Structural characterization of the core oligosaccharide isolated from the lipopolysaccharide of the psychrophilic bacterium Colwellia psychrerythraea strain 34H

Article abstract of DOI:10.1002/ejoc.201300005

Cold-adapted bacteria are microorganisms that thrive at very low temperatures in permanently cold environments (0-10 °C). Their ability to survive under these harsh conditions is the result of molecular evolution and adaptations, which include the structural modification of the phospholipid membrane. To give insight into the role of the membrane in the mechanisms of adaptation to low temperature, the characterization of other cell-wall components is necessary. Among these components, the lipopolysaccharides are complex amphiphilic macromolecules embedded in the outer leaflet of the external membrane, of which they are the major constituents. The cold-adapted Colwellia psychrerythraea 34H bacterium, living in deep sea and Arctic and Antarctic sea ice, was cultivated at 4 °C. The lipooligosaccharide (LOS) was isolated and analysed by means of chemical analysis. Then it was degraded either by mild hydrazinolysis (O-deacylation) or hot KOH (4 M; N-deacylation). Both products were investigated in detail by ¹H and ¹³C NMR spectroscopy and by ESI FT-ICR mass spectrometry. The oligosaccharide portion consists of a unique and very short species with the following general structure: α-L-Col-(1→2)-α-D-GalA-(1→2)-α-D-Man-[3-P-D-Gro] -(1→5)-α-D-Kdo-4-P-Lipid-A. The structural characterization of the lipooligosaccharide from the steno-psychrophilic bacterium Colwellia psychrerythraea strain 34H has been achieved by means of chemical analysis, mass spectrometry, and NMR spectroscopy experiments. The data revealed a very short, negatively charged, and unique oligosaccharide, lacking heptose residues. Copyright

Full text of DOI:10.1002/ejoc.201300005

FULL PAPER
DOI: 10.1002/ejoc.201300005
Structural Characterization of the Core Oligosaccharide Isolated from the
Lipopolysaccharide of the Psychrophilic Bacterium Colwellia psychrerythraea
Strain 34H
Sara Carillo,[a] Giuseppina Pieretti,[a] Buko Lindner,[b] Ermenegilda Parrilli,[a]
Sannino Filomena,[a] Maria Luisa Tutino,[a] Rosa Lanzetta,[a] Michelangelo Parrilli,[a] and
Maria Michela Corsaro*[a]
Dedicated to the memory of Ernesto Fattorusso
Keywords: Bacteria / Lipopolysaccharides / Carbohydrates / Oligosaccharides / Structure elucidation / NMR spectroscopy
Cold-adapted bacteria are microorganisms that thrive at very
low temperatures in permanently cold environments (0–
10 °C). Their ability to survive under these harsh conditions
is the result of molecular evolution and adaptations, which
include the structural modification of the phospholipid mem-
brane. To give insight into the role of the membrane in the
mechanisms of adaptation to low temperature, the charac-
terization of other cell-wall components is necessary. Among
these components, the lipopolysaccharides are complex am-
phiphilic macromolecules embedded in the outer leaflet of
the external membrane, of which they are the major constitu-
ents. The cold-adapted Colwellia psychrerythraea 34H bac-
terium, living in deep sea and Arctic and Antarctic sea ice,
was cultivated at 4 °C. The lipooligosaccharide (LOS) was
isolated and analysed by means of chemical analysis. Then
it was degraded either by mild hydrazinolysis (O-deacyl-
ation) or hot KOH (4
M; N-deacylation). Both products were
investigated in detail by 1H and 13C NMR spectroscopy and
by ESI FT-ICR mass spectrometry. The oligosaccharide por-
tion consists of a unique and very short species with the fol-
lowing general structure: α- -Col-(1Ǟ2)-α-
L
D-GalA-(1Ǟ2)-α-
D
-Man-[3-P- -Gro]-(1Ǟ5)-α-D-Kdo-4-P-Lipid-A.
D
have the ability to grow at temperatures below 15 °C, but
have maximum growth rates at temperature optima above
18 °C.[3,4] To survive extremes of pH, temperature, and sa-
linity, microorganisms have been found to develop unique
defenses against their environment, leading to the biosyn-
thesis of unusual molecules ranging from simple osmolytes
to complex secondary metabolites. In addition, the cell en-
velope shows adaptive changes in the face of the extreme
environmental conditions, particularly in its lipid composi-
tion.
