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DOI: 10.1002/ejoc.201201702

Structure and Immunological Activity of the Lipopolysaccharide Isolated from

the Species Alkalimonas delamerensis

Alba Silipo,*[a] Flaviana Di Lorenzo,[a] Luigi Lembo Fazio,[b] Ida Paciello,[b]

Luisa Sturiale,[c] Angelo Palmigiano,[c] Michelangelo Parrilli,[a] William D. Grant,[d]
Domenico Garozzo,[c] Rosa Lanzetta,[a] Maria Lina Bernardini,[b,e] and Antonio Molinaro[a]

Dedicated to the memory of Professor Ernesto Fattorusso

Keywords: Natural products / Carbohydrates / Oligosaccharides / Structure–activity relationships / Medicinal chemistry /

Inflammation

We have defined the complete structure and assessed the
biological activity of the lipopolysaccharide (LPS) from Alka-
limonas delamerensis, an alkaliphilic bacterium isolated from
soda lakes in China and East Africa. The structural determi-
nation, which was achieved by the use of chemical, spectro-
scopic and spectrometric approaches, indicates a novel
Rough type lipopolysaccharide that is rich in negatively
charged groups in the lipid A inner core region. The proin-
flammatory activity of the pure lipopolysaccharide has also
been defined by testing the LPS as a stimulant in human
HEK293 cells, which express the CD14/MD2/TLR4 complex
(HEK293-hTLR4), and in bone marrow-derived macro-
phages.

Introduction grow optimally at alkaline pH (usually between pH 9 and

Some bacterial species have the ability to live in habitats
that are characterized by one or more environmental ex-
tremes. There are prokaryotes abounding in boiling hot
springs, in ice, in extremely salty bodies of water, and in
soils and water having extreme pH values. Some of these
species are not only tolerant of these extremes but actually
require the environmental extreme in order to grow. Be-
cause of this, these prokaryotes have been referred to as
extremophiles. Alkaliphiles are defined as organisms that
[a] Dipartimento di Scienze Chimiche, Università di Napoli
Federico II, Complesso Universitario Monte S. Angelo,
Via Cintia 4, 80126 Napoli, Italy
Fax: +39-081-674393
E-mail: silipo@unina.it
Homepage: www.unina.it
[b] Dipartimento di Biologia e Biotecnologie “Charles Darwin”,
Sapienza-Università di Roma,
Via Dei Sardi, 70, 00185 Roma, Italy
Homepage: http://bbcd.bio.uniroma1.it/bbcd/
[c] Istituto di Chimica e Tecnologia dei Polimeri – ICTP – CNR,
Via P. Gaifami 18, 95126 Catania, Italy
Homepage: http://www.ictp.cnr.it/
[d] Department of Infection, Immunity and Inflammation,
University of Leicester,
University Road, Leicester LE1 9HN, United Kingdom
Homepage: http://www2.le.ac.uk/departments/iii
[e] Isituto Pasteur-Fondazione Cenci Bolognetti,
Piazzale Aldo Moro 5, 00185 Roma, Italy
http://www.istitutopasteur.it/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201201702.
10), with some being capable of cultivation at pH values
higher than pH 11.[1,2] Recently, the study of alkaliphiles
has attracted increasing interest due to their physiological
adaptations to high pH and their potential uses in biotech-
nological applications.[2] Their potential industrial use has
led to increased systematic studies and expansion in the
numbers and types of alkaliphiles isolated from a variety of
environments. Alkalimonas delamerensis is an alkaliphilic
and slightly halophilic bacterium that was originally iso-
lated from Lake Elmenteita in East Africa and is also pres-
ent in inner Mongolian soda lakes.[3] Gram-negative bacte-
ria possess an asymmetric outer membrane that represents
the first and most immediate line of defense against a harsh
environment and antimicrobial agents. The outer mem-
brane is composed of an asymmetric bilayer, the external
leaflet of which is formed by lipopolysaccharides (LPS),
which are complex macromolecules that cover approxi-
mately 75 % of the external cell envelope. The LPS layer
that coats the external surface acts a key mediator of the
interaction of bacteria with the external milieu and contrib-
utes to form a defensive barrier that helps bacteria resist
antimicrobial compounds and environmental stresses. LPSs
are also called endotoxins because they are cell-bound and,
once released, can play a key role in the pathogenesis of
Gram-negative infections in both plant and animal hosts.[4]
The LPS component in extremophiles may be particularly
important in the resistance of organisms to the unique ex-

