10.1 Hz, J3,4 8.5 Hz, H-3), 3.24 (s, 3H, OCH3),
3
3.01 (dd, 1H, 3J4,5 9.2 Hz, H-4), 2.42 (s, 3H, CH3C6H4),
1.79 (s, 3H, COCH3); 13C NMR (125.76 MHz, Me2SO-
d6): d 169.24 (COCH3), 145.10 (p-C6H4), 132.47 (i-
C6H4), 130.31 (m-C6H4), 127.78 (o-C6H4), 101.75 (C-
1), 74.05 (C-3), 73.47 (C-5), 70.27 (C-6), 70.15 (C-4),
55.68 (OCH3), 55.12 (C-2), 23.25 (COCH3), 21.27
(CH3C6H4); CIMS (m/z, %) 390 (2) [M+H]+. Anal.
Calcd for C16H23NO8S (389.11): C, 49.35; H, 5.95;
N, 3.60; S, 8.23. Found: C, 49.55, H, 6.25; N, 3.44; S,
8.36.
Starting from the anomeric mixture of 1a,b
(10.0 g, 43.0 mmol)4 compounds 3a,b (16,7 g, 83%) were
obtained according to a procedure of Kajihara et al.5
Both anomers were separated by flash-chromatography
(ethyl acetate) to provide analytical samples.
3.5. Methyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-6-O-p-
tolysulfonyl-a-D-glucopyranoside (3a)
3.7. Methyl 2-acetamido-3,4-di-O-acetyl-2,6-dideoxy-a-
D-glucopyranoside (5a)
21
23
Mp 67–70 °C; ½aꢃD +87.6 (c 1.0, CHCl3); Rf 0.48 (ethyl
Mp 104–109 °C; ½aꢃD +76.4 (c 1.0, CHCl3); Rf 0.42
1
acetate); 1H NMR (250.13 MHz, CDCl3): d 7.77 (m,
2H, o-C6H4), 7.34 (m, 2H, m-C6H4), 5.65 (d, 1H,
(ethyl acetate); H NMR (300.13 MHz, CDCl3): d 5.69
(d, 1H, JNH,2 9.5 Hz, NH), 5.14 (dd, 1H, J3,4 9.5 Hz,
H-3), 4.82 (‘t’, 1H, J4,5 9.9 Hz, H-4), 4.63 (d, 1H, J1,2
3.6 Hz, H-1), 4.28 (ddd, 1H, J2,3 10.7 Hz, H-2), 3.79
3
3
3
3
3
3JNH,2 9.5 Hz, NH), 5.15 (dd, 1H, J3,4 9.5 Hz, H-3),
3
3
3
4.91 (‘t’, 1H, J4,5 10.0 Hz, H-4), 4.61 (d, 1H, J1,2
3.7 Hz, H-1), 4.21 (ddd, 1H, J2,3 10.7 Hz, H-2), 4.10–
3
3
(dq, 1H, J5,6 6.3 Hz, H-5), 3.36 (s, 3H, OCH3),
3
3
4.01 (m, 2H, H-6), 3.94 (ddd, 1H, J5,6b 3.4 Hz, J5,6a
5.3 Hz, H-5), 3.33 (s, 3H, OCH3), 2.44 (s, 3H,
CH3C6H4), 2.02, 1.98 (2s, 2 ꢂ 3H, 2 ꢂ COCH3), 1.92
(s, 3H, NHCOCH3); 13C NMR (62.89 MHz, CDCl3):
d 171.25, 169.84, 169.34 (3 ꢂ CO), 145.06 (p-C6H4),
132.54 (i-C6H4), 129.81 (m-C6H4), 128.04 (o-C6H4),
97.97 (C-1), 71.00 (C-3), 68.37 (C-4), 67.93 (C-6),
67.52 (C-5), 55.45 (OCH3), 51.66 (C-2), 23.16
(NHCOCH3), 21.64 (CH3C6H4), 20.65, 20.52
2.01, 1.99 (2s, 2 ꢂ 3H, 2 ꢂ COCH3), 1.93 (s, 3H,
NHCOCH3), 1.17 (d, 3H, H-6); 13C NMR
(62.89 MHz, CDCl3):
d
171.42, 169.83, 169.57
(3 ꢂ CO), 98.09 (C-1), 73.30 (C-3), 71.35 (C-4), 65.40
(C-5), 55.17 (OCH3), 52.16 (C-2), 23.20 (NHCOCH3),
20.72, 20.68 (2 ꢂ COCH3), 17.30 (C-6); CIMS (m/z,
%) 304 (87) [M+H]+. Anal. Calcd for C13H21NO7
(303.13): C, 51.48; H, 6.98; N, 4.62. Found: C, 51.42;
H, 7.21; N, 4.45.