Microorganisms that thrive in permanently or seasonally
very cold habitats solve the “freezing risk” by adopting
heterogeneous strategies, such as the maintenance of mem-
brane fluidity by increasing the synthesis of unsaturated
fatty acids, and by changing the chain length of the fatty
acids,[5–7] and also the quality and quantity of phosphoryl-
ation.[8,9] The outer membrane of Gram-negative bacteria
forms a barrier for the cell, and it is made up of phospho-
lipids, outer-membrane proteins (OMP), and lipopolysac-
charides (LPS). The lipopolysaccharides are complex am-
phiphilic macromolecules embedded in the outer leaflet of
the external membrane of which they are the major con-
stituents. Smooth-form lipopolysaccharides (S-LPS) consist
of three covalently linked regions, the glycolipid lipid A,
Introduction
Microorganisms can thrive in what we call extreme envi-
ronments on Earth. Macelroy named these lovers (“philos”
to Greeks) of extreme environments “extremophiles”.[1]
They had to adapt to one or several extreme physicochemi-
cal parameters: thermophiles and hyperthermophiles live
above 60 °C near geysers and hydrothermal vents; halo-
philes thrive in hyper-saline environments; alkaliphiles pre-
fer high pH; and acidophiles thrive at low pH.[2] Cold-
adapted microorganisms include both steno-psychrophilic
(formerly “true psychrophile”) and eury-psychrophilic (for-
merly “psychrotolerant” or “psychrotrophic”) organisms.
The former show an optimal growth temperature of 15 °C,
and a maximum temperature for growth of 20 °C; the latter
[a] Dipartimento di Scienze Chimiche, Università degli Studi di
Napoli Federico II, Complesso Universitario Monte S. Angelo,
Via Cintia 4, 80126 Napoli, Italy
Fax: +39-081-674393
E-mail: corsaro@unina.it
Homepage: http://www.unina.it
[b] Division of Immunochemistry, Research Center Borstel,
Leibniz-Center for Medicine and Biosciences
Parkallee 10, 23845 Borstel, Germany
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300005.
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also known as the endotoxin for human pathogens, the oli-
gosaccharide region (core region), and the O-specific poly-
saccharide (O-chain, O-antigen). Rough-form lipopolysac-
charides (R-LPS), also named lipooligosaccharides (LOS),
lack the polysaccharide portion.[10,11] In order to check
whether a role is played by the lipopolysaccharides in the
molecular mechanism of adaptation to low temperatures,
the complete structural determination of these molecules
must be undertaken.
Colwellia psychrerythraea strain 34H, a Gram-negative
bacterium isolated from Arctic marine sediments, is an in-
tensively investigated steno-psychrophilic bacterium.[12] It
has cardinal growth temperatures (optimum of 8 °C, maxi-
mum of 19 °C, and extrapolated minimum of –14.5 °C)[12]
that rank among the lowest of all characterized bacteria,
which makes this bacterium an attractive model to study
the adaptive strategies of the cellular envelope to a sub-zero
lifestyle.
Figure 1. 14% DOC-PAGE analysis of Colwellia psychrerythraea
strain 34H LOSPCP (lane A) and Escherichia coli O55:B5 LPS used
as standard (lane B).
In this paper, we report the structural characterization
of the carbohydrate backbone of the LOS of Colwellia
psychrerythraea 34H. The lipooligosaccharide was degraded
both by mild hydrazinolysis (O-deacylation) and hot KOH
(4 m; N-deacylation). Both products were investigated by
GC–MS analysis of the fatty acid methyl esters revealed
the presence of decanoic, dodecanoic, 3-hydroxydodecan-
oic, tetradecenoic, tetradecanoic, pentadecenoic, pentade-
canoic, 3-hydoxytetradecenoic, esadecenoic, esadecanoic,
octadecenoic, and octadecanoic acids.
1
chemical analysis, by H and 13C NMR spectroscopy, and
by electrospray-ionization Fourier transform ion cyclotron
resonance mass spectrometry.
