Smith degradation of the O-antigenic polysaccharide of Salmonella Dakar: structural studies of the products

Article abstract of DOI:10.1016/j.carres.2008.01.027

Two different oligosaccharides were obtained from the Smith degradation of the O-polysaccharide isolated from the lipopolysaccharide of Salmonella Dakar. The structures of these oligosaccharides were investigated by chemical analysis, NMR spectroscopy and MALDI-TOF mass spectrometry. The following structures of these products were determined: α-d-GalpNAc-(1→4)-α-d-Quip3NAc-(1→3)-α-l-Rha p-(1→2)-threitol and {A figure is presented}. where Quip3NAc is 3-acetamido-3,6-dideoxyglucose. The reaction products confirmed the structure of the repeating unit of the Salmonella Dakar O-polysaccharide reported previously [Kumirska, J.; Szafranek, J.; Czerwicka, M.; Paszkiewicz, M.; Dziadziuszko, H.; Kunikowska, D.; Stepnowski, P. Carbohydr. Res. 2007, 342, 2138-2143].

Full text of DOI:10.1016/j.carres.2008.01.027

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1120–1125
Note
Smith degradation of the O-antigenic polysaccharide
of Salmonella Dakar: structural studies of the products
Jolanta Kumirska,a,* Janusz Szafranek,a Małgorzata Czerwicka,a Marek Gołe˛biowski,a
Monika Paszkiewicz,a Halina Dziadziuszko,b Danuta Kunikowskab and Piotr Stepnowskia
aFaculty of Chemistry, University of Gdan´sk, Sobieskiego 18, 80-952 Gdan´sk, Poland
bDepartment of Molecular Microbiology and Serology, National Salmonella Centre, Medical University of Gdan´sk,
Do Studzienki 38, 80-227 Gdan´sk, Poland
Received 11 October 2007; received in revised form 14 January 2008; accepted 21 January 2008
Available online 2 February 2008
Abstract—Two different oligosaccharides were obtained from the Smith degradation of the O-polysaccharide isolated from the
lipopolysaccharide of Salmonella Dakar. The structures of these oligosaccharides were investigated by chemical analysis, NMR
spectroscopy and MALDI-TOF mass spectrometry. The following structures of these products were determined:
a-D-GalpNAc-(1?4)-a-D-Quip3NAc-(1?3)-a-L-Rhap-(1?2)-threitol and
where Quip3NAc is 3-acetamido-3,6-dideoxyglucose. The reaction products confirmed the structure of the repeating unit of the Sal-
monella Dakar O-polysaccharide reported previously [Kumirska, J.; Szafranek, J.; Czerwicka, M.; Paszkiewicz, M.; Dziadziuszko,
H.; Kunikowska, D.; Stepnowski, P. Carbohydr. Res. 2007, 342, 2138–2143].
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Smith degradation; Salmonella Dakar; O-Polysaccharide; NMR; Mass spectrometry
This paper presents a method for the isolation and full
structural investigation of two products obtained from
the Smith degradation of the O-polysaccharide chain
of the Salmonella Dakar lipopolysaccharide (S. Dakar
OPS); the specific degradation technique most fre-
quently used in structural investigations of polysaccha-
rides.1–3 The periodate oxidation of the OPS was
performed according to the Pritchard and Rener’s pro-
cedure4 to substantiate its assigned structure.5
revealed the presence of ethane-1,2-diol, threitol, rhamn-
itol, 3-acetamido-3,6-dideoxyglucitol, galactosaminitol
and glucitol in a molar ratio of 0.3:1.2:1.0:1.0:1.0:0.2.
The substitution patterns of Rha at C-3, GalNAc at
C-3 and C-4 and Qui3NAc at C-4 protected those sugars
from periodate oxidation. A trace of Glc was probably
derived from the glucosyl residue present in the core
region.
The red.–ox. S. Dakar OPS was mildly hydrolysed.
The reaction products were fractionated by GPC on a
Bio-Gel P-2 column. The isolated oligosaccharide
fraction was additionally separated by HPLC and two
products were obtained; their MALDI mass spectra
are shown in Figures 1 and 2. An additional sugar anal-
ysis of the minor product was also performed.
Sugar analysis of the periodate-oxidised and NaBH4-
reduced S. Dakar OPS (red.–ox. S. Dakar OPS)
*
Corresponding author. Tel.: +48 58 523 54 70; fax: +48 58 523 54
54; e-mail: kumirska@chem.univ.gda.pl
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.01.027