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ternal surroundings and, thus, an understanding of its pri-
mary structure is important to comprehend the mechanisms
and the chemical tricks that these organisms use to survive
in harsh environments.
Lipopolysaccharides are built up according to a common
structural architecture. They are composed of a hydrophilic
heteropolysaccharide (formed by core oligosaccharide and
O-specific polysaccharide or O-chain) covalently linked to
a lipophilic domain termed lipid A, which is embedded in
the outer leaflet and anchors these macromolecules to the
membrane through electrostatic and hydrophobic interac-
tions. Bacteria can also biosynthesize LPS without O-spe-
cific chains and, in this case, LPS is defined as R-type
(Rough type) or lipooligosaccharide (LOS). These three
major domains are genetically, biologically and chemically
distinct. The core region[5] consists of typical monosaccha-
ride residues such as heptoses and an acidic sugar termed
Kdo (3-deoxy-d-manno-oct-2-ulosonic acid), which is a
characteristic marker of LPS and can be decorated with
negatively charged substituents, often present in nonstoi-
chiometric amount, such as phosphate (P), pyrophosphate
(PP), pyrophosphorylethanolamine (PPEtN), phospho-ara-
binosamine (PAra4N) and uronic acids (as GalpA). The li-
pid A[6] is most conservative portion of the LPS and has
crucial functions of protection and defense. The lipid A
structure is made of a β-(1씮6) disaccharide backbone con-
sisting of glucosamine (GlcN), phosphorylated at positions
1 and 4⬘ and acylated with 3-hydroxy fatty acids at posi-
tions 2 and 3 of both GlcNs through amide and ester link-
ages. These acyl chains, defined as primary because they are
directly linked to the sugar backbone, are further acylated
to their hydroxy groups by secondary acyl moieties. Kdo,
or a derivative of this sugar, is linked to the distal glucos-
amine of the lipid A backbone through position 6⬘. The
lipid A is the endotoxic portion of LPS and is responsible
for transmembrane signaling that stimulates the immune re-
sponse. Many LPS species with low or nonendotoxic ac-
tivity have been identified that can operate as “antagonists”
reducing or, in a dose-dependent manner, completely in-
hibiting the cytokine production induced by strongly endo-
toxic species.[5,7] Considerable efforts have been made to
identify natural or synthetic molecules that are able to inter-
fere with the interaction between LPS and inflammatory
cells and that, depending upon the responding cell species,
may exhibit agonistic and/or antagonistic properties in
LPS-responsive cells. In fact, the antagonist species elimin-
ate the disease-related biological events provoked by LPS,
strongly reducing the endotoxin-induced immune acti-
vation. The study of LPS molecules from nonhuman associ-
ated/environmental bacteria can have several therapeutic
implications. It is extremely important to identify LPS/lipid
A molecules that in mammalian and, in particular, in hu-
man cells, display inhibitory activity on cytokine pro-
duction stimulated by LPS from toxic Gram-negative bacte-
ria. We have previously ascertained that lipid A from Halo-
monas magadiensis antagonizes cytokine production in-
duced by LPS and lipid A from E. coli on both human and
murine cells.[8] The cytokine inhibition occurs in a dose-
2654 www.eurjoc.org © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
A. Silipo et al.
dependent manner and this suggests that the antagonist li-
pid A inhibits LPS binding to the target cells in a competi-
tive way. These antagonist lipid A molecules can be used
therapeutically to block uncontrolled activation of the in-
nate response and consequent septic shock induced by
massive circulation of endotoxin in the host. Work is in
progress to check the biological activity of the other lipid
A species from nonhuman pathogen Gram-negative bacte-
ria that could have therapeutic activity and clarify the
mechanisms of activation of the immune response. How-
ever, a complete understanding of the structure-function re-
lationship of lipid A immunogenicity is still to come. Pursu-
ing our study of LPS from extremophiles as a way to gain
structural insights into the structure-function relationships,
we have fully elucidated the LPS structure from Alka-
limonas delamerensis.
Results and Discussion
Isolation and Compositional Analysis of LPS Isolated from
Alkalimonas delamerensis
The LPS fraction was extracted from Alkalimonas dela-
merensis and analyzed by SDS electrophoresis. The isolated
and purified LPS was found to have the typical run to the
bottom of the gel as R-type LPS (LOS). The compositional
analysis revealed the presence of d-GlcN, d-Glc, l-glycero-
d-manno-heptose (l,d-Hep), and 3-deoxy-manno-oct-2-ulo-
pyranosonic acid (Kdo). Methylation analysis revealed the
presence of terminal Glc, 6-substituted Glc, 4,6-substituted
Glc, 3,4,7-substituted Hep, 4,7-substituted Hep, 2-substi-
tuted Hep, 7-substituted Hep, terminal Hep, and 6-substi-
tuted GlcN. Fatty acids analysis revealed the presence of
(R)-3-hydroxydodecanoic [C12:0(3-OH)] acid, dodecanoic
acid (C12:0), decanoic acid (C10:0), octadecenoic acid
(18:1), hexadecenoic acid (16:1), and hexadecanoic acid
(C16:0).
To establish the primary structure of the oligosaccharide
moiety of Alkalimonas delamerensis, LOS was fully deacyl-
ated to obtain an oligosaccharide fraction OS that, by gel-
permeation chromatography, was further purified. Compo-
sitional analysis of the isolated OS confirmed the presence
of the sugar residues found in the intact LOS fraction.
Structural Characterization of OS Product
The 1H NMR spectrum of OS is shown in Figure 1.
Combinations of homo- and hetero-nuclear 2D NMR ex-
periments were performed to define the oligosaccharide
structure of OS. In the anomeric region of the 1H NMR
spectrum, ten anomeric signals were identified (A–L,
Table 1, Figure 1); furthermore, signals resonating at 2.05/
2.35 ppm were identified as the H-3 methylene proton sig-
nals of the Kdo K. Both 3JH1,H2 coupling constants and the
intraresidual NOE contacts allowed the identification of the
anomeric configuration of each monosaccharide unit,
Eur. J. Org. Chem. 2013, 2653–2665
Lipopolysaccharide from Alkalimonas delamerensis

