Structure of the O-polysaccharide of Providencia alcalifaciens O2 containing ascarylose and N-(l-alanyl)-d-glucosamine

Article abstract of DOI:10.1016/j.carres.2014.10.016

The O-polysaccharide was obtained by degradation of the lipopolysaccharide of Providencia alcalifaciens O2 under mild acidic conditions followed by GPC. The polysaccharide was found to contain two unusual components: 3,6-dideoxy-l-arabino-hexose (ascarylo

Full text of DOI:10.1016/j.carres.2014.10.016

Carbohydrate Research 401 (2015) 11–15
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Carbohydrate Research
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Note
Structure of the O-polysaccharide of Providencia alcalifaciens O2
containing ascarylose and N-(L-alanyl)-D-glucosamine
Olga G. Ovchinnikova a, , Magdalena Moryl b, Alexander S. Shashkov a, Alexander O. Chizhov a,
Nikolay P. Arbatsky a, Anna M. Shpirt a, Antoni Rozalski b, Yuriy A. Knirel a,
⇑
a N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation
b Department of Immunobiology of Bacteria, Institute of Microbiology, Biotechnology and Immunology, University of Lodz, PL 90-237 Lodz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 August 2014
Received in revised form 17 October 2014
Accepted 20 October 2014
Available online 27 October 2014
The O-polysaccharide was obtained by degradation of the lipopolysaccharide of Providencia alcalifaciens
O2 under mild acidic conditions followed by GPC. The polysaccharide was found to contain two unusual
components: 3,6-dideoxy-L-arabino-hexose (ascarylose, Asc) and 2-(L-alanyl)amino-2-deoxy-D-glucose
(GlcNAla). Ascarylose was partially split off during lipopolysaccharide degradation and could be elimi-
nated completely by selective acid hydrolysis, which also partially cleaved the b-GalNAc-(1?6) linkage.
The following structure of the branched pentasaccharide repeating unit was established by 1H and 13C
NMR spectroscopy of the O-polysaccharide and O-deacetylated polysaccharide, as well as products of
partial acid hydrolysis:
Keywords:
Providencia alcalifaciens
O-Antigen
Lipopolysaccharide
Bacterial polysaccharide structure
Ascarylose
Ó 2014 Elsevier Ltd. All rights reserved.
Bacteria of the genus Providencia are widespread in nature and
oligosaccharide fractions (IIa and IIb). Sugar analysis using GLC of
the alditol acetates derived after full acid hydrolysis of PS-1 revealed
may cause urinary tract infections and enteric diseases in humans.1
A
combined O-antigen (O-polysaccharide)-based serotyping
ascarylose (3,6-dideoxy-L-arabino-hexose), glucose, GlcN, and
scheme for medically important Providencia species, Providencia
alcalifaciens, Providencia stuartii and Providencia rustigianii, consists
of 63 O-serogroups.2 Aiming at elaboration of the chemical basis
for this classification, O-polysaccharide structures have been
established for 38 O-serogroups.3–5 In this work, we elucidated
the structure of the O-polysaccharide of the lipopolysaccharide
(LPS) of P. alcalifaciens O2.
The LPS was isolated from bacterial cells by the phenol–water
procedure6 and degraded with aq 2% acetic acid. Fractionation of
the carbohydrate portion by GPC on Sephadex G-50 Superfine
resulted in an O-polysaccharide fraction (I) (PS-1) and two
GalN. GLC of the acetylated methyl glycosides showed the presence
of glucuronic acid (GlcA). Further studies showed that the
O-polysaccharide did not contain glucose, which was evidently a
component of the LPS core. The L configuration of the 3,6-dideoxy-
hexose and the
D configuration of the other monosaccharides were
determined by GLC of the acetylated (S)-2-octyl glycosides7 and
were in agreement with the glycosylation effects on 13C NMR
chemical shifts.8 In addition to the monosaccharides,
L-alanine
was identified by GLC of the acetylated ester with (+)-2-octanol.
The 13C NMR spectrum of PS-1 (Fig. 1, top) showed a structural
heterogeneity, likely owing to non-stoichiometric O-acetylation
(there was a signal for an O-acetyl group at d 21.9). Mild alkaline
treatment of PS-1 resulted in an O-deacetylated polysaccharide
(PS-2), which was still irregular (Fig. 1, bottom) owing to partial
loss of ascarylose during mild acid degradation of the LPS (see
⇑
Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328.