Mass Spectrometric Analysis of the O-Deacylated LOSPCP
The LOSPCP was O-deacylated with anhydrous hydraz-
ine,[17] and the product obtained (LOS-OH) was analysed
by ESI FT-ICR MS. The charge-deconvoluted spectrum
showed the presence of one main species (M) at
1844.607 Da. Other signals were attributed to potassium
and sodium adducts (Figure 2). To obtain further structural
information, the LOS-OH was subjected to capillary skim-
mer dissociation (CSD), which generated the Y and B frag-
ments[18] resulting from the cleavage of the Kdo/lipid A
linkage.[19]
The CSD spectrum (Figure 3) showed the presence of a
fragment at 922.176 Da, which was assigned to the core oli-
gosaccharide, and a second fragment at 922.423 Da, which
was assigned to the lipidA-OH. In particular, the following
composition was assigned to the core oligosaccharide: Gro-
ColGalAManKdoP2 (accurate mass 922.175 Da), where
Gro represents a glycerol residue. To the lipid A-OH was
assigned the following composition: GlcN2P2[C12:0(3-
OH)][C14:1(3-OH)] (accurate mass 922.426 Da), in agree-
ment with the information obtained by chemical analysis.
Results and Discussion
LPS Extraction and Preliminary Analysis
Colwellia psychrerythraea strain 34H cells were grown
aerobically, and the recovered cell pellet was extracted using
phenol/chloroform/light petroleum (PCP) to obtain the
crude LPS.[13] When it was analysed by DOC-PAGE elec-
trophoresis, the crude LPS showed positive silver staining.
In particular, a rough LPS (LOSPCP) was revealed (Fig-
ure 1). Subsequent extraction by the phenol/water
method[14] yielded only a low amount of the lipooligosac-
charide (LOSW), the purity of which was lower than that
of the LOSPCP. The sugar composition of the LOSPCP was
obtained by GC–MS analysis of the acetylated methyl
glycosides. Thus, the occurrence of d-galacturonic acid
(GalA), 2-amino-2-deoxy-d-glucose (GlcN), d-mannose
(Man), 3,6-dideoxy-l-xylo-hexose (colitose, Col), and 3-de-
oxy-d-manno-oct-2-ulopyranosonic acid (Kdo) was re-
vealed. The latter residue was revealed only after de-
phosphorylation of the LOSPCP, which was achieved by HF
treatment. This result suggested the presence of a phosphate
group on this residue, which prevented the detection of Kdo
by GC–MS.[15] Methylation analysis indicated the presence
of terminal Col, 6-substituted GlcN, 2-substituted GalA,
and 2,3-disubstituted Man. The methylation data also re-
vealed that all the residues were in the pyranose form.
NMR Spectroscopic Analysis of the Fully Deacylated
LOSPCP
To characterize the core oligosaccharide, the LOS-OH
was further N-deacylated by strong alkaline hydrolysis, and
The absolute configurations of the sugar residues were the resulting oligosaccharide (OS) was analysed by one- and
determined by GC–MS analysis of the corresponding acet- two-dimensional NMR spectroscopy (Table 1, Figures 4, 5,
ylated 2-octyl glycosides.[16]
6, and S1).
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Core Oligosaccharide from Colwellia psychrerythraea Strain 34H
Figure 2. Charge-deconvoluted ESI FT-ICR mass spectrum of the LOS-OH isolated from Colwellia psychrerytraea strain 34H. The
spectrum was acquired in negative-ion mode.
Figure 3. Charge-deconvoluted CSD mass spectrum of the LOS-OH isolated from Colwellia psychrerytraea strain 34H. The spectrum was
acquired in negative-ion mode.
1
Table 1. H and 13C NMR assignments of the fully deacylated oligosaccharide of the LOSPCP from Colwellia psychrerythraea strain 34H.
The values are referenced to acetone as internal standard (1H: δ = 2.225 ppm; 13C: δ = 31.45 ppm). The spectra were recorded at 302 K.