Figure 1. 1H NMR spectrum of product OS. Anomeric signals of spin system are designated as listed in Table 1.

Table 1. 1H, 13C and 31

P NMR chemical shifts of the oligosaccharide derived from alkaline treatment of the LOS from Alkalimonas
delamerensis.
Chemical shift δ (1H/13C)
Unit Nucleus 1 2 3 4 5 6 7 8
A H 5.77 3.50 4.00 3.57 4.27 4.43/3.93
6-α-GlcN C 91.16 54.5 69.8 69.9 72.5 69.8
P 2.87
B H 4.90 3.12 3.90 4.03 3.86 3.67/3.79
6-β-GlcN C 100.15 56.4 72.4 73.8 74.0 62.7
P 1.59
C H 5.35 4.29 4.25 4.32 3.96 4.04 3.94
3,4,7-α-Hep C 100.17 70.3 74.1 74.05 71.9 69.8 70.6
D H 5.28 3.98 4.02 4.17 ND 4.02 3.95
4,7-α-Hep C 99.4 71.1 69.9 79.7 ND 69.8 70.21
E H 5.29 4.109 3.94 ND ND 4.02 3.87
t-α-Hep C 101.3 70.8 71.7 ND ND 69.8 62.2
F H 5.92 4.30 4.02 4.31 4.12 3.94 3.87
2 -α-Hep C 98.2 79.9 69.6 68.7 70.9 68.18 62.7
P 2.06
G H 4.70 3.63 3.59 3.77 3.47 4.22/3.84 H5 3.63 H6 4.01/3.86
6-β-Glc/t-β-Glc C 103.8 73.8 73.8 69.5 77.2 64.7 73.7 59.9
H H 5.30 3.65 3.94 4.05 4.12 3.86/3.91
t-α- Glc C 101.2 72.2 72.1 71.9 69.3 61.09
I H 5.08 4.28 4.04 3.78 3.79 4.04 4.14/3.97
t-α-Hep C 99.3 69.3 69.0 71.3 72.1 69.2 62.4
L H 4.85 3.80 3.83 4.15 4.06 3.72/3.92
4,6-pyr-α-Glc C 99.3 71.04 72.7 67.2 71.7 64.4
Pyr: COOH CH3 O-C-O
H/C 177.1 1.55/21.7 102.4
K H – – 2.05/2.35 4.71 4.37 3.94 4.05 3.87/3.97
5-α-Kdo C 174.7 99.7 34.5 69.7 72.8 68.2 62.9
P 2.3

