Synthesis and identification in bacterial lipopolysaccharides of 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto- and -D-glycero-D-talo-non-2-ulosonic acids

Article abstract of DOI:10.1016/S0008-6215(01)00041-6

5,7-Diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto- and -D-glycero-D-talo-non-2-ulosonic acids were synthesized by condensation of 2,4-diacetamido-2,4,6-trideoxy-d-mannose with oxalacetic acid. Comparison of the ¹H and ¹³C NMR data and the specific optical rotation values of these monosaccharides and the corresponding l-glycero-d-galacto and l-glycero-d-talo isomers synthesized earlier [Tsvetkov, Y. E.; Shashkov, A. S.; Knirel, Y. A.; Backinowsky, L. V.; Zaehringer, U. Mendeleev Commun. 2000, 90-92] with data of the natural compounds enabled the identification in bacterial lipopolysaccharides of derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic (legionaminic) acid and epimers of legionaminic acid at C-4 and C-8.

Full text of DOI:10.1016/S0008-6215(01)00041-6

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Carbohydrate Research 331 (2001) 233–237
Rapid Communication
Synthesis and identification in bacterial
lipopolysaccharides of
5,7-diacetamido-3,5,7,9-tetradeoxy-
D
-glycero- -galacto-
D
and - -glycero- -talo-non-2-ulosonic acids
D
D
Yury E. Tsvetkov,a,b Alexander S. Shashkov,a Yuriy A. Knirel,a,b Ulrich Za¨hringerb,*
aN. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47,
Moscow 117913, Russian Federation
bForschungszentrum Borstel, Zentrum fu¨r Medizin und Biowissenschaften, Parkallee 22, D-23845 Borstel, Germany
Received 4 December 2000; accepted 29 January 2001
Abstract
5,7-Diacetamido-3,5,7,9-tetradeoxy-
sized by condensation of 2,4-diacetamido-2,4,6-trideoxy-
13C NMR data and the specific optical rotation values of these monosaccharides and the corresponding
galacto and -glycero- -talo isomers synthesized earlier [Tsvetkov, Y. E.; Shashkov, A. S.; Knirel, Y. A.;
Backinowsky, L. V.; Za¨hringer, U. Mendelee6 Commun. 2000, 90–92] with data of the natural compounds enabled
the identification in bacterial lipopolysaccharides of derivatives of 5,7-diamino-3,5,7,9-tetradeoxy- -glycero-
D
-glycero-
D
-galacto- and -
-mannose with oxalacetic acid. Comparison of the H and
-glycero-D-
D-glycero-D-talo-non-2-ulosonic acids were synthe-
1
D
L
L
D
D
D-
galacto-non-2-ulosonic (legionaminic) acid and epimers of legionaminic acid at C-4 and C-8. © 2001 Elsevier Science
Ltd. All rights reserved.
Keywords: 5,7-Diamino-3,5,7,9-tetradeoxynon-2-ulosonic acid; Synthesis; Legionaminic acid, identification of; Lipopolysaccharide
components; Legionella pneumophila
N-Acyl and O-acetyl derivatives of 5,7-di-
amino-3,5,7,9-tetradeoxynon-2-ulosonic acids
have been reported as components of the lipo-
polysaccharides (LPS) of gram-negative bacte-
ria, which play a role in serological specificity
and endow the bacterial surface with peculiar
physicochemical properties.1–3 The first
monosaccharide of this class discovered in the
mid-1980s in the LPS of Pseudomonas aerugi-
nosa and Shigella boydii was identified as the
L
-glycero-
L
-manno
isomer
(pseudaminic
acid).4,5 The
D
-glycero-
L
-galacto configuration
that was ascribed to the second isomer found
first in P. aeruginosa,6 was later revised to the
L
-glycero-
-galacto configuration.7,8 A homo-
D
polymer of a derivative of a 5,7-diamino-
3,5,7,9-tetradeoxynon-2-ulosonic acid, called
legionaminic acid, was described as the O-
chain of the LPS in Legionella pneumophila
serogroup 13,9 and Pseudomonas fluorescens
ATCC 49271,10,11 and it was suggested that
legionaminic acid has the same
L
-glycero- -
D
* Corresponding author. Tel.: +49-4537-188462; fax: +
49-4537-188612.
E-mail address: uzaehr@fz-borstel.de (U. Za¨hringer).
galacto configuration.7 While the configura-
tions of the higher sugars in these and some
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00041-6