Residue
1-H
C-1
2-H
C-2
3-H
C-3
4-H
C-4
5-H
C-5
6a-H
C-6
6b-H/7-H
C-7
7b-H/8-H
C-8
A
5.56
92.8
5.30
100.2
5.16
101.7
4.97
101.5
4.82
99.9
–
3.38
55.3
4.03
80.8
3.84
76.9
3.95
64.7
3.07
56.8
–
3.88
70.6
3.93
71.5
4.09
70.0
1.93
34.2
3.81
72.9
1.95–2.15
35.5
3.39
71.1
3.69
68.6
4.24
72.3
3.81
69.6
3.81
75.5
4.49
71.2
4.09
73.9
4.14
73.6
4.41
73.4
4.13
68.2
3.70
75.3
4.20
74.1
3.80
70.3
3.88
62.4
–
174.8
1.13
16.8
3.46
63.7
3.76
72.8
4.21
α-GlcpN-1-P
B
2-α-Manp
C
2-α-GalpA
D
α-Colp
E
3.65
3.69
6-β-GlcpN-4-P
F
5-α-Kdop-4-P
3.76
70.4
3.64–3.86
64.9
174.2
101.0
1
1
In particular, H–1H DQF-COSY (double quantum-fil- relation spectroscopy), H–1H ROESY (rotating-frame nu-
tered correlation spectroscopy), H–1H TOCSY (total cor- clear Overhauser enhancement spectroscopy), 1H–13C
1
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Figure 4. 1H NMR spectrum of the oligosaccharide (OS) obtained by strong alkaline hydrolysis of the LOSPCP. The spectrum was
recorded in D2O at 302 K at 600 MHz. The letters refer to the residues as described in Table 1 and Scheme 1.
1
Figure 5. Alcohol signals region (δ = 3.0–4.6 ppm) of the H–13C DEPT-HSQC spectrum of OS. The spectrum was recorded in D2O at
302 K at 600 MHz using acetone as an internal standard (δH = 2.225 ppm and δC = 31.45 ppm). The letters refer to the residues as
described in Table 1 and Scheme 1.
DEPT-HSQC (distortionless enhancement by polarization
Residue A was assigned as the 6-substituted α-GlcpN-1-
1
transfer-heteronuclear single quantum coherence), H–13C P of lipid A on the basis of the multiplicity of the anomeric
HSQC-TOCSY, 1H–13C HMBC (heteronuclear multiple proton signal due to its phosphorylation (3JH,P = 6.1 Hz).
bond correlation), 2D F2-coupled HSQC, and 31P NMR Moreover, the C-2 resonance occurred at δ = 55.3 ppm,
spectroscopy were performed.
which indicated a nitrogen-bearing carbon atom, and the
The 1H NMR spectrum (Figure 4) of the fully deacylated C-6 resonance was shifted downfield by glycosylation to δ
LOSPCP showed the presence of five anomeric proton sig- = 70.3 ppm.
nals (A–E) between δ = 4.7 and 5.7 ppm. By taking into
account all the 2D NMR experiments, the spin systems of (1JC-1,1-H = 166.3 Hz), was assigned as the lipid A 6-substi-
all the monosaccharides were identified. tuted β-GlcpN-4-P residue, as a result of its C-2 chemical
Residue E, with C-1/1-H signals at δ = 99.9/4.82
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Core Oligosaccharide from Colwellia psychrerythraea Strain 34H
Figure 6. Anomeric region of the 1H–13C HMBC spectrum of OS. The spectrum was recorded in D2O at 302 K at 600 MHz using acetone
as an internal standard (δH = 2.225 ppm and δC = 31.45 ppm). The letters refer to the residues as described in Table 1 and Scheme 1.
shift at δ = 56.8 and its linkage to O-6 of residue A. In fact with standard values,[24] and the α-anomeric configuration
1-H of residue E showed a long-range scalar coupling with was established by the JC-1,1-H coupling-constant value,
C-6 of residue A in the HMBC spectrum. The β-anomeric which was 176.4 Hz. The HMBC spectrum (Figure 6)
configuration was corroborated by the intraresidue NOE showed the presence of a long-range scalar coupling be-
correlations observed between 1-H and both 3-H and 5-H tween the 1-H proton of this residue and C-5 of Kdo, thus
in the ROESY spectrum. Moreover, the downfield shifts of indicating that this position was substituted by residue B.
4-H and C-4 are diagnostic for the presence of a phosphate
Residue C, with 1-H/C-1 signals at δ = 5.16/101.7 ppm,
was identified as a 2-substituted α-galactopyranuronic acid,
group linked at O-4.[20]
The Kdo (residue F) proton and carbon chemical shifts since its C-6 signal occurred at δ = 174.8 ppm, and its C-2
were identified starting from the diastereotopic protons 3ax- signal was shifted downfield to δ = 76.9 ppm. The α-anom-
H and 3eq-H. The chemical-shift difference between these eric configuration was inferred from its JC-1,1-H coupling-
protons depends on the configuration of the C-1 carbon constant value of 176.3 Hz. This residue was linked to resi-
atom, being different for the α and β anomers. In this case, due B at the O-2 position, as shown by the presence of a
the difference of Δ(3ax-H – 3eq-H) = 0.2 ppm allowed us to correlation between 1-H of residue C and C-2 of residue B
assign an α configuration to the residue.[21] Moreover, both in the HMBC spectrum (Figure 6).