S. Borowski et al. / Carbohydrate Research 343 (2008) 1004–1011
1009
3
3
3.8. Methyl 2-acetamido-3,4-di-O-acetyl-2,6-dideoxy-
b-D-glucopyranoside (5b)
(d, 1H, JOH,3 5.2 Hz, OH-3), 4.17 (d, 1H, J1,2 8.5 Hz,
3
H-1), 3.43 (ddd, 1H, J2,3 10.3 Hz, H-2), 3.28 (s, 3H,
OCH3), 3.26–3.11 (m, 2H, H-3, H-5), 2.82 (ddd, 1H,
23
23
D
3
Mp 169–171 °C (dec); ½aꢃD ꢀ2.2 (c 1.0, CHCl3); lit.5 ½aꢃ
3J4,5 9.3 Hz, J3,4 8.8 Hz, H-4), 1.79 (s, 3H, COCH3),
1
3
ꢀ38.0 (c 0.3, Me2SO); Rf 0.28 (ethyl acetate); H NMR
1.17 (d, 3H, J5,6 6.2 Hz, H-6); 13C NMR (62.89 MHz,
3
(300.15 MHz, CDCl3): d 5.56 (d, 1H, JNH,2 9.0 Hz,
Me2SO-d6): d 169.20 (COCH3), 101.84 (C-1), 76.06
(C-3), 74.26 (C-5), 71.78 (C-4), 55.62 (OCH3), 55.51
(C-2), 23.28 (COCH3), 18.09 (C-6); CIMS (m/z, %)
220 (100) [M+H]+. Anal. Calcd for C9H17NO5
(219.11): C, 49.31; H, 7.82; N, 6.39. Found: C, 49.45;
H, 8.07; N, 6.18.
3
NH), 5.17 (dd, 1H, J3,4 9.5 Hz, H-3), 4.80 (‘t’, 1H,
3J4,5 9.5 Hz, H-4), 4.49 (d, 1H, J1,2 8.4 Hz, H-1), 3.87
3
3
3
(ddd, 1H, J2,3 10.7 Hz, H-2), 3.55 (dq, 1H, J5,6
6.2 Hz, H-5), 3.47 (s, 3H, OCH3), 2.02, 2.01 (2s,
2 ꢂ 3H, 2 ꢂ COCH3), 1.93 (s, 3H, NHCOCH3), 1.23
(d, 3H, H-6); 13C NMR (75.47 MHz, CDCl3): d
171.07, 170.21, 169.64 (3 ꢂ CO), 101.42 (C-1), 73.57
(C-3), 72.59 (C-5), 69.95 (C-4), 56.50 (OCH3), 54.66
(C-2), 23.35 (NHCOCH3), 20.69 (2 ꢂ COCH3), 17.42
(C-6); CIMS (m/z, %) 304 (82) [M+H]+. Anal. Calcd
for C13H21NO7 (303.13): C, 51.48; H, 6.98; N, 4.62.
Found: C, 51.19; H, 6.68; N, 4.39.
3.12. Selective acetylation of compounds 6a and 6b
A soln of acetyl chloride (0.4 mL) in dry toluene
(2.0 mL) was added dropwise to a stirred soln of com-
pound 6a or 6b (each 1.0 g, 4.56 mmol) in dry pyridine
(20 mL) at ꢀ40 °C. The mixture was kept at that tem-
perature for additional 30 min, then at ꢀ20 °C for
30 min, and finally stored at 5 °C for 48 h (TLC, solvent
B1), and concentrated. The only observed side-products
were the diacetylated compounds 5a and 5b which were
removed easily by column chromatography (ethyl ace-
tate) to give crystalline 7a (480 mg, 40%) and 7b (456,
38%), respectively.