234
Y.E. Ts6etko6 et al. / Carbohydrate Research 331 (2001) 233–237
other LPS12–14 (Table 1) were ascribed by
NMR spectroscopy without their isolation as
bis-azidation with Bu4NN3, resulted in diazide
7 having the manno configuration. After
debenzoylation (7“8), reduction of the azido
groups was performed by hydrogenation over
Pd(OH)2–C without affecting the benzyl
group19 and followed by N-acetylation (8“9).
Removal of the benzyl aglycon by hy-
monosaccharides,
two
5,7-diacetamido-
3,5,7,9-tetradeoxynon-2-ulosonic acids were
released by mild acid hydrolysis from the LPS
of Vibrio alginolyticus15 and L. pneumophila,16
and their configurations were tentatively as-
drogenolysis
gave
2,4-diacetamido-2,4,6-
signed to be
ero- -talo, respectively.
L
-glycero-
D
-galacto and
L
-glyc-
trideoxy- -mannose (10) in 38% overall yield
D
D
from 5. All new compounds were fully charac-
terized by NMR spectroscopy and elemental
analysis.
Aiming at unambiguous identification of
these higher sugars, including determination
of their absolute configuration, we have syn-
thesized, for the first time, 5,7-diacetamido-
Condensation of 10 with oxalacetic acid in
the presence of sodium tetraborate at pH
10.520 gave a mixture of 3 and 4. The individ-
ual compounds 3, [h]D +27.2° (c 1, water),
and 4, [h]D −12.5° (c 1, water), were isolated
in 7 and 10% yield, respectively, by anion-ex-
change chromatography on Dowex 1×8 (aq
0.3 M formic acid) followed by reversed-phase
3,5,7,9-tetradeoxy-
L
-glycero-
-talo-non-2-ulosonic acids (2)
2,4-diacetamido-2,4,6-trideoxy- -gu-
D
-galacto-
(1)
and -
L
-glycero-
D
from
L
lose.17 In this paper, we report the first synthe-
sis of the corresponding
D
-glycero- -galacto
D
(3) and -glycero- -talo (4) isomers, which
D
D
1
enabled determination of the configurations of
the natural monosaccharides, legionaminic
acid (Leg) and two of its epimers, 4-epi- and
8-epi-legionaminic acid (4eLeg and 8eLeg,
respectively).
C18 HPLC (aq 0.05% CF3CO2H). The H and
13C NMR spectroscopic data (Table 2)
showed that 3 and 4 are mixtures of a and b
anomers in the ratio 1:18 and 1:5.4, respec-
tively, and proved their configurations. Partic-
ularly, the J3a,4, J4,5, and J5,6 coupling constant
values clearly indicated that the substituents at
C-4,5,6 in 3 and C-5,6 in 4 are equatorial,
whereas the hydroxy group at C-4 in 4 is
axial.
Synthesis was performed starting from ben-
zyl b-
-fucopyranoside 5,18 which afforded 3-
D
benzoate 6 on Bu2SnO-mediated selective
benzoylation (Scheme 1). Conversion of 6 into
the corresponding 2,4-ditriflate, followed by
Table 1
Some naturally occurring isomers of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acid
Source (lipopolysaccharide of)
Configuration
Originally ascribed
Previously revised to7,8
Revised to/confirmed in this work
Legionaminic acid (Leg)
Legionella pneumophila
Pseudomonas fluorescens
Vibrio salmonicida
Acinetobacter baumannii
Vibrio alginolyticus
8-Epilegionaminic acid
(8eLeg)
D
D
-glycero-
-glycero-
L
L
-galacto9
L
L
-glycero-
-glycero-
D
D
-galacto
-galacto
D
D
D
D
D
-glycero-
-glycero-
-glycero-
-glycero-
-glycero-
D
D
D
D
D
-galacto
-galacto
-galacto
-galacto
-galacto
-galacto10,11
-galacto8
L
L
-glycero-
-glycero-
D
D
-galacto12
-galacto15
D
-glycero-
L
L
-glycero-
D
-galacto
Pseudomonas aeruginosa
Salmonella arizonae
Yersinia ruckerii
D
D
D
-glycero-
-glycero-
-glycero-
L
L
L
-galacto6
-galacto13
-galacto14
L
L
L
-glycero-
-glycero-
-glycero-
D
D
D
-galacto
-galacto
-galacto
L
L
L
-glycero-
-glycero-
-glycero-
D
D
D
-galacto
-galacto
-galacto
4-Epilegionaminic acid
(4eLeg)
Legionella pneumophila
L
-glycero-
D
-talo16
D-glycero-D-talo

Y.E. Ts6etko6 et al. / Carbohydrate Research 331 (2001) 233–237
235
Scheme 1. i, Bu2SnO, benzene, reflux; ii, BzCl, rt; iii, Tf2O, pyridine, CH2Cl2, 0 °C; iv, Bu4NN3, toluene, 100 °C; v, MeONa,
MeOH, rt; vi, H2, Pd(OH)2-C, MeOH, rt; vii, Ac2O, MeOH, rt; viii, oxalacetic acid, Na2B4O7, pH 10.5, rt.
1
Comparison of the H and 13C NMR data
of synthetic and natural compounds (Table 2)
suggested that 5,7-diacetamido-3,5,7,9-tetra-
deoxynon-2-ulosonic acids from LPS of V.
alginolyticus (Leg5Ac7Ac)15 and L. pneu-
mophila (4eLeg5Ac7Ac)16 have the same rela-
tive configurations as 3 and 4 and differ from
those of 1 and 2, respectively. In addition to
the JH,H coupling constant values, this conclu-
sion was based on the 13C NMR chemical
shifts for C-6 and C-8, which are indicative of
the configuration at the side chain. The spe-
cific optical rotation values of the natural
compounds, [h]D +25.2° (c 0.5, water) for
Leg5Ac7Ac15 and [h]D −3.9° (c 0.2, water)
for 4eLeg5Ac7Ac (this work), compared with
the data for 3 and 4, respectively (see above),
indicated that they have the same absolute
configurations as well. The identity of the
monosaccharide from L. pneumophila was
confirmed by GLC–MS of the acetylated (+)-
2-butyl ester (+)-2-butyl glycosides.

236
Y.E. Ts6etko6 et al. / Carbohydrate Research 331 (2001) 233–237
1
13

Y.E. Ts6etko6 et al. / Carbohydrate Research 331 (2001) 233–237
237
The data obtained enabled also determina-
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We thank Mr H. Moll for expert help with
GLC–MS. This work was supported by grant
436 RUS 113/314/0 of the Deutsche
Forschungsgemeinschaft and grants 99-03-
32955 and 00-04-04009 of the Russian Foun-
dation for Basic Research.
.