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whereas the relative configuration of each residue was as-
signed based on vicinal 3JH,H coupling constants.
Residues A and B were both identified as composing the
disaccharide backbone of the lipid A moiety. The gluco con-
figuration was indicated by high 3JH,H ring proton values
(ca. 8–10 Hz). In detail, residue A was plainly identified as
the α-GlcN of the lipid A skeleton by the correlation of H-
2 with a nitrogen bearing carbon signal at δ = 54.5 ppm;
the high-field shift of proton resonances of H-2 was indica-
tive of the absence of acylation at these positions. Moreover,
the downfield shift of C-6 A at δ = 69.8 ppm was proof of
glycosyl substitution at this position (Table 1). The chemi-
cal shift values of H-1/C-1, the low 3JH1,H2 coupling con-
stant (3 Hz), and the intraresidual NOE contact of H-1
with H-2 were all in agreement with an α-anomeric configu-
ration of A. Similar considerations allowed us to identify
spin system B as GlcN. The chemical shift values of H-1/
C-1 B, the 3JH1,H2 coupling constant (8.7 Hz) and the intra-
residual NOE contact of H-1 with H-3 and H-5 were all in
agreement with a β-anomeric configuration of B.
Because of the absence of the anomeric proton signal,
the spin system of Kdo residue K was assigned starting
from the diastereotopic H-3 methylene protons (H-3ax and
H-3eq, respectively, Figure 1 and Table 1). The α-configura-
tion was attributed on the basis of the chemical shift values
of H-3 and of the 3JH7,H8a and 3JH7,H8b coupling con-
stants.[9,10]
Figure 2. Expansion of the TOCSY (grey) and T-ROESY (black) spectra of product OS in which the key interresidual NOE contacts
are indicated.
2656 www.eurjoc.org © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
A. Silipo et al.
Spin systems C, D, E, F, I (Table 1, Figure 1), were all
identified as α-heptose residues, as indicated by their
3
JH1,H2 and 3JH2,H3 coupling constants (below 3 Hz) and
by the intraresidual NOE of H-1 with H-2. The 13C NMR
chemical shift value of C-6 of these heptose residues (all
below 70 ppm) allowed them to be identified as l-glycero-d-
manno-heptose,[10] in accordance with the chemical analysis.
Spin systems G and H were both identified as glucose,
as indicated by the large ring 3JH,H coupling constants. The
strong intraresidue NOE contacts of H-1 with H-3 and H-
5, together with the 3JH1,H2 coupling constant (7.9 Hz), was
diagnostic of the β-configuration of G, whereas the intrares-
idue NOE contact of H-1 with H-2 and the 3JH1,H2 cou-
pling constant (3 Hz) were indicative of an α-anomeric con-
figuration of residue H.
Residue L was recognized as an α-Glc residue; the α-
configuration was assigned on the basis of the intraresidual
NOE contact of H-1 with H-2 and by the upfield chemical
shift of its H-5 and C-5 nuclei.
Moreover, the 1H NMR spectrum showed the presence
of a methyl signal resonating at δ = 1.55 ppm (Figure 1,
Table 1 and Figure S1) belonging to a noncarbohydrate
spin system. The nature of this group was revealed by
analysis of the HMBC spectrum, which showed that the
methyl group signal at δ = 1.55 ppm correlated to two dif-
ferent carbon signals at δ = 102.4 and 177.1 ppm, which
were evidently fully substituted because they were not pres-
Eur. J. Org. Chem. 2013, 2653–2665
Lipopolysaccharide from Alkalimonas delamerensis
Figure 3. Expansion of the HMBC (black) and HSQC (grey) spectra of product OS in which the key interresidual long-range contacts
are indicated.
ent in the HSQC spectrum. These data pointed to a cyclic
ketal of pyruvic acid (Figure S1 and Figure 3).
The downfield shift of carbon resonances allowed the
glycosylated positions to be identified, and these were in
full agreement with the methylation analysis data; O-6 of
residues A and B, O-3, O-4 and O-7 of C, O-4 and O-7 of
D, O-2 of F, O-6 of G, O-5 of K1, whereas residues H, I, L,
E were nonreducing terminal sugars. The interresidual
NOE contacts (Figure 2) and the long-range correlations
(Figure 3) present in the HMBC spectrum yielded the oligo-
saccharide sequence.
The linkage between glucosamine residues A and B of
the lipid A backbone was validated by the interresidual
NOE contact of H-1 B with H-6 A and by the correspond-
ing long-range correlation (Figure 3). The weak downfield
shift of C-6 B was in agreement with the expected α-(2씮6)
ketosidic linkage of Kdo K with residue B of β-GlcN.
The linkage of heptose C to O-5 of Kdo K was proven
by the NOE contact between H-1 of heptose C and H-5 of
Kdo K and by the corresponding long-range connectivity
(Figure 2). Residue C was substituted at O-3, O-4 and O-7.
In detail, heptose D was located at position 3 of C, as
shown by the expected NOE and long-range correlations
(Figures 2 and 3). Moreover, the NOE contact (Figure 2) of
H-4 and H-6 C with H-1 G evidenced that the O-4 of α-
heptose C was glycosylated by the β-glucose G that was, in
turn, glycosylated at O-6 by terminal heptose I, as evident
from the correlations present in both ROESY and HMBC
spectra (Figures 2 and 3). Residue L, α-glucose, was located
Eur. J. Org. Chem. 2013, 2653–2665 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
at O-7 of Heptose C, as shown by the NOE contact of L1
with C7 and C6. Analysis of the HMBC spectra was crucial
to locate the pyruvate residue because the ketal carbon sig-
nal resonating at δ = 102.4 ppm correlated to H-4 and H-6
signals of L, which indeed experienced a downfield dis-
placement. Furthermore, it has been shown that chemical
shifts of the pyruvate moiety are indicative of the absolute
configuration at C-2;[11] in particular, in the case of 4,6-S-
configured pyruvic acetals on d-Glcp, the signal of the
methyl group resonates characteristically at 24.5–25 ppm,
whereas in the R enantiomer it resonates at 15–18 ppm.
These data suggested the S configuration for the pyruvic
group located on residue L.[12,13]
Residue D was identified as a 4,7-Heptose, substituted at
O-7 by heptose F, as evident by the NOE of H-7 D with H-
1 F and by the related HMBC correlation, and at O-4 by
α-glucose H (Figures 2 and 3). Residue F, identified as the
2-substituted α-heptose, was substituted with the terminal
α-Heptose E, as indicated by the NOE contacts of H-1 E
with H-1 and H-2 F and by the long-range correlation E1–
F2 (Figures 2 and 3).
The 31P-1H HSQC spectrum (Figure 4) showed several
cross peaks, the 31P chemical shifts of which were in accord-
ance with the presence of four phosphate groups linked at
O-1 of α-GlcN A, O-4 of β-GlcN B, at O-4 of Kdo K and
at O-4 α-Hep F. A MALDI mass spectrum of the oligosac-
charide mixture OS confirmed the above structural hypo-
theses (not shown). Thus, all data were consistent with the
shown oligosaccharide structure.
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 A. Silipo et al.
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31
Figure 4. P-1H HSQC spectrum of OS.

