First synthesis of β-D-Galf-(1→3)-D-Galp - The repeating unit of the backbone structure of the O-antigenic polysaccharide present in the lipopolysaccharide (LPS) of the genus Klebsiella

Article abstract of DOI:10.1016/S0008-6215(03)00076-4

β-D-Galactofuranosyl-(1→3)-D-galactopyranose (1), the repeating unit of the backbone structure of the O-antigenic polysaccharide present in the lipopolysaccharide (LPS) of the genus Klebsiella, has been efficiently synthesized using 1,2:5,6-di-O-isopropylidine-α-D-galactofuranose (3) as the glycosyl acceptor and 2,3,5,6-tetra-O-benzoyl-β-D-galactofuranosyl trichloroacetimidate (6) as the glycosyl donor with TMSOTf as catalyst by the well-known Schmidt glycosylation method. The preparation of 3 was improved by increasing the ratio of DMF to acetone and employing a solid-supported catalyst.

Full text of DOI:10.1016/S0008-6215(03)00076-4

Carbohydrate Research 338 (2003) 1033–1037
www.elsevier.com/locate/carres
First synthesis of b-
D
-Galf-(1“3)- -Galp—the repeating unit of
D
the backbone structure of the O-antigenic polysaccharide present
in the lipopolysaccharide (LPS) of the genus Klebsiella
Hairong Wang,b Guohua Zhang,a Jun Ninga,*
aResearch Center for Eco-En6ironmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing 100085, China
bDepartment of Chemistry, Tsinghua Uni6ersity, Beijing 100084, China
Received 11 November 2002; accepted 12 February 2003
Abstract
b-
ride present in the lipopolysaccharide (LPS) of the genus Klebsiella, has been efficiently synthesized using 1,2:5,6-di-O-isopropyl-
idine-a- -galactofuranose (3) as the glycosyl acceptor and 2,3,5,6-tetra-O-benzoyl-b- -galactofuranosyl trichloroacetimidate (6) as
D-Galactofuranosyl-(1“3)-D-galactopyranose (1), the repeating unit of the backbone structure of the O-antigenic polysaccha-
D
D
the glycosyl donor with TMSOTf as catalyst by the well-known Schmidt glycosylation method. The preparation of 3 was
improved by increasing the ratio of DMF to acetone and employing a solid-supported catalyst. © 2003 Elsevier Science Ltd. All
rights reserved.
Keywords: Oligosaccharides; Galactofuranose; Synthesis
While a number of microorganisms produce polysac-
charides containing the Galf residue, among the most
important are the Klebsiella. This genus of bacteria
contains a number of opportunistic pathogens that
cause serious infections, including pneumonia, bac-
teremia, and urinary-tract infections.5 A major compo-
nent of the outer membrane of many bacteria is
lipopolysaccharide (LPS) which comprises a hydropho-
bic lipid A portion, a core oligosaccharide, and an
O-antigenic polysaccharide. The O-polysaccharide
varies in structure from strain to strain, giving rise to
unique antigenic epitopes. In the genus Klebsiella, there
exists a family of structurally related galactose-contain-
ing O-polysaccharides. These are based on a backbone
structure consisting of a disaccharide repeating unit
1. Introduction
Although unknown in human biology, polysaccharides
containing galactofuranosyl residues are important con-
stituents of glycoconjugates from many lower organ-
isms including bacteria,1 protozoa,2 and fungi.3 Glycans
containing Galf moieties are generally found in the
extracellular glycocalyx (cell-wall complex) and conse-
quently play critical roles in the survival and patho-
genicity of these microorganisms. A well-established
method for the treatment of bacterial disease is the use
of antibiotics that act by inhibiting cell-wall biosynthe-
sis.4 Given the xenobiotic nature of Galf-containing
polysaccharides to humans, the biosynthetic pathways
leading to their formation are particularly attractive
targets for drug action. However, the processes by
which these polysaccharides are assembled in nature are
not well understood, and much additional research in
this area is needed before this goal can be achieved.