3.9. Deacetylation of 5a,b
Methanolic sodium methoxide soln (0.5 M, 1.0 mL) was
added to a soln of compounds 5a,b (5.0 g, 16.45 mmol)
in dry methanol (60 mL). After stirring at ambient tem-
perature for 2 h (TLC, solvent B1), the reaction mixture
was neutralized with IR120 (H+) Amberlite resin, fil-
tered, dried and concentrated. Column chromatography
(solvent B3) of the residue provided the separated iso-
mers 6a (2.05 g, 57%) and 6b (323 mg, 9%).
3.13. Methyl 2-acetamido-3-O-acetyl-2,6-dideoxy-a-D-
glucopyranoside (7a)
24
Mp 160–163 °C; ½aꢃD +71.2 (c 1.0, CHCl3); Rf 0.20
1
3.10. Methyl 2-acetamido-2,6-dideoxy-a-D-glucopyran-
oside (6a)
(ethyl acetate); H NMR (250.13 MHz, CDCl3): d 5.85
3
3
(d, 1H, JNH,2 9.5 Hz, NH), 4.99 (dd, 1H, J3,4 9.2 Hz,
3
H-3), 4.60 (d, 1H, J1,2 3.6 Hz, H-1), 4.22 (ddd, 1H,
24
3
Mp 172–173 °C (ethanol); ½aꢃD +55.5 (c 0.9, CHCl3); Rf
3J2,3 10.7 Hz, H-2), 3.69 (dq, 1H, J5,6 6.2 Hz, H-5),
1
3
0.45 (solvent B2); H NMR (300.13 MHz, Me2SO-d6): d
3.36 (s, 3H, OCH3), 3.35 (t, 1H, J4,5 9.5 Hz, H-4),
3
3
7.70 (d, 1H, JNH,2 8.5 Hz, NH), 5.01 (d, 1H, JOH,4
5.7 Hz, OH-4), 4.68 (d, 1H, JOH,3 5.7 Hz, OH-3), 4.47
(d, 1H, J1,2 3.6 Hz, H-1), 3.67 (ddd, 1H, J2,3 10.7 Hz,
2.06 (s, 3H, COCH3), 1.93 (s, 3H, NHCOCH3), 1.31
(d, 3H, H-6); 13C NMR (75.47 MHz, CDCl3): d
172.43, 170.1 (2 ꢂ CO), 98.26 (C-1), 74.54 (C-3), 74.18
(C-4), 67.48 (C-5), 55.04 (OCH3), 52.03 (C-2), 23.18
(NHCOCH3), 20.98 (COCH3), 17.54 (C-6); CIMS
(m/z, %) 262 (63) [M+H]+. Anal. Calcd for
C11H19NO6 (261.12): C, 50.57; H, 7.33; N, 5.36. Found:
C, 50.59; H, 7.65; N, 5.09.
3
3
3
H-2), 3.51–3.34 (m, 2H, H-3, H-5), 3.22 (s, 3H,
3
3
OCH3), 2.85 (ddd, 1H, J4,5 9.3 Hz, J3,4 8.5 Hz, H-4),
3
1.82 (s, 3H, NHCOCH3), 1.15 (d, 3H, J5,6 6.3 Hz,
H-6); 13C NMR (62.89 MHz, Me2SO-d6): d 169.55
(NHCOCH3), 98.23 (C-1), 76.63 (C-3), 70.64 (C-4),
67.36 (C-5), 54.55 (OCH3), 54.10 (C-2), 22.79
(NHCOCH3), 18.08 (C-6); CIMS (m/z, %) 220 (100)
[M+H]+. Anal. Calcd for C9H17NO5 (219.11): C,
49.31; H, 7.82; N 6.39. Found: C, 49.25; H, 7.98; N,
6.21.