Structural Characterization by MALDI Mass Spectrometry
of the Intact LOS and Lipid A
The negative ion MALDI mass spectrum of intact LOS
is reported in Figure 5 (a). It showed at higher molecular
masses (between m/z 3000 and 4200) a series of [M – H]–
ions related to the native LOS mixture, mainly composed
of species differing by the composition of the acyl moieties
on the lipid A backbone (see detail in Figure 5, b). In ad-
dition to these peaks, ion fragments arising from the labile
glycoside bond cleavage between Kdo and the lipid A moi-
ety[14] were also present at lower mass ranges (Figure 5, a).
Such very useful LOS fragmentation (β-elimination) yields
both oligosaccharide(s) as B-type ions and lipid A as Y-
type ions.[15] Thus, the ion fragments in Figure 5 (a) at m/z
1895.2 and 1850.8 (the latter arising from the well-known
neutral loss of CO2 from the Kdo unit), corresponded to

Figure 5. Negative ion MALDI TOF mass spectrum of the intact LOS from Alkalimonas delamerensis (part a); ion fragments, mainly
attributable to the core oligosaccharide, have been indicated. The composition of each LOS species in terms of acylation pattern is shown
in the expanded mass-range (part b).

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Lipopolysaccharide from Alkalimonas delamerensis