protons showed a correlation in the DQF-COSY spectrum
The last residue (D) of the core oligosaccharidic chain
with a signal at δ = 4.49 ppm, assigned as Kdo 4-H, which was identified as a terminal α-colitose, since its 3-H/C-3 and
was in turn correlated to a carbon atom with signal at δ = 6-H/C-6 signals occurred at δ = 1.93/34.2 and 1.13/
71.2 ppm in the DEPT-HSQC spectrum. Both 4-H and C- 16.8 ppm, respectively. The α-anomeric configuration was
4 resonances were shifted downfield relative to reference inferred from its JC-1,1-H value of 173.9 Hz. Its 1-H showed
values,[22] and the observed chemical shifts were diagnostic an interresidue NOE correlation with 2-H of residue C in
for the presence of a phosphate in that position.[23] The the ROESY spectrum, and a long-range scalar coupling
Kdo 5-H proton was identified by vicinal scalar coupling with the signal at δ = 101.7 ppm of a carbon atom in the
with 4-H in the DQF-COSY spectrum, and the correspond- HMBC spectrum (Figure 6). Thus, it was shown to be
ing carbon atom was downfield shifted to δ = 74.1 ppm linked to residue C at the O-2 position.
indicating glycosylation at this position. In addition, the
To sum up, the above data allowed the identification of
Kdo anomeric carbon atom showed a long-range corre- the main carbohydrate backbone of the lipopolysaccharide
lation with 6-H of residue E, thus confirming its linkage to from Colwellia psychrerythraea, as shown in Scheme 1.
the lipid A backbone.
The 31P NMR spectrum of OS confirmed the presence
Residue B was assigned as a 2-substituted α-mannopyr- of only three phosphomonoester signals (Kdo-4-P at δ =
anose on the basis of the small J1-H,2-H and J2-H,3-H cou- 3.9 ppm, GlcN-4-P at δ = 3.5 ppm, and GlcN-1-P at δ =
pling-constant values. The glycosylation at the C-2 position 2.6 ppm), in contrast with the results obtained from the
was inferred by comparing the carbon chemical shift values analysis of the LOS-OH mass spectra, which indicated the
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Scheme 1.
presence of four phosphate groups. This, together with the NMR Spectroscopic Analysis of the LOS-OH
lack of the glycerol residue in the OS structure, suggested
that these groups did not withstand the alkaline treatment,
To establish the structure of the core oligosaccharide in-
which is consistent with the lability of diesters under these cluding the labile groups lost during the harsh alkaline
hydrolysis conditions.[25] In addition, the finding of a 2,3- treatment, the LOS-OH was analysed by NMR spec-
disubstituted mannose in the methylation analysis did not troscopy (Figures 7, S2 and S3). In particular 1H–1H DQF-
fit with the above reported structure.
COSY, 1H–1H TOCSY, 1H–1H ROESY, and 1H–13C
1
Figure 7. Carbinol (a) and anomeric (b) regions of the H–13C DEPT-HSQC spectrum of LOS-OH from Colwellia psychreritraea strain
34H. The spectrum was recorded in D2O at 298 K at 600 MHz using acetone as an internal standard (δH = 2.225 ppm and δC
=
31.45 ppm). The letters refer to the residues as described in Table 2 and Scheme 2.
Table 2. 1H and 13C NMR assignments of the LOS-OH from Colwellia psychrerythraea strain 34H. All the values are referenced to
acetone as internal standard (1H: δ = 2.225 ppm; 13C: δ = 31.45 ppm). The spectra were recorded at 298 K.