E-mail address: yknirel@gmail.com (Y.A. Knirel).
Present address: Science Complex, University of Guelph, Guelph, Ontario N1G
2W1, Canada.
below). In addition, the sample was contaminated with
a
http://dx.doi.org/10.1016/j.carres.2014.10.016
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

12
O.G. Ovchinnikova et al. / Carbohydrate Research 401 (2015) 11–15
Figure 1. 13C NMR spectra of the O-polysaccharide (PS-1) (top) and O-deacetylated polysaccharide (PS-2) (bottom) from P. alcalifaciens O2. Arabic numerals refer to carbons
in alanine and sugar residues denoted by letters as follows: A, Asc; Gl, GlcN; Ga, GalN; aG,
with asterisk.
a
-GlcA; bG, b-GlcA. Signals for the peptidoglycan-like polysaccharide8 are marked
peptidoglycan-like polysaccharide reported earlier to be common
for a number of Providencia serogroups.9 In an attempt to obtain
a regular Asc-lacking polymer, PS-2 was subjected to partial acid
hydrolysis with 0.1 M CF3CO2H, and the products were fraction-
ated by GPC on TSK HW-40 (S) to give fractions I-V.
hexamer and octamer with dominant dodecasaccharide and eico-
sasaccharide, respectively (data not shown).
As compared with the 13C NMR spectrum of TS, in the spectrum
of the fraction I polysaccharide (PS-3) the signal for C-6 of GlcNAla
shifted downfield from d 61.8 to d 69.0. In the 1H, 13C HMBC
spectrum of PS-3, this signal showed a correlation with H-1 of
b-GalNAc, thus indicating the GalpNAc-(b1?6)-GlcpNAla linkage.
These data suggested that the repeat of PS-3 corresponded to TS
and had the structure shown in Chart 1.
The 1H and 13C NMR spectra of fractions I-V were assigned using
2D COSY, TOCSY, ROESY, 1H, 13C HSQC, HSQC-TOCSY, and HMBC
experiments, and the NMR chemical shifts for fractions I, IV, and
V are tabulated in Table 1. Fraction V was identified as free
ascarylose.
Therefore, partial acid hydrolysis of PS-2 completely split off
ascarylose, the terminal monosaccharide residue of a polysaccha-
ride side chain, and selectively cleaved some GalNAc linkages in
the main chain.
As compared with the NMR spectra of PS-3, the spectra of PS-2
showed additional signals for an ascarylose residue. A comparison
with the 1H and 13C NMR data of the free monosaccharide (Table 1)
Fraction IV was a tetrasaccharide (TS) composed of one linked
residue each of
a-GlcpA, b-GlcpA, and b-GlcpN, a GalN residue at
the reducing end, as well as one alanyl and one N-acetyl group.
The 1H, 13C HMBC spectrum of TS showed a cross-peak between
C-1 (CO) of Ala and H-2 of GlcN at d 172.7/3.81, thus demonstrating
N-alanylglucosamine (GlcNAla); hence, GalN is N-acetylated. The
HMBC spectrum also showed the following correlations between
indicated that ascarylose occurs as the
attachment of Asc was inferred by a downfield displacement of
the signal for C-4 of -GlcA from d 73.1 in PS-3 to d 80.1 in PS-2
and the presence of a Asc C-1/ -GlcA H-4 cross-peak at d 100.9/
3.62 in the 1H,13C HMBC spectrum of PS-2. Therefore, Asc is
attached to -GlcA at position 4, and PS-2 has the structure shown
a-pyranoside. The site of
anomeric protons and linkage carbons and vice versa:
a-GlcA
H-1/GlcNAla C-4, GlcNAla H-1/b-GlcA C-4, b-GlcA H-1/
a- and
a
b-GalNAc C-3,
a-GlcA C-1/GlcNAla H-4, GlcNAla C-1/b-GlcA H-4,
a
and b-GlcA C-1/
a- and b-GalNAc H-3. These data showed that TS
is linear and has the structure shown in Chart 1, which was con-
firmed by low-field positions of the signals for linkage carbons
(Table 1) and a ROESY experiment (data not shown).