“3)-b-D-Galf-(1“3)-a-
D-Galp-(1“ known as
D-
galactan I.6 Synthesis of the galactofuranosyl residue
containing oligosaccharides present in the cell wall of
bacteria is very important. On one hand, these oligosac-
charides could be valuable models for immunoassay
studies; on the other hand, they can be used as syn-
thetic substrates for fundamental biochemical studies
that may lead to the isolation and purification of the
appropriate biosynthetic enzymes. This, together with
* Corresponding author. Fax: +86-10-62923563
E-mail address: jning@mail.rcees.ac.cn (J. Ning).
0008-6215/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00076-4

1034
H. Wang et al. / Carbohydrate Research 338 (2003) 1033–1037
the fact that the synthesis of b-
D
-Galf-(1“3)-
D
-Galp
yield.8 Recently, Rauter’s group found that by using
Zeolite HY as the catalyst, the furanose diketal 3 was
formed in 40% yield, together with the pyranose diketal
4, obtained only in 20% yield, which significantly im-
proved the preparation of 3.9 Some reports showed that
high temperature is a factor known to favor furanose
formation in solutions of reducing sugars.10 Inspired by
this, we increased the ratio of N,N-dimethylformamide
(DMF) to acetone to 2:1 in our synthesis of 3, attempt-
ing to increase the reaction reflux temperature in order
to obtain more furanose diketal 3. To simplify the
purification procedure, an anhydrous Dowex 50 [H+]
resin was used as the solid-supported catalyst (Scheme
(1) was not previously reported, prompted us to synthe-
size the disaccharide.
2. Results and discussion
In our synthesis, 1,2:5,6-di-O-isopropylidene-a-
tofuranose (3) is a key starting material. So far, 1,2:5,6-
-galactofuranose (3) has found
D-galac-
di-O-isopropylidene-a-
D
only limited use in synthetic carbohydrate chemistry
because it is not easily accessible. For example, 3 has
been obtained from
D
-glucose derivatives in three- or
-galactose in a maximum 22%
,
1). Furthermore, 4 A molecular sieves were used in our
six steps,7 or from
D
reaction as the water scavenger in order to increase the
yield. As a result of our reaction conditions, the ratio of
1,2:5,6-di-O-isopropylidene-a-
D
-galactofuranose
to
1,2:3,4-di-O-isopropylidene-a-
D-galactopyranose in the
reaction product can reach more than 4:1, and the
-galactofuran-
desired 1,2:5,6-di-O-isopropylidene-a-
D
ose can be easily isolated by flash chromatography,
followed by crystallisation in 50% yield.11
Scheme 1. Reagents and conditions: (a) 2:1 DMF–acetone,
Dowex 50 [H ] (dry), 4 A MS, reflux, 48 h, 50%.
The glycosyl donor 6 was synthesized from 1-O-acet-
+
,
yl-2,3,5,6-tetra-O-benzoyl-
-galactofuranose (5)12 by
D
selective 1-O-deacetylation with ammonia in THF–
CH3OH, and then trichloroacetimidation with
trichloroacetonitrile in the presence of K2CO3 (Scheme
2). The structure of 6 was confirmed by 1H NMR
spectral analysis. The J1,2 is small (0–2 Hz) for the b
anomers13 and larger (4–5 Hz) for the a anomers.13
Condensation14 of 3 and 6 with TMSOTf as catalyst
Scheme 2. Reagents and conditions: (a) (i) 3:1 THF–CH3OH,
1.5 N NH3, rt, 3 h; (ii) CH2Cl2, CCl3CN (2.0 equiv), K2CO3
(2.0 equiv), rt, 12 h, 82% (over two steps).
,
and 4 A molecular sieves in CH2Cl2 at room tempera-
ture smoothly afforded the disaccharide 7 in an excel-
lent yield (85%) (Scheme 3). The structure of 7 was
1
unambiguously confirmed by H and 13C NMR data.