3.14. Methyl 2-acetamido-3-O-acetyl-2,6-dideoxy-b-D-
glucopyranoside (7b)
26
Mp 181–184 °C; ½aꢃD ꢀ90.6 (c 1.0, CH3OH); Rf 0.10
1
(ethyl acetate); H NMR (300.13 MHz, CDCl3): d 5.57
3
3
3.11. Methyl 2-acetamido-2,6-dideoxy-b-D-glucopyran-
oside (6b)
(d, 1H, JNH,2 9.2 Hz, NH), 4.99 (dd, 1H, J3,4
3
10.5 Hz, H-3), 4.41 (d, 1H, J1,2 8.4 Hz, H-1), 3.86
3
(ddd, 1H, J2,3 8.8 Hz, H-2), 3.47 (s, 3H, OCH3), 3.45–
26
Mp 155–157 °C (ethanol); ½aꢃD ꢀ27.6 (c 1.0, CH3OH);
3.30 (m, 2H, H-4, H-5), 2.10 (s, 3H, COCH3), 1.94 (s,
23
1
3
lit.5 ½aꢃD ꢀ46.3 (c 2.0, H2O); Rf 0.23 (solvent B2); H
3H, NHCOCH3), 1.36 (d, 3H, J5,6 5.8 Hz, H-6); 13C
3
NMR (300.13 MHz, Me2SO-d6): d 7.66 (d, 1H, JNH,2
NMR (75.47 MHz, CDCl3):
d
172.21, 170.20
3
9.0 Hz, NH), 5.02 (d, 1H, JOH,4 5.3 Hz, OH-4), 4.86
(2 ꢂ CO), 101.54 (C-1), 75.98 (C-3), 74.35 (C-5), 71.96

1010
S. Borowski et al. / Carbohydrate Research 343 (2008) 1004–1011
(C-4), 56.45 (OCH3), 54.45 (C-2), 23.40 (NHCOCH3),
20.94 (COCH3), 17.60 (C-6); CIMS (m/z, %) 262
(100) [M+H]+. Anal. Calcd for C11H19NO6 (261.12):
C, 50.57; H, 7.33; N, 5.36. Found: C, 50.83; H, 7.67;
N, 5.06.
3.17. Methyl 2-acetamido-3-O-acetyl-2,6-dideoxy-b-D-
xylo-hexopyranosid-4-ulose (8b)
24
Mp 120–122 °C; ½aꢃD +11.2 (c 1.0, CHCl3); Rf 0.16
1
(ethyl acetate); H NMR (300.13 MHz, CDCl3): d 6.07
3
(d, 1H, JNH,2 8.0 Hz, NH), 5.78 (d, 1H, H-3), 5.10 (d,
3
3.15. Methyl 2-acetamido-3-O-acetyl-2,6-dideoxy-a- and
b-D-xylo-hexopyranosid-4-ulose (8a and 8b)
1H, J1,2 7.2 Hz, H-1), 4.19 (q, 1H,J5,6 6.5 Hz, H-5),
3
3.86 (ddd, 1H, J2,3 10.8 Hz, H-2), 3.51 (s, 3H, OCH3),
2.15 (s, 3H, COCH3), 1.99 (s, 3H, NHCOCH3), 1.36
(d, 3H, H-6); 13C NMR (75.47 MHz, CDCl3): d 198.28
(C-4), 170.47, 170.05 (2 ꢂ CO), 100.58 (C-1), 74.10,
73.93 (C-3, C-5), 57.99 (C-2), 56.84 (OCH3), 23.42
(NHCOCH3), 20.49 (COCH3), 14.51 (C-6); CIMS
(m/z, %) 260 (100) [M+H]+. Anal. Calcd for
C11H17NO6 (259.11): C, 50.96; H, 6.61; N, 5.40. Found:
C, 50.96; H, 6.53; N, 5.09.
3.15.1. Via pyridinium dichromate (PDC) oxidation.
A
catalytic amount of glacial acetic acid (100 lL) and
˚
freshly activated and powdered 3-A molecular sieves
(800 mg) were added to a stirred suspension of com-
pound 7a or 7b (261 mg, 1.0 mmol) and PDC (564 mg,
1.5 mmol) in dry dichloromethane (5 mL). After stirring
for 18 h at ambient temperature (TLC, ethyl acetate),
Celite was added (500 mg) and, 20 min later, the insolu-
ble constituents were filtered off and washed with dichlo-
romethane (2 ꢂ 3 mL). The combined filtrates were
dried, concentrated and the residue was repeatedly
evaporated with toluene to remove traces of acetic acid.