the intact core oligosaccharide composed of five Hep, one
Kdo, three Hex (hexoses), two phosphates, and one pyr-
uvate residue. The main LOS species were consistent with
a combination of this core OS ion peak and differently acy-
lated lipid A species, namely hepta-, hexa-, penta- and tetra-
acylated lipid A.
The fine structure of lipid A was obtained by MS and
MS/MS analysis of the lipid A split by the core oligosaccha-
ride. To this end, lipid A was isolated following acid hydrol-
ysis of the LOS and analyzed by MALDI TOF/TOF MS,
taking into consideration lipid A compositional analysis
(see above). The negative ion mass spectrum obtained in
reflection mode (Figure 6), showed a mixture of bis-phos-
phorylated lipid A species differing by the acylation pattern.
The highest mass ion peak (m/z 1892.24) was consistent
with a hepta-acyl lipid A with a fatty acid composition
composed of four 12:0(3-OH), one 10:0, one 12:0, and one
18:1. A second peak resulted from the overlapped isotopic
clusters arising from the hepta-acylated species at m/z
1864.21 (Δm/z = 28) having four 12:0(3-OH), one 10:0, one
12:0, and one 16:1, and the lipid A at m/z 1866.25 (Δm/z =
26) with four 12:0(3-OH), one 10:0, one 12:0 and one 16:0.
Likewise, the same fatty acid distribution was observable
for the hexa-acyl lipid A moieties at m/z 1694.10, 1666.06
and 1668.08, differing from the previously described hepta-
acyl series by a missing 12:0(3-OH) (see Figure 6, insets).
In the lower mass range, a peak at m/z 1429.85 corre-
sponded to the penta-acyl species with three 12:0(OH), one
10:0 and one 12:0, whereas the ion at m/z 1231.69 was con-
sistent with this last lipid A minus a 12:0(3-OH) residue.
The location of fatty acid residues was precisely defined
by a study of the fragmentation spectra of the lipid A ion
at m/z 1429.85 (as reported in Figure 7), and of lipid A at
m/z 1694.10 and 1892.24 (Figures S2 and S3, respec-
tively).[12] As a result, the fully acylated lipid A glucosamine
backbone (hepta-acyl lipid A) was found to carry four pri-
mary 12:0(3-OH) groups bearing 12:0 and 10:0 as second-
ary fatty acids on the amide linked 12:0(3-OH), and a vari-
able long chain fatty acid (18:1, 16:0 or 16:1) located on
the primary acyl chain linked to the nonreducing GlcN;
moreover, the high heterogeneity of the lipid A mixture,
ranging from hepta- to tetra-acylated species, suggested a
nonstoichiometric substitution of both the primary O-
linked 12:0(3-OH), and of the long chain fatty acid.
In summary, the complete structure of the lipo-oligosac-
charide from A. delamerensis is reported in Figure 8.

Figure 6. Reflectron MALDI TOF mass spectrum (in negative polarity) of lipid A from Alkalimonas delamerensis; significant components
of the heterogeneous lipid A mixture, differing in the acylation pattern, were also assigned by analyzing the ion isotopic distribution (see
insets).

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 A. Silipo et al.
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Figure 7. MALDI TOF/TOF MS2 analysis of the penta-acylated lipid A at m/z 1429.85 from Alkalimonas delamerensis. The molecular
fragment at m/z 610.3 can be assigned as Y1 ion, arising from the rupture of the glycosidic linkage, which generates a monophosphoryl
reducing GlcN bearing a hydroxydodecanoic acid [C12:0(3-OH)] and a decanoic acid (C10:0). The specific fatty acid location at this
GlcN residue was suggested by the cross-ring fragmentation A21,3 at m/z 1018.5, occurring exclusively when the C3 position at this
terminal monosaccharide is free. Therefore, the fragment ion at m/z 610.3 carries a N-acyloxyacyl moiety composed of a C12:0(3-OH)
as amide-linked primary fatty acid, and of a secondary C10:0. Additional fragmentations generated ions that allowed positioning of the
acyl moieties at the non-reducing GlcN unit, the fatty acid composition of which accounted for two C12:0(3-OH) and one dodecanoic
acid (C12:0). The ion peak at m/z 1229.5 and the most intense ion at m/z 1213.6 were the result of the loss of C12:0 and of C12:0(3-
OH), respectively. The absence of a peak originating from the loss of a whole O-acyloxyacyl moiety, composed of C12:0(3-OH) linked
to C12:0, suggested that the latter is a secondary acyl chain bound to the N-linked primary C12:0(3-OH). Further ions originated from
two combined fragmentations, as indicated in the spectrum.

HEK293-hTLR4/MD2-CD14 Activation by A. delamerensis
LPS
The biological activity of A. delamerensis LPS was as-
sessed in vitro in the model of HEK293 cells, which express
human cognate molecules of the innate immune system
committed to recognize LPS, i.e., CD14, MD2 and TLR4.
HEK293 hTLR4/MD2-CD14 were exposed to different
concentrations (1, 10 and 100 ng/mL) of Alkalimonas LPS.
Escherichia coli LPS, containing fully hexa-acylated lipid A,
was used at the same concentrations as above and evaluated
as a control. Activation of NF-κB (Nuclear Factor kappa
B) and CXCL-8 (Chemokine ligand 8) production were
used as the read-out of this experiment. NF-κB activation
was measured after 4 h of LPS stimulation through the as-
sessment of luciferase activity, as described in the Experi-
mental Section. CXCL-8 production was evaluated at the
same time point through the use of an ELISA system. We
found that Alkalimonas LPS showed the same ability as E.
coli LPS to induce the activation of NF-κB at all concentra-
tions tested. In accordance with this result, CXCL-8 pro-
duction was similar upon stimulation with either of the two
LPS forms (Figure 9, A).
Several natural or synthetic lipid A structures have been
reported to exhibit a high potential as antagonists for endo-
toxically active LPS.[16] We assessed whether Alkalimonas
LPS could interfere with TLR4-mediated signaling induced