Residue
1-H
C-1
2-H
C-2
3-H
C-3
4-H
C-4
5-H
C-5
6a-H
C-6
6b-H/7-H
C-7
7b-H/8-H
C-8
A
5.27
94.1
5.16
100.2
5.47
99.7
4.98
100.3
4.51
102.5
–
3.80
55.1
4.40
77.3
3.85
75.1
3.87
64.9
3.71
56.2
–
3.67
72.4
4.40
75.1
4.06
70.0
1.88–1.98
34.3
3.67
72.4
1.89–2.16
35.5
3.49–3.56
63.4
3.47
70.7
3.75
68.1
4.20
72.2
3.97
69.7
3.67
75.5
4.47
70.1
3.91
72.7
4.27
74.1
4.42
73.4
4.07
68.2
3.57
75.1
4.19
74.5
3.76
69.4
3.87
62.4
–
4.06
α-GlcpN-1-P
B
2-α-Manp-3-P
C
2-α-GalpA
D
α-Colp
E
6-β-GlcpN-4-P
F
5-α-Kdop-4-P
t-Gro-1-P
3.61
nd
1.13
17.1
3.49
64.2
3.76
72.9
3.62
3.91
69.8
3.68–3.78
64.3
nd
3.79–3.83
67.7
nd
3.77
71.9
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Core Oligosaccharide from Colwellia psychrerythraea Strain 34H
Scheme 2.
DEPT-HSQC spectra were acquired. Analysis of all the
spectra (Table 2) confirmed that the main carbohydrate
backbone was that shown in Scheme 1, but that additional
signals due to the glycerol residue were present.
Colwellia psychrerythraea strain 34H, a strictly psychro-
philic marine bacterium isolated from deep sea and Arctic
and Antarctic sea ice.[12] The molecules were extracted by
the PCP method, and they had a lipooligosaccharide frac-
tion. The complete structural determination was achieved
by chemical analysis, NMR spectroscopy, and ESI mass
spectrometry. It is worth noting the lack of heptose residues
in the inner core of C. psychreritraea. Actually, in their
place, an α-mannose residue is linked to the Kdo. This
structural feature is commonly found in the Rhizobiaceae
family, but has never before been found in extremophiles.
Moreover, colitose and glycerol are present in the Colwellia
LOS. Both these residues have been already found in several
O-polysaccharides and K-antigens from Gram-negative
bacteria, but to the best of our knowledge, this is the first
time that they have been found in a core oligosaccharide.
Until now, only a few structures of LPSs from other cold-
adapted microorganisms have been characterised. By com-
paring the C. psychrerythraea 34H LOS structure with those
obtained from P. haloplanktis TAC125[27,28] and TAB23,[29]
and from P. arctica,[15] it turned out that they have in com-
mon the lack of an O-chain and the presence of a high
charge density in the core region due to the presence of
acidic monosaccharides and phosphate groups. In addition,
the core regions have been found to be made up of only a
few sugar units. These structural features seem to be com-
In more detail, the signal of C-1 of the Gro residue oc-
1
curred at δ = 67.7 ppm in the H–13C DEPT-HSQC spec-
trum (Figure 7), thus indicating its phosphorylation at C-1.
Furthermore, the signals of 3-H and C-3 of residue B were
shifted downfield to δ = 4.40/75.1 ppm, thus indicating that
Gro was linked to the mannose residue at position O-3 by
a phosphodiester linkage, consistent with the presence of
the 2,3-disubstituted mannose in the GC–MS methylation
analysis. To determine the relative configuration of the Gro
residue, it was oxidized to glyceric acid. The product was
hydrolysed, the free glyceric acid was esterified with chiral
2-octanol, and the resulting octyl ester derivative was ana-
lysed by GC–MS.[26] By comparison of its retention time
with that of a standard sample, it was found to be d-config-
ured.
In conclusion, the complete structure of the core lipid A
saccharidic backbone of the LPS from Colwellia psychrer-
ythraea strain 34H is as shown in Scheme 2.
Conclusions
In this paper, we reported the complete structure of the
sugar backbone of the lipopolysaccharide fraction from
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120 °C for 16 h. The KOH was neutralized with HCl (2 m aq.) until
pH = 6, and the mixture was extracted three times with CHCl3.
The aqueous phase was recovered and desalted on a Sephadex G-
10 column (Amersham Biosciences, 2.5ϫ43 cm, 31 mLh–1, frac-
tion volume 2.5 mL, eluent NH4HCO3 10 mm). The eluted oligo-
saccharide mixture was then lyophilized (3.5 mg, 44% w/w).
mon to cold-adapted microorganisms. Recently, O-chain
polysaccharides were found in LPS from Psychrobacter
muricolla and cryohalentis. Although the latter micro-
organisms had been isolated at –9 °C, the LPS was ex-
tracted from bacterial cells grown at 24 °C.[30,31] It would
be worth investigating the LPS produced at a lower growth
temperature.