The two anomeric proton signals in the 1H NMR
spectrum of 7 were located at 5.91 ppm as a doublet
with J1,2=3.9 Hz, and at 5.44 ppm as a singlet, respec-
tively, whereas the 13C NMR spectrum revealed15
anomeric carbons at 105.1 and 104.8 ppm. Removal of
the 1,2:5,6-di-O-isopropylidene group of 7 in 10:1
CHCl3–CF3COOH (v/v) at room temperature did not
give satisfactory results because the reaction products
are too complex. However, selective removal of the
5,6-O-isopropylidene group of 7 with 90% HOAc at
40 °C, followed by acetylation using Ac2O in pyridine
and subsequent 1,2-O-deisopropylidenation with 10:1
CHCl3–CF3COOH (v/v) at room temperature,
smoothly gave the required compound 9 in 84.6% yield
(over three steps). Deprotection of 9 with an ammonia-
saturated CH3OH gave the desired disaccharide 1 in
96% yield. The 13C NMR spectrum revealed anomeric
carbons at 109.01 (C-1 for Galf ), 105.98 (C-1 for
b-Galp) and 96.0 (C-1 for a-Galp) ppm, and thin-layer
chromatography (TLC) (methanol) analysis showed
two spots, indicating that compound 1 was a mixture of
Scheme 3. Reagents and conditions: (a) TMSOTf (cat.),
CH2Cl2, MS 4 A Powder, rt, 3 h, 85%. (b) (i) 90% HOAc,
40 °C, 4 h; (ii) AcO2, pyridine, rt, 2 h, 94% (over two steps).
(c) 10:1 HCCl3–CF3COOH, rt, 2 h, 90%. (d) ammonia-satu-
rated CH3OH, rt, 72 h, 96%.
,

H. Wang et al. / Carbohydrate Research 338 (2003) 1033–1037
1035
a and b isomers. O-Deacylation of blocked oligosac-
3.3. 2,3,5,6-Tetra-O-benzoyl-b-D-galactofuranosyl
trichloroacetimidate (6)
charides with methanolic sodium methoxide sometimes
could be complicated by a b-elimination reaction typi-
cal of 3-substituted reducing aldoses;16,17 however, this
problem can be avoided by using ammonia-saturated
CH3OH.17,18
A
solution of 1-O-acetyl-2,3,5,6-tetra-O-benzoyl-D-
galactofuranose (5)12 (3.6 g, 5.64 mmol) in 1.5 N NH3
solution of 3:1 THF–CH3OH (80 mL) was kept at
room temperature (rt) for 3 h, at the end of which time
TLC (3:1 petroleum ether–EtOAc) showed that the
reaction was complete and the solution was concen-
trated. The resultant residue without purification was
dissolved in dry CH2Cl2 (50 mL), and then CCl3CN
(2.6 mL, 12.4 mmol) and anhydrous K2CO3 (3 g, 21.8
mmol) were added. The reaction mixture was stirred at
rt for 12 h, and solid material was filtered off. Concen-
tration of the filtrate, followed by purification on a
silica-gel column with 3:1 petroleum ether–EtOAc as
eluent, gave the monosaccharide donor 6 (3.42 g, 82%
In conclusion, a highly concise synthesis of the re-
peating unit disaccharide 1 present in LPS of the genus
Klebsiella was achieved. The simple and efficient prepa-
ration of the galactofuranose-containing oligosaccha-
ride present in the bacterial outer membrane should
promote studies on bacteria diseases.
3. Experimental
3.1. General methods
1
over the two steps): [h]D +28.3° (c 1.0, CHCl3); H
Optical rotations were determined at 20 °C with a
Perkin–Elmer Model 241-Mc automatic polarimeter.