2660 www.eurjoc.org © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem. 2013, 2653–2665
Lipopolysaccharide from Alkalimonas delamerensis
Figure 8. Structure of the LOS from Alkalimonas delamerensis.
by hexa-acylated lipid A of E. coli. For this purpose,
HEK293-hTLR4 cells were preincubated for 1 h with dif-
ferent amounts (1, 10 and 100 ng/mL) of Alkalimonas LPS
and then exposed to 10 ng of hexa-acyl E. coli LPS for 4 h.
NF-κB activation and CXCL-8 production were evaluated
as above. We found that cell priming with Alkalimonas LPS
did not change either NF-κB activation or CXCL-8 pro-
duction with respect to only E. coli LPS stimulation shown
in part A of Figure 8 (see also Figure 9, B).
Finally, we investigated whether Alkalimonas LPS could
stimulate the TLR2, as already observed with LPS of Coxi-
ella burnetii[17] and Porphyromonas gingivalis.[18] To this end,
HEK293 cells expressing hTLR2 were exposed to Alka-
Eur. J. Org. Chem. 2013, 2653–2665 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
limonas LPS or E. coli LPS for 6 h and analyzed as de-
scribed above for the HEK293-hTLR4/MD2-CD14 stimu-
lation assay. E. coli LPS was used as a negative control and
the synthetic triacylated lipoprotein Pam3CSK4 (0.5 μg/
mL), which mimics a natural TLR2 agonist, was used as a
positive control. As expected, no significant TLR2 acti-
vation was observed with either Alkalimonas LPS or E. coli
LPS (Figure 9, C–D).
Bone Marrow-Derived Macrophages (BMDMs) Stimulation
BMDMs expressing the entire TLRs repertoire were col-
lected and differentiated from five-week old wild-type
C57BL/6 female mice. BMDMs were stimulated with either
Alkalimonas LPS or E. coli LPS at concentrations of 1, 10
and 100 ng/mL during 6 h. The production of TNF-α
(Tumor Necrosis Factor alfa) and KC (Keratinocyte-de-
rived cytokine) was evaluated by using an ELISA system.
No significant difference was observed between the amount
of cytokines produced by either of the two LPS forms (Fig-
ure 10).
Conclusions
We have reported a novel lipo-oligosaccharide structure
and its proinflammatory activity from Alkalimonas dela-
merensis, an extremophile alkaliphilic Gram-negative bacte-
rium isolated from soda lakes in East Africa and Mongolia.
This work follows up our previous studies on the struc-
ture activity relationship of endotoxins and their ability to
elicit innate immune response.[17] In case of human LPS re-
sponsive cells, the structural features of E. coli lipid A, com-
posed of a bis-phosphorylated hexa-acylated disaccharide
backbone with an asymmetric distribution of the acyl resi-
dues, represents the most stimulatory agonistic structure
and thus expresses the highest endotoxic activity. Structural
variations on this pattern are typically less or not agonisti-
cally active. In particular, the limited ability to trigger cyto-
kine release and the acquisition of antagonist activity oc-
curs with changes in the disaccharide backbone, such as the
absence of one or both phosphate groups, alterations in the
hydrophobic region, the symmetric distribution of the acyl
chains, or either a lower or higher acylation pattern. The
nature of acyl residues is a critical determinant for bacterial
pathogenicity and can reduce the host innate immune re-
sponse; several types of branched, unsaturated or further
substituted acyl chains have often been found in less agonis-
tic species. We have demonstrated that the structurally novel
lipid A from Halomonas magadiensis, constituted by a blend
of species in which the under-acylated derivatives are the
major part, weakly induces TNF-α synthesis by human
monocytes and significantly antagonizes the synthesis and
release of cytokine induced in these cells by E. coli LPS. In
A. delamerensisis we have found that lipid A is characterized
by a mixture of differently acylated species, the synergic ef-
fect of which confers the LPS an agonist activity exerted
through TLR4. Furthermore, in A. delamerensisis LPS, a
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 A. Silipo et al.
FULL PAPER