J. Kumirska et al. / Carbohydrate Research 343 (2008) 1120–1125
1121
Figure 1. MALDI mass spectrum of the major product obtained from
the Smith degradation of the Salmonella Dakar O-polysaccharide.
Figure 2. MALDI mass spectrum of the minor product obtained from
the Smith degradation of the Salmonella Dakar O-polysaccharide.
The results of the sugar analysis of the red.–ox. S.
Dakar OPS as well as the m/z values of 681.1
(M1+Na) and 697.1 (M1+K) given by the MALDI spec-
trum of the major product (Fig. 1) revealed the presence
of a trisaccharide built from Rha, GalNAc and Qui3-
NAc and threitol (derived from the degraded Gal resi-
due). The 13C NMR spectrum of this product (Fig. 3)
contained signals for three anomeric carbons, three
HOCH2–C groups, two CH3–C groups, two nitrogen-
bearing carbons, twelve non-anomeric oxygen-bearing
sugar-ring carbons and resonances for two N-acetyl
groups. The absence of signals at 84–88 ppm indicated
76.32) and C-2 of threitol (d 77.77), demonstrated the
glycosylation pattern of these sugar residues.6 The lack
of the down field carbon signals for GalNAc in the
13C NMR spectrum (Table 1) indicated that this sugar
residue was present at the non-reducing end of this tri-
3
saccharide. The JH-1,H-2 coupling constants (4.0 Hz
for GalNAc and 3.6 Hz for Qui3NAc), the chemical
shift of the H-1 protons (d 5.29 for GalNAc, and d
5.06 for Qui3NAc) and the chemical shift of the C-5 sig-
nal of Rha at d 69.51,9,10 confirmed a configurations for
these monosaccharide residues. The monosaccharide se-
quence within the chain was established by NOESY and
HMBC experiments (data not shown). In the NOESY
spectrum, the following inter-residual NOE correlations
were observed: GalNAc H-1, Qui3NAc H-4 at d 5.29/
3.54; Rha H-1, Threitol H-2 at d 5.08/3.81; Qui3NAc
H-1, Rha H-3 at d 5.06/3.87, whereas in the HMBC
spectrum between the following anomeric protons and
linkage carbons: Rha H-1, Threitol C-2 at d 5.08/77.77
1
that all the sugars were in the pyranose form.6 The H
NMR spectrum (Fig. 4) contained three anomeric reso-
nances at almost equal intensities, and signals for two N-
acetyl and two methyl groups. The complete assignment
of the chemical shift spin systems of the sugar residues
and threitol presented in this product (Table 1) was
achieved from the results of 2D NMR experiments,
according to the published methodology.7,8 The chemi-
cal shifts for C-4 of Qui3NAc (d 76.31), C-3 of Rha (d
Figure 3. 13C NMR spectrum (100 MHz) of the major product obtained from the Smith degradation of the Salmonella Dakar O-polysaccharide. The
detailed description of the abbreviations used is presented in Table 1.