Melting points (mp) were determined with a ‘Mel-
Temp’ apparatus. 1H and 13C NMR spectra were
recorded with Bruker ARX 400 spectrometers (400
NMR (400 MHz, CDCl3):
l
8.72 (s,
1
H,
OC(NH)CCl3), 8.09–7.26 (m, 20 H, 4 PhH), 6.70 (s, 1
H, H-1), 6.13 (m, 1 H, H-5), 5.78 (d, 1 H, J 4.3 Hz,
H-3), 5.75 (s, 1 H, H-2), 4.86 (t, 1 H, J 4.3 Hz, H-4),
4.78–4.75 (m, 2 H, H-6a, 6b). Anal. Calcd for
C36H28Cl3NO10: C, 58.35; H, 3.81. Found: C, 58.56; H,
3.74.
1
MHz for H, 100 MHz for 13C) for solutions in CDCl3
or CD3OD as indicated. Chemical shifts are given in
ppm downfield from internal Me4Si. TLC was per-
formed on silica gel HF254 with detection by charring
with 30% (v/v) H2SO4 in MeOH or in some cases by a
UV detector. Electrospray-ionization mass spectra were
recorded on an Autospec instrument. Column chro-
matography was conducted by elution of a column
(16×240, 18×300, 35×400 mm) of silica gel (100–
200 mesh) with EtOAc–petroleum ether (60–90 °C) as
the eluent. Solutions were concentrated at B60 °C
under reduced pressure.
3.4. 2,3,5,6-Tetra-O-benzoyl-b-D-galactofuranosyl-(1“
3)-1,2:5,6-di-O-isopropylidene-a-D-galactofuranose (7)
A solution of 3 (1.36 g, 5.25 mmol) and 6 (3.89 g, 5.25
mmol) in dry CH2Cl2 (50 mL) was stirred with acti-
,
vated 4 A molecular sieves (1 g) at rt under an atmo-
sphere of nitrogen for 20 min. To the solution was
added MeSiOTf (12 mL). The reaction mixture was
stirred for 2 h, at the end of which time TLC (2.5:1
petroleum ether–EtOAc) indicated that the reaction
was complete. The reaction mixture was neutralized
with Et3N and filtered, and the filtrate was concen-
trated. The resultant residue was subjected to the
column chromatography with 2:1 petroleum ether–
EtOAc as eluent to afford the disaccharide 7 (3.74 g,
85%): [h]D −26.5° (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3): l 8.08–7.26 (m, 20 H, 4 PhH), 6.05 (m, 1 H,
H-5%), 5.92 (d, 1 H, J 3.9 Hz, H-1), 5.71 (d, 1 H, J 4.5
Hz, H-3%), 5.50 (s, 1 H, H-1%), 5.44 (s, 1 H, H-2%),
4.81–4.66 (m, 4 H, H-2, 4%, 6%a, 6%b), 4.33 (m, 1 H, H-5),
4.20 (d, 1 H, J 4.5 Hz, H-3), 4.04–3.96 (m, 2 H, H-4,
6a), 3.86 (t, 1 H, J 7.5 Hz, H-6b), 1.55, 1.41, 1.36, 1.34
(4 s, 12 H, 2 C(CH3)2); 13C NMR (100 MHz, CDCl3):