The structure of TS was also corroborated by ESI MS; the nega-
tive ion mass spectrum showed the [MÀH]À ion peak at m/z
804.2532 (calculated m/z 804.2528 for C29H46N3OÀ23). Similar MS
analysis showed that fraction III represented an octasaccharide
consisting of two TS repeats ([MÀH]À: experimental m/z
1591.4994; calculated m/z 1591.5022 for C58H91N6OÀ45). Fractions
II and I were found to be mixtures of higher oligomers up to
a
in Chart 1. In addition to the signals for PS-2, the NMR spectra of
fraction I showed signals for PS-3, which was evidently due to par-
tial loss of ascarylose during mild acid degradation of the LPS.
A comparison of the 1H, 13C HSQC spectra of PS-2 and PS-1
showed a partial displacement of the GlcNAla H-3/C-3 cross-peak
from d 3.74/74.3 in PS-2 to d 5.19/76.2 in PS-1, which was
evidently due to a deshielding effect of the O-acetyl group10 and
indicated O-acetylation of GlcNAla at position 3 (Chart 1). This con-
clusion was confirmed by upfield displacements (b-effects of

O.G. Ovchinnikova et al. / Carbohydrate Research 401 (2015) 11–15
13
Table 1
1H and 13C NMR chemical shifts (d, ppm) and JH,H coupling constants (Hz)
3
Residue
C-1
H-1
(J1,2
C-2
H-2
(J2,3ax
C-3
C-4
H-4
(J3ax,4
C-5
H-5
(J4,5
C-6
H-6 (6a,6b)
(J5,6)
H-3 (3equiv, 3ax)
(J2,3equiv; J3equiv,4; J3equiv,3ax
)
)
)
)
)
O-Deacetylated polysaccharide (PS-2)
?3)-b-
D
-GalpNAc-(1?
102.9
4.41
105.6
4.56
103.1
4.52
102.4
5.18
52.4
4.05
73.7
3.34
56.2
3.76
73.1
3.67
68.9
3.94
81.8
3.80
75.3
3.62
74.3
3.74
73.0
3.73
34.6
1.89, 2.02
69.2
4.09
83.1
3.65
80.4
3.63
80.1
3.62
68.1
3.58
76.1
3.63
76.8
3.82
74.8
3.78
73.6
4.12
71.5
3.99
62.3
3.72, 3.79
175.0
?4)-b-
D
-GlcpA-(1?
?4,6)-b-
?4)- -GlcpA-(1?
-Ascp-(1?
Asc-lacking polysaccharide (PS-3)
D-GlcpNAla-(1?
68.9
3.75, 4.21
175.7
a-
D
a
100.9
4.57
18.0
1.21
?3)-b-
D
-GalpNAc-(1?
103.0
4.40
105.6
4.55
103.2
4.50
102.3
5.17
52.3
4.06
73.8
3.33
56.2
3.75
73.0
3.64
82.0
3.79
75.3
3.60
74.9
3.77
74.2
3.68
69.2
4.10
83.4
3.61
80.1
3.63
73.1
3.50
76.1
3.65
77.5
3.72
74.3
3.72
74.7
3.99
62.3
3.73, 3.78
176.1
?4)-b-
D
-GlcpA-(1?
?4,6)-b-
D-GlcpNAla-(1?
69.0
3.74, 4.22
177.4
a-
D-GlcpA-(1?
Tetrasaccharide (TS)
?3)- -GalpNAc
a-
D
92.5
5.20
96.4
4.67
105.6,
105.4
4.56,
4.50
103.1
4.54,
4.53
50.1
4.27
53.6
3.98
73.6,
73.7
3.36
78.7
3.98
81.7
3.80
75.4
69.8
4.17
69.1
4.11
83.6
71.6
4.10
76.2
3.68
77.5
62.5
3.72
62.3
3.74
176.2
?3)-b-
D
D
-GalpNAc
?4)-b-
-GlcpA-(1?
3.62
3.65,
3.68
78.0
3.71
3.70
?4)-b-
D
-GlcpNAla-(1?
56.4
3.81
75.2
3.81
75.9
3.67
61.8
3.79, 3.93
a-
D-GlcpA-(1?
100.7
5.42
72.7
3.60
73.9
3.68
73.1
3.49
74.3
3.97
177.6
Ascarylose
-Ascp
a
93.93
4.958
(1.4)
95.84
4.866
(1.1)
103.30
5.270
(<1)
101.01
5.209
(4.4)
69.31
3.918
(3.2)
69.19
3.934
(3.6)
76.44
4.205
(n.d.)