1
NMR Spectroscopy: H and 13C NMR spectra were recorded with
a Bruker Avance 600 MHz spectrometer equipped with a cryo-
probe. All two-dimensional homo- and heteronuclear experiments
(COSY, TOCSY, ROESY, HSQC-DEPT, HSQC-TOCSY, 2D F2-
coupled HSQC, and HMBC) were performed by using standard
Experimental Section
Bacteria Growth and LOS Isolation: Colwellia psychrerythraea 34H pulse sequences available in the Bruker software. The mixing time
was grown aerobically at 4 °C in marine broth medium (DIFCOTM
2216). When the liquid culture reached the late exponential phase
(OD600 = 2), cells were harvested by centrifugation at 3000 g at
4 °C for 20 min. Dried bacteria cells (4.8 g) were extracted by the
PCP method[13] to give LOSPCP (52 mg, yield 1.1% w/w of dried
cells).
for TOCSY, ROESY, and HSQC-TOCSY experiments was 100 ms.
Chemical shifts were measured in D2O at 302 K and 298 K for OS
and LOS-OH, respectively, by using acetone as an internal standard
(δH = 2.225 ppm and δC = 31.45 ppm).
Mass Spectrometry Analysis: Electrospray-ionization Fourier trans-
form ion cyclotron (ESI FT-ICR) mass spectrometry was per-
formed in the negative-ion mode with an APEX QE (Bruker Dal-
tonics) instrument equipped with a 7 T actively shielded magnet.
The LOS-OH sample was dissolved at a concentration of ca.
10 ngμL–1 and analysed as described previously.[33] Mass spectra
were charge-deconvoluted, and the mass numbers given refer to the
monoisotopic masses of the neutral molecules.
Sugar and Fatty Acids Analysis: A sample of LOSPCP (0.5 mg) was
treated first with HF (48% aq.; 100 μL); then methanolysis was
performed. The monosaccharides obtained were acetylated and
analysed by GC–MS as described previously,[15] while the fatty ac-
ids were analysed as methyl esters. The sugars were identified by
comparison with standard samples. In particular, the colitose stan-
dard was obtained from Escherichia coli O55:B5 LPS (Sigma). The
absolute configurations of the sugars were determined by gas
chromatography of the acetylated (S)-2-octyl glycosides.[16] The ab-
solute configuration of Gro was determined by GC–MS analysis
of its 2-octyl ester derivative. Briefly, it was oxidized using 2,2,6,6-
tetramethylpiperidine-1-oxyl (TEMPO), then hydrolysed with TFA
(trifluoroacetic acid; 2 m), and esterified with chiral 2-octanol.[26]
All the sugar derivatives were analysed with an Agilent Technol-
ogies 6850A gas chromatography apparatus equipped with a mass-
selective detector 5973N and a Zebron ZB-5 capillary column
(Phenomenex, 30 mϫ0.25 mm i.d., flow rate 1 mLmin–1, He as
carrier gas). Acetylated methyl glycosides were analysed using the
following temperature program: 150 °C for 3 min, 150Ǟ240 °C at
3 °Cmin–1. Fatty acids were analysed as follows: 140 °C for 3 min,
140Ǟ280 °C at 10 °Cmin–1, 280 °C for 20 min. The analysis of
acetylated octyl glycosides was performed as follows: 150 °C for
5 min, then 150Ǟ240 °C at 6 °Cmin–1, 240 °C for 5 min. The gly-
ceric acid octyl ester derivatives were analysed with the following
temperature program: 80 °C for 5 min, 80Ǟ200 °C at 5 °Cmin–1,
200Ǟ300 °C at 10 °Cmin–1.
Supporting Information (see footnote on the first page of this arti-
cle): The whole 1H-13C DEPT-HSQC spectra of OS and LOS-OH,
as well as the proton spectra of LOS-OH are reported in this sec-
tion. The main signals are described in the main text of the article.
Moreover the elemental analysis of both the products are reported.
Acknowledgments
The authors thank the Centro Interdipartimentale Metodologie
Chimico Fisiche Università di Napoli and BioTekNet for the use
of the 600 MHz NMR spectrometer. This work was supported by
the Programma Nazionale di Ricerca in Antartide 2010 (grant
PNRA 2010/A1.05).
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3778
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Received: January 7, 2013
Published Online: April 26, 2013
Eur. J. Org. Chem. 2013, 3771–3779
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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