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J. Kumirska et al. / Carbohydrate Research 343 (2008) 1120–1125
Figure 4. 1H NMR spectrum (400 MHz) of the major product obtained from the Smith degradation of the Salmonella Dakar O-polysaccharide. The
detailed description of the abbreviations used is presented in Table 1.
Table 1. 1H and 13C NMR chemical shifts (d) for the major product obtained from the Smith degradation of the Salmonella Dakar O-polysaccharide
Sugar 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
a-D-GalpNAc-(1?
?4)-a-D-Quip3NAc-(1?
?3)-a-L-Rhap-(1?
?2)-Threitol
A
B
C
T
5.29
97.17
4.21
49.56
3.86
67.55
4.03
68.66
3.89
71.53
3.85, 3.75
60.28
5.06
95.18
3.60
70.66
4.34
54.13
3.54
76.31
4.23
67.38
1.33
18.24
5.08
99.02
4.22
67.69
3.87
76.32
3.65
70.44
3.89
69.51
1.36
16.96
3.73, 3.69
3.81
4.08
3.78
62.86
77.77
71.84
61.44
1
There are additional chemical shifts for NAc in the H NMR spectrum at d 2.01 and 2. 07 for CH3; also in the 13C NMR spectrum at d 22.40 and
22.57 for CH3, and at d 174.67 and 175.54 for C@O.
and Qui3NAc H-1, Rha C-3 at d 5.06/76.32. Based on
all the data, the major product obtained from the Smith
degradation of the S. Dakar O-polysaccharide has the
following structure:
The 13C NMR spectrum (Fig. 5) of the second prod-
uct of the Smith degradation of S. Dakar OPS contained
two additional signals: one in the anomeric region and
one at d 60.0–78.00 if compared to the 13C NMR spec-
a-d-GalpNAc-ð1!4Þ-a-d-Quip3NAc-ð1!3Þ-a-l-Rhap-ð1!2Þ-threitol
Analogous structural studies were performed on the
second product (23.4%) obtained from the Smith degra-
dation of the S. Dakar O-polysaccharide. The MALDI
spectrum of this compound (Fig. 2) showed two major
ions: with m/z 723.1 (M2+Na) and with m/z 739.1
(M2+K), whereas the sugar analysis of this oligosaccha-
ride revealed the presence of ethane-1,2-diol, threitol,
rhamnitol, 3-acetamido-3,6-dideoxyglucitol and N-
acetyl-galactosaminitol, in the ratios 0.3:1.0:1.1:0.9:1.0,
respectively.
trum of the previous product. The absence of signals at
84–88 ppm (Fig. 5) again indicated six-membered rings
in all the monosaccharides.6 In the anomeric region of
1
the H NMR spectrum (Fig. 6), one additional reso-
nance at d 4.92 was also shown; the TOCSY spectrum
(data not shown) indicated that it was correlated only
with the resonance at d 3.66. The 1D NMR data and
2D NMR experiments enabled the complete chemical
shift spin systems of three monosaccharide residues—
GalNAc, Qui3NAc and Rha (Table 2). The TOCSY

J. Kumirska et al. / Carbohydrate Research 343 (2008) 1120–1125
1123
Figure 5. 13C NMR spectrum (100 MHz) of the minor product obtained from the Smith degradation of the Salmonella Dakar O-polysaccharide. The
detailed description of the abbreviations used is presented in Table 2.
Figure 6. 1H NMR spectrum (400 MHz) of the minor product obtained from the Smith degradation of the Salmonella Dakar O-polysaccharide. The
detailed description of the abbreviations used is presented in Table 2.
Table 2. 1H and 13C NMR chemical shifts (d) for the minor product obtained from the Smith degradation of the Salmonella Dakar O-polysaccharide
Sugar 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
a-D-GalpNAc-(1?
?4)-a-Quip3NAc-(1?
?3)-a-L-Rhap-(1?
A
B
C
5.29
97.16
4.21
49.55
3.88
67.36
4.03
68.66
4.08
71.82
3.76
61.42
5.09
95.28
3.60
70.63
4.34
54.12
3.55
76.28
4.24
67.54
1.33
18.24
5.06
4.22
3.86
3.65
3.72
1.35
96.33
67.59
76.49
70.32
69.79
16.95
3.96, 4.36
(T1a), (T1b
66.36
4.16
(T2)
79.14
3.84
(T3)
67.03
3.79
(T4)
61.62
4.92
(D1)
100.95
3.66
(D2)
62.32
)
1
There are additional chemical shifts for NAc in the H NMR spectrum at d 2.00 and 2.07 for CH3; also in the 13C NMR spectrum at d 22.39 and
22.56 for CH3, and at d 174.66 and 175.53 for C@O.
spectrum contained also a signal at d 3.84 that was cor-
related with the resonances at d 4.36, 4.16 and 3.79.
Moreover, in the TOCSY spectrum, there were correla-
tions from the proton at d 4.36 to the protons at d 3.96
and 3.84. Identification of threitol and ethane-1,2-diol in
sugar analysis and 2D NMR experiments clearly indi-
cated that the minor product of the Smith degradation
of S. Dakar OPS contained 1,3-O-(20-hydroxyethylid-
ene)threitol formed from the degraded Gal residue
(Table 2). In the HMBC spectrum (data not shown), in-
tra-residual correlations between H-1b of threitol (T1b)
and the carbons C-2 (T2), C-3 (T3) and C-10 of the 20-
hydroxyethylidene group (D1) at d 4.36/79.14, 4.36/
67.03 and 4.36/100.95, respectively were observed. In
addition, the cross-peak H-10 (D1), C-2 (T2) at d 4.92/
79.14 was also shown. The 13C NMR data (Table 2)
revealed down field signals at d 76.28, 76.49 and 79.14
for C-4 of Qui3NAc, C-3 of Rha and C-2 (T2) of