72.27
4.273
(9.9)
34.11
68.35
3.610
(11.1)
68.08
3.565
(11.6)
83.13
4.192
(8.2)
70.93
3.862
(9.1)
77.18
3.482
(9.3)
69.31
3.936
(5.5)
70.19
3.896
(n.d.)
18.10
1.252
(6.3)
18.39
1.276
(6.2)
19.39
1.174
(6.6)
19.03
1.169
(6.4)
2.049, 1.856
(4.3; 4.3, 13.6)
38.03
2.201, 1.706
(4.3; 4.3; 13.9)
32.66
2.389, 1.838
(6.4; 8.2; 14.0)
31.36
2.299, 1.821
(7.4; 6.9; 12.3)
b-Ascp
a-Ascf
b-Ascf
81.96
3.950
(8.9)
1H NMR chemical shifts are shown in italics.
n.d., not determined.
Additional chemical shifts for the N-alanyl group are dC 17.7–18.0 (C-3), 50.9 (C-2), and 172.6–172.7 (C-1), dH 1.52–1.53 (H-3) and 4.04–4.05 (H-2); for the N-acetyl group dC
23.7–23.8 (Me) and 176.0 (CO), dH 2.04–2.05 in the polysaccharides; dC 23.3 and 23.5 (both Me), 176.0 and 176.1 (both CO), dH 2.01 in TS; for the O-acetyl group dC 21.9 (Me)
and 174.7 (CO), dH 2.08 in PS-1.
Chart 1. Structures of the O-polysaccharide (PS-1), O-deacetylated polysaccharide (PS-2), Asc-lacking polysaccharide (PS-3), and tetrasaccharide (TS) from P. alcalifaciens O2.
In PS-1 and PS-2, ascarylose is present in a non-stoichiometric amount owing to its partial cleavage during mild acid degradation of the LPS. The degree of O-acetylation in
PS-1 is ꢀ60%.

14
O.G. Ovchinnikova et al. / Carbohydrate Research 401 (2015) 11–15
Figure 2. Parts of charge-deconvoluted negative ion ESI mass spectra of fractions IIa (bottom) and IIb (top) isolated from the degraded LPS. Shown m/z values refer to [MÀH]À
ions.
O-acetylation9) of the signals for the neighboring carbons C-2 and
C-4 from d 56.2 and 80.4 in PS-2 to d 55.3 and 76.3 in PS-1, respec-
tively. As judged by the ratio of integral intensities of the signals
for OAc and NAc groups in the 1H NMR spectra of PS-1, the degree
of O-acetylation was ꢀ60%.
mine has been hitherto reported only once as a component of
the O-polysaccharide of Proteus penneri 25 (O69)15, a representa-
tive of a genus closely related to Providencia.
1. Experimental
Therefore, the O-polysaccharide of P. alcalifaciens O2 has the oli-
gosaccharide repeat (O-unit) with the structure shown in Chart 1.
This structure was confirmed by negative ion ESI MS analysis of
fractions IIa and IIb isolated from the degraded LPS. The
charge-deconvoluted spectrum of IIb (Fig. 2, top) showed the
[MÀH]À ion peak at m/z 2199.5341 for a Hex4GalA1Hep3Ara4N1-
anhKdo1P1PEtN3 core oligosaccharide (calculated m/z 2199.5347
for C70H123N4O66PÀ4 ), which has been reported earlier in the LPS
of P. alcalifaciens O19 and O21.11 In the spectrum of IIa (Fig. 2,
bottom), the major [MÀH]À ion peak at m/z 3158.8583 belonged
evidently to the core bearing one O-unit, the O-unit having thus
the molecular mass of 959.3242 Da (calculated m/z 3158.8578 for
1.1. Bacterial strain and isolation of the lipopolysaccharide
Providencia alcalifaciens O2:H2 strain 67002 obtained from the
Hungarian National Collection of Medical Bacteria (National Insti-
tute of Hygiene, Budapest) was cultivated under aerobic conditions
in tryptic soy broth supplemented with 0.6% yeast extract. The bac-
terial mass was harvested at the end of the logarithmic growth
phase, centrifuged, washed with distilled water, and lyophilized.
LPS was isolated in a yield of ꢀ6.2% of dry bacterial mass by
phenol–water extraction6 followed by dialysis of the extract with-
out layer separation and freed from insoluble contaminations by
centrifugation. The resultant solution was treated with cold
(4 °C) aq 50% CCl3CO2H; after centrifugation the supernatant was
dialyzed against distilled water and freeze-dried.