The brown residue was then extracted with diethyl ether.
The ethereal extracts were combined, dried and evapo-
rated to give colourless, crystalline and analytically pure
8a (235 mg, 91%) and 8b (155 mg, 60%), respectively.
3.18. Deacetylation of compounds 8a and 8b
´
3.18.1. Via Zemplen procedure. A methanolic sodium
methoxide soln (0.5 M, 0.75 mL) was added to a soln
of compound 8a or 8b (65 mg, 0.25 mmol) in dry meth-
anol (2 mL). After stirring at ambient temperature for
2 h (TLC, solvent B1), the soln was neutralized by the
addition of IR120 (H+) Amberlite resin, filtered, and
concentrated. The purification of the crude material by
column chromatography (solvent B3) afforded 9a
(39 mg, 72 %) and 9b (22 mg, 40%), respectively.
3.15.2. Via Swern-oxidation. Dimethyl sulfoxide
(214 lL, 3.0 mmol) was added dropwise to a stirred soln
of oxalyl chloride (128 lL, 1.5 mmol) in dry dichloro-
methane (3 mL) at ꢀ78 °C. After the mixture had been
stirred for 15 min, a soln of compound 7a or 7b
(261 mg, 1.0 mmol) in dry dichloromethane (9 mL)
was added dropwise at ꢀ78 °C. Stirring was continued
for another 1 h before triethylamine (690 lL, 5.0 mmol)
was added dropwise. The mixture was warmed to room
temperature and then concentrated. The slightly yellow
coloured crude product was purified by column chroma-
tography (ethyl acetate) to provide 8a (241 mg, 93%)
and 8b (148 mg, 57%), respectively.
3.18.2. Via methanolic HCl soln. Compound 8a or 8b
(130 mg, 0.5 mmol) was dissolved in methanolic HCl
(0.28 M, 19 mL, prepared by adding of 0.4 mL acetyl
chloride to 18.6 mL ice-cold dry methanol) and the soln
was stirred at ambient temperature for 12 h (TLC,
solvent B1). The soln was then neutralized with
PbCO3 ꢂ Pb(OH)2 (1.6 g), filtered, and concentrated.
The residue was purified by column chromatography
(solvent B3) to provide 9a (48 mg, 44%) and 9b
(13 mg, 12%), respectively.
Under both deacetylation conditions compounds 6a,
10 and 6a, 6b and 10 were determined as side-products
starting from 8a and 8b, respectively. The overall yield
of isolated side-products in each case was about 10%.
3.16. Methyl 2-acetamido-3-O-acetyl-2,6-dideoxy-a-D-
xylo-hexopyranosid-4- ulose (8a)
25
Mp 136–139 °C; ½aꢃD +126.2 (c 1.0, CHCl3); Rf 0.24
1
(ethyl acetate); H NMR (250.13 MHz, CDCl3): d 5.85
3.19. Methyl 2-acetamido-2,6-dideoxy-a-D-xylo-hexo-
pyranosid-4-ulose (9a)
3
3
(d, 1H, JNH,2 9.5 Hz, NH), 5.44 (d, 1H, J2,3 11.5 Hz,
3
H-3), 4.82 (d, 1H, J1,2 3.5 Hz, H-1), 4.64 (ddd, 1H,
H-2), 4.28 (q, 1H, J5,6 6.5 Hz, H-5), 3.48 (s, 3H,
22
3
Mp 121–123 °C; ½aꢃD +141.7 (c 1.0, CH3OH); Rf 0.52
OCH3), 2.14 (s, 3H, COCH3), 1.98 (s, 3H, NHCOCH3),
1.30 (d, 3H, H-6); 13C NMR (75.47 MHz, CDCl3): d
197.94 (C-4), 170.69, 169.62 (2 ꢂ CO), 98.28 (C-1),
74.35 (C-3), 69.42 (C-5), 55.99 (OCH3), 53.74 (C-2),
23.20 (NHCOCH3), 20.52 (COCH3), 13.75 (C-6); CIMS
(m/z, %) 260 (97) [M+H]+. Anal. Calcd for C11H17NO6
(259.11): C, 50.96; H, 6.61; N, 5.40. Found: C, 50.89; H,
6.92; N, 5.13.