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J. Kumirska et al. / Carbohydrate Research 343 (2008) 1120–1125
1,3-O-(20-hydroxyethylidene)threitol, respectively, dem-
onstrating the substitution pattern of these sugar
residues and the cyclic acetal in this compound.6 The
lack of the down field carbon signals for GalNAc in
the 13C NMR spectrum (Table 2) indicated the terminal
position of this sugar residue in the oligosaccharide
chain. The a anomeric configuration of all residues were
also established. The sequence of the sugar residues in
this oligosaccharide was determined by NOESY and
HMBC experiments. In addition, in the HMBC spec-
trum, the correlation between the H-1 of Rha and C-2
(T2) of 1,3-O-(20-hydroxyethylidene)threitol at d 5.06/
79.14 was also observed. Therefore, the minor product
obtained from the Smith degradation of the S. Dakar
O-polysaccharide has the following structure:
5 min. The GLC–MS analysis was carried out on a
TRIO-2000 instrument (VG Biotech, UK) with an elec-
tron impact ionisation energy of 70 eV, coupled with a
Hewlett–Packard 5890 gas chromatograph using the
same capillary column and the same temperature pro-
gramme. For sugar identifications, GLC analyses with
standard co-injections and mass spectra were employed.
The red.–ox. S. Dakar OPS (16.6 mg) was hydrolysed
with aq 2% HOAc (100 °C, 2 h). The oligosaccha-
ride fraction was isolated by GPC on a Bio-Gel P-2
(200–400 mesh, BioRad, Richmond, USA) column
(100 ꢁ 0.9 cm) in pyridine–acetic acid buffer (pyridine–
HOAc–water, 2:5:493, v/v/v) with flow rate 3.5 mL hꢀ1
,
and then rechromatographed by HPLC on a semi-
preparative LiChrosorb—100 NH2 column (300
This cyclic acetal is probably formed during the mild
acid hydrolysis of the red.–ox. S. Dakar OPS, wherein
the hydrated hydroxyethanal moiety (from C-1 and C-
2 of reduced and oxidised Gal residue) can appear and
its non-hydrated form undergoes acid-catalysed transac-
etalation to give this product. The mechanism of this
reaction was presented by Aspinall,3 and, for example,
the formation of the hydrated form of glyceraldehyde
moiety from 2-substituted periodate-oxidised and
NaBH4-reduced Rha was observed by Perepelov et al.11
mm ꢁ 8 mm, 5 lm, Knauer) using CH3CN–H2O
(70:30, v/v) as eluent at a flow rate of 1 mL minꢀ1
.
The chromatograph was equipped with a differential
refractometric detector (RIDK 102, Prague, Czech
Republic). As a result, this fraction was separated into
two products (23.4% and 52.1% of the oligosaccharide
fraction, respectively); sugar analysis of the minor prod-
uct was performed. Both the products were subjected to
NMR and MALDI mass spectrometric studies accord-
ing to the described conditions.5 The mass spectra were
carried out from m/z 200 to 1000 and were reported as