C
107H180N7O92PÀ4 ; calculated molecular mass 959.3230 Da for
Asc1GlcA2GalNAc1GlcNAla1Ac1-H2O). Both mass spectra also
showed ion peaks for compounds lacking one and two PEtN groups
(D
m ca. 123 Da) and that of Ia for compounds lacking Asc and/or
O-acetyl group ( m ca. 130 and ca. 42 Da, respectively).
A peculiar feature of the O-polysaccharide of P. alcalifaciens O2
is the presence of ascarylose and N-( -alanyl)- -glucosamine.
D
1.2. Preparation of the O-polysaccharide
L
D
A portion of the LPS (445 mg) was heated with 2% HOAc (9 mL)
for 2.5 h at 100 °C, a lipid precipitate was removed by centrifuga-
tion at 13,000g for 20 min. The carbohydrate-containing superna-
tant was fractionated on a column (60 Â 2.5 cm) of Sephadex G-
Ascarylose has been known as a component of a few other bacterial
polysaccharides, including Yersinia pseudotuberculosis 5a12,13 and
Vibrio cholerae O:3.14 To our knowledge, N-(
L-alanyl)-D-glucosa-

O.G. Ovchinnikova et al. / Carbohydrate Research 401 (2015) 11–15
15
50 Superfine in 0.05 M pyridinium acetate buffer pH 4.5 to give one
polysaccharide (PS-1) and two oligosaccharide fractions in yields
20.6, 31.0, and 108.9 mg, respectively.
long-range couplings to optimize the 2D 1H,13C HMBC experiment
for coupling constant JH,C 8 Hz.
1.6. Mass spectrometry
1.3. O-Deacetylation and partial acid hydrolysis
High-resolution ESI MS was performed in the negative ion mode
using a maXis instrument (Bruker Daltonics). Oligosaccharide sam-
PS-1 was subjected to O-deacetylation with 12% aqueous
ammonia (0.5 mL, 37 °C, 16 h). Ammonia was removed with a
stream of nitrogen at 20 °C, and the residual solution was lyophi-
lized to give O-deacetylated polysaccharide (PS-2).
ples (ca. 50 ng
trile mixture and injected with a syringe at a flow rate of
L minÀ1. Capillary entrance voltage was set at 3 kV and shield
l
LÀ1) were dissolved in a 1:1 (v/v) water–acetoni-
3
l
PS-2 (18.4 mg) was hydrolyzed with 0.1 M CF3CO2H (1 mL) at
100 °C for 2 h. Following acid evaporation the residue was fraction-
ated by GPC on a column (80 Â 1.6 cm) of TSK HW-40 (S) in 1%
HOAc to give fractions I (PS-3), II, III, IV (TS), and V (Asc) in yields
3.3, 4.1, 3.6, 2.0, and 1.0 mg, respectively
voltage at À500 V. Nitrogen was used as the drying gas, and the
interface temperature was set at 180 °C. Internal calibration was
done with ESI Calibrant Solution (Agilent).
Acknowledgments
1.4. Composition analysis
This work was supported by the Ministry of Science and Higher
Education, Poland (core funding for statutory research activity,
grant 1132 from the Department of Immunobiology of Bacteria,
University of Lodz).
A PS-1 sample was subjected to hydrolysis with 0.5 M CF3CO2H
(100 °C, 30 min) or 2 M CF3CO2H (120 °C, 2 h) followed by reduc-
tion with a solution of NaBH4 (0.4 mL, 10 mg mLÀ1) in 1 M NH4OH
(20 °C, 2 h), or to methanolysis (1 mL MeOH, 0.1 mL AcCl, 85 °C,
16 h). The products were acetylated with a 1:1 Ac2O-pyridine mix-
ture (100 °C, 1 h) and analyzed by GLC using a Maestro (Agilent)
7820 GC (Interlab, Russia) equipped with a HP-5 ms column and
a temperature gradient of 7 °C minÀ1 from 160 to 290 °C. For deter-
mination of the absolute configurations of the monosaccharides, a
PS-1 sample was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h) and
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were obtained using standard Bruker software, and Bruker TopSpin
2.1 program was used to acquire and process the NMR data. A mix-
ing time of 150 and 200 ms was used in TOCSY and ROESY exper-
iments, respectively. A 60-ms delay was used for evolution of