(solvent B1); 1H NMR (250.13 MHz, CDCl3): d 6.04
3
(br s, 1H, NH), 4.82 (d, 1H, J1,2 3.5 Hz, H-1), 4.42–
3
4.31 (m, 2H, H-2, H-3), 4.31 (q, 1H, J5,6 6.5 Hz,
H-5), 3.48 (s, 3H, OCH3), 2.05 (s, 3H, COCH3), 1.33
(d, 3H, H-6); 13C NMR (75.47 MHz, CDCl3): d 203.91
(C-4), 170.46 (COCH3), 98.37 (C-1), 74.21 (C-3), 68.53
(C-5), 57.36 (C-2), 56.12 (OCH3), 23.30 (COCH3),
13.60 (C-6); CIMS (m/z, %) 218 (100) [M+H]+. Anal.

S. Borowski et al. / Carbohydrate Research 343 (2008) 1004–1011
1011
Calcd for C9H15NO5 (217.10): C, 49.76; H, 6.96; N,
6.45. Found: C, 49.68; H, 7.15; N, 6.58.
Calcd for C8H9NO4 (183.16): C, 52.46; H, 4.95; N,
7.65. Found: C, 52.39; H, 5.01; N, 7.55.
3.20. Methyl 2-acetamido-2,6-dideoxy-b-D-xylo-hexopyr-
anosid-4-ulose (9b)
References
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22
Colourless syrup; ½aꢃD ꢀ45.9 (c 0.5, CH3OH); Rf 0.47
1
´ _
(solvent B1); H NMR (300.13 MHz, CD3OD): d 4.35
(s, 1H, J1,2 10.7 Hz, H-1), 3.83–3.75 (m, 1H, H-2),
Brade, L.; Rozalski, A.; Bartodziejska, B.; Mayer, H. Acta
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4.21–4.14, 3.72–3.62 (2m, 2 ꢂ 1H, H-3, H-5), 3.39 (s,
3
3H, OCH3), 1.95 (s, 3H, COCH3), 1.27 (d, 3H, J5,6
6.5 Hz, H-6); 13C NMR (75.47 MHz, CD3OD): d
205.31 (C-4), 173.52 (COCH3), 102.94 (C-1), 75.66
(C-3), 74.49 (C-5), 60.94 (C-2), 57.05 (OCH3), 22.98
(COCH3), 14.59 (C-6); CIMS (m/z, %) 218 (57)
[M+H]+. Anal. Calcd for C9H15NO5 (217.10): C,
49.76; H, 6.96; N, 6.45. Found: C, 49.60; H, 7.21; N,
6.66.
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3.21. N-(5-Hydroxy-6-methyl-4-oxo-4H-pyran-3-yl)acet-
amide (10)
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kumaran, K. Tetrahedron Lett. 1985, 26, 1699–1702.
11. Mancuso, A. J.; Swern, D. Synthesis 1981, 165–185.
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ratory Chemicals, 3rd ed.; Pergamon Press: Oxford, 1988.
14. Walker, E.; Roussel, P.; Jeanloz, R. W. Carbohydr. Res.
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Mp 172 °C (dec); Rf 0.67 (solvent B1); 1H NMR
(500.13 MHz, CDCl3): d 9.13 (s, 1H, H-2), 8.01 (br s,
1H, NH), 6.99 (br s, 1H, OH), 2.38 (s, 3H, CH3), 2.18
(s, 3H, COCH3); 13C NMR (125.76 MHz, CDCl3): d
168.75 (COCH3), 166.39 (C-4), 150.33 (C-6), 144.23
(C-2), 140.77 (C-5), 126.03 (C-3), 23.86 (COCH3),
14.63 (CH3); CIMS (m/z, %) 184 (100) [M+H]+. Anal.