l 165.9, 165.6, 165.5, 165.4 (4 COPh), 113.9, 109.9 (2
C(CH3)2), 105.1, 104.8 (2 C-1), 85.1, 84.2, 82.3, 82.0,
80.2, 77.1, 75.9, 70.3, 65.6, 63.3, 27.4, 26.7, 26.5, 25.3 (2
C(CH3)2). Anal. Calcd for C46H46O15: C, 65.86; H,
5.53. Found: C, 65.66; H, 5.47.
3.2. Preparation of 1,2:5,6-di-O-isopropylidene-a-D-
galactofuranose (3)
A solution of -galactose (2) (10 g, 55.56 mmol) in dry
D
and hot N,N-dimethylformamide (400 mL) was added
to stirred acetone (200 mL), which contained dry
+
,
Dowex 50 [H ] resin (15 g) and 4 A molecular sieves (8
g). The reaction mixture was stirred under reflux for 48
h. Solid material was filtered off, and the solution was
concentrated to a syrup under vacuum. TLC (3:1
petroleum ether–EtOAc) showed that the ratio of
1,2:5,6-di-O-isopropylidene-a-
1,2:3,4-di-O-isopropylidene-a-
D
-galactofuranose
to
D-galactopyranose in the
resultant residue was \4:1. After the residue was
decolorized and, in some degree, purified by subjection
to a flash chromatography with 3:1 petroleum ether–
EtOAC as eluent, compound 3 (7.2 g, 50%) was crystal-
lized from petroleum ether–EtOAc as white crystals:
mp 96.5–98 °C; (Lit.8 97–98 °C), [h]D −33.8° (c 0.8,
CH3OH) (Lit.8 −34°).

1036
H. Wang et al. / Carbohydrate Research 338 (2003) 1033–1037
3.5. 2,3,5,6-Tetra-O-benzoyl-b-D-galactofuranosyl-(1“
afford 1 (624 mg, 96%) as a mixture of a and b isomers
1
3)-5,6-di-O-acetyl-1,2-O-isopropylidene-a-
D-galacto-
(mainly b): [h]D +28.5° (c 0.8, H2O); H NMR (400
pyranose (8)
MHz, D2O): l 5.12, 4.96 (2 H-1); 13C NMR (100 MHz,
D2O): l 109.01 (C-1 for Galf ), 105.98 (C-1 for b-
Galp), 96.0 (C-1 for a-Galp). ESIMS: Anal. Calcd for
C12H22O11: [M] 342.3. Found: [M+H+]+ 343.2.
Compound 7 (3.8 g, 4.53 mmol) was dissolved in 90%
HOAc (100 mL). The mixture was kept at 40 °C for 4
h, and then concentrated under reduced pressure. The
resulting residue was treated with Ac2O and Py at rt for
2 h and then purified by flash chromatography (1:1
petroleum ether–EtOAc) to give 8 (3.76 g, 94% over
Acknowledgements
1
two steps): [h]D −12.2° (c 1.0, CHCl3); H NMR (400
MHz, CDCl3): l 8.10–7.23 (m, 20 H, 4 PhH), 6.15 (m,
1 H, H-5%), 5.87 (d, 1 H, J 4.1 Hz, H-1), 5.64 (d, 1 H,
J 5.0 Hz, H-3%), 5.49 (s, 1 H, H-1%), 5.42 (s, 1 H, H-2%),
5.42 (m, 1 H, H-5), 4.83 (dd, 1 H, J 3.4, 12 Hz, H-6a%),
4.77–4.72 (m, 3 H, H-2, 4%, 6b%), 4.41 (dd, 1 H, J 3.9, 12
Hz, H-6a), 4.28 (dd, 1 H, J 1.5, 6.1 Hz, H-3), 4.20 (dd,
1 H, J 7.4, 12.0 Hz, H-6b), 4.08 (dd, 1 H, J 4.4, 6.0 Hz,
H-4), 2.14, 1.96 (2 s, 6 H, C(CH3)2), 1.59, 1.38 (2 s, 6 H,
2 COCH3). Anal. Calcd for C47H46O17: C, 63.94; H,
5.25. Found: C, 63.76; H, 5.30.
This work was supported by the Beijing Natural
Science Foundation (6021004) and National Natural
Science Foundation of China (59973026 and 29905004).
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1
90%): [h]D −7.8° (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3): l 8.09–7.23 (m, 20 H, 4 PhH), 6.11 (m, 1 H,
H-5%), 5.75 (s, 0.75 H, H-1% for b isomer), 5.68 (d, 0.25
H, J 5.0 Hz, H-3% for a isomer), 5.62 (d, 0.75 H, J 5.0
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isomer), 5.48 (d, 0.75 H, J 1.1 Hz, H-2% for b isomer),
5.43 (d, 0.25 H, J 1.1 Hz, H-2% for a isomer), 5.37 (m,
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