the results of 60–180 laser shots. The NMR spectra for
both samples were recorded with a Varian Mercury
400 MHz spectrometer in D2O solutions at 30 °C. The
mixing times of 110 ms and 300 ms were used in the
TOCSY and NOESY experiments, respectively.
1. Experimental
The S. Dakar strain, growth conditions, isolation of the
lipopolysaccharide and the separation of the O-polysac-
charide chain from LPS were described in the previous
paper.5 The polysaccharide (24.1 mg) was oxidised with
40 mM NaIO4 (3.6 mL) (Lancaster, Germany) in the
dark at 4 °C for 120 h according to the mentioned
procedure.4 The reaction was terminated with ethylene
glycol (0.5 mL), and the product was dialysed against
distilled water (Spectra-Por 1000 Da, Spectrum Labora-
tories Inc., USA), then lyophilised. After reduction and
neutralisation of the excess of NaBH4 with aq 50%
HOAc, the product was dialysed again and lyophilised
(17.1 mg of the product was obtained). A portion of
0.5 mg was subjected to sugar analysis5 and analysed
by GLC and GLC–MS. The GLC analysis was
performed on a GC 8000 TOP (CE Instruments) gas
chromatograph equipped with a capillary column Sol–
Gel (30 m, 0.25 mm I.D., 0.25 lm film thickness, SGE)
using a linear temperature programme from 100 °C to
260 °C at 4 °C minꢀ1, then isothermally at 260 °C for
Acknowledgement
Financial support was provided by the Polish Ministry
of Research and Higher Education in the form of
Grants BW/8000-5-0392-7 and DS/8200-4-0085-7.
References
1. Lindberg, B.; Lo¨nngren, J.; Svensson, S. Adv. Carbohydr.
Chem. Biochem. 1975, 31, 185–240.
2. Rauvala, H.; Finne, J.; Krusius, T.; Ka¨rkka¨inen, J.;
Ja¨rnefelt, J. Adv. Carbohydr. Chem. Biochem. 1981, 38,
389–416.
3. Aspinall, G. O. Chemical Characterization and Structure
Determination of Polysaccharide. In The Polysaccharides;
Aspinall, G. O., Ed.; Academic Press: New York, London,
1984; Vol. 1, pp 35–131.

J. Kumirska et al. / Carbohydrate Research 343 (2008) 1120–1125
1125
4. Pritchard, D. G.; Rener, B. P. Carbohydr. Res. 1988, 173,
255–262.
8. Duus, J. Ø.; Gotfredsen, C. H.; Bock, K. Chem. Rev. 2000,
100, 4589–4614.
5. Kumirska, J.; Szafranek, J.; Czerwicka, M.; Paszkiewicz,
M.; Dziadziuszko, H.; Kunikowska, D.; Stepnowski, P.
Carbohydr. Res. 2007, 342, 2138–2143.
6. Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem.
1983, 41, 27–66.
9. Brandbury, H. J.; Jenkins, G. A. Carbohydr. Res. 1984,
126, 125–156.
10. Jannson, P.-E.; Kenne, L.; Wildmalm, G. Carbohydr. Res.
1989, 188, 169–191.
11. Perepelov, A. V.; Han, W.; Senchenkova, S. N.; Shevelev,
S. D.; Shashkov, A. S.; Feng, L.; Liu, Y.; Knirel, Y. A.;
Wang, L. Carbohydr. Res. 2007, 342, 648–652.
7. Hounsell, E. F. Prog. Nucl. Magn. Reson. Spectrosc. 1995,
27, 445–474.