Figure 9. NF-κB activity and CXCL-8 production of HEK293-hTLR4 and HEK293-hTLR2 stimulated with Alkalimonas and E. coli
LPS. (A) Fold of NF-κB activation (left panel) upon stimulation of HEK293-hTLR4 after 4 h with Alkalimonas LPS (1, 10 and
100 ng/mL) and commercial hexa-acylated E. coli LPS; (right panel) CXCL-8 secretion after stimulation for 4 h as above. (B) TLR4-
dependent antagonistic activity of Alkalimonas LPS on hexa-acylated E. coli LPS. Fold of NF-κB activation (left panel) and CXCL-8
release (right panel) upon stimulation of HEK293-hTLR4 after 1 h with Alkalimonas LPS (1, 10 and 100 ng/mL) and then exposed to
E. coli LPS (10 ng/mLl, 4 h). (C) Fold of NF-κB activation upon stimulation of HEK293-hTLR2 (6 h) with Alkalimonas LPS (1, 10 and
100 ng/mL) and commercial hexa-acylated E. coli LPS. (D) CXCL-8 secretion after stimulation for 6 h as above. HEK293-hTLR2
treated for 6 h with PAM3CSK4 (0.5 μg/mL) (C and D, right panels) were used as a positive control. Report assay data corresponds to
the mean ⫾S. D. (triplicate determinations) and are representative of three independent assays. *P ⬍ 0.05, ***P ⬍ 0.001 after Student’s
t-test.

2662 www.eurjoc.org © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem. 2013, 2653–2665
Lipopolysaccharide from Alkalimonas delamerensis
Figure 10. Cytokine release in BMDMs stimulated with Alkalimonas and E. coli LPS. TNF-α and KC released by BMDMs after stimula-
tion with Alkalimonas and E. coli LPS (1, 10 and 100 ng/mL) measured by ELISA at 6 h. Mean values ⫾S. D. of three representative
experiments are shown. *P ⬍ 0.05, ***P ⬍ 0.001 after Student’s t-test.
key role must be also played by the total negative charge,
which is very high and carried by four phosphate and two
carboxyl groups.
From the structural point of view, the core oligosaccha-
ride turned out to be constituted by a novel decasaccharide
characterized by high negative charge density provided by
two phosphate groups, the carboxylic group on the Kdo
moiety, and by the pyruvate residue. These anionic substitu-
ents are uniformly distributed along the saccharide moiety
and strongly increase the anionic character of the whole
LPS molecule. The Kdo unit, which always carries another
negatively charged substituent at its O-4,[4] represented in
A. delamerensis by a phosphate group, is substituted at O-
5 by a heptose tetrasaccharide in which a second phosphate
is located on the 2-linked Hep residue, while the pyruvic
acid is located on the α-glucose sitting at O-7 of the first
heptose proximal to the Kdo. Although pyruvate residues
are found in bacterial exopolysaccharides[19] and in O-poly-
saccharides[20–22] as a post polymerization decoration, their
presence in the core region of lipopolysaccharides has rarely
been observed.[11] In particular, in the core oligosaccharide
from Pseudomonas stutzeri OXI, which is a Gram-negative
microorganism able to grow in media containing aromatic
hydrocarbons,[11] for the first time, the presence of two moi-
eties of pyruvic acid were identified that possessed different
absolute configuration. Their presence in P. stutzeri OX1
was attributed to its exposure to solvents and was thought
to be a new, structurally mediated, biochemical response to
a harsh and unusual environment. Likewise, we can suppose
that the negative charges of the pyruvate moiety could fur-
ther contribute to increase the polyanionic character of A.
delamerensis LPS, furnishing an increased capacity to bind
cations that might favor better packing of the membrane,
thus favoring bacterial adaptation to the external environ-
ment and constituting a selective barrier to the entrance of
organic molecules. Such structural features may increase the
capacity to bind naturally available cations, thus allowing
the molecules to overcome the electrostatic repulsion that
would otherwise arise. In fact, the LPS leaflet is stabilized
by electrostatic interactions of the negatively charged
groups present in lipid A and in the core region (phosphate
groups, uronic acids) with divalent cations (Mg2+, Ca2+),
which contribute to linking the LPS molecules to each
Eur. J. Org. Chem. 2013, 2653–2665 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
other, providing higher resistance to physical stress. The
presence of negatively charged substituents in close proxim-
ity seems to be functionally important and the accumu-
lation of vicinal carboxylic functions (derived from the Kdo
and the pyruvic acid) could further stabilize such a struc-
ture through the formation of additional electrostatic inter-
actions. These features could also contribute resistance to
mechanical pressures acting on the cell, further enhancing
the stability of the external membrane with the formation
of a strong, rigid, and protective barrier that is important
for the integrity of an outer membrane that is exposed to
potentially harmful external surroundings.
Experimental Section
LPS Extraction: Freeze-dried cells were extracted three times with
a mixture of aq. 90 % phenol/chloroform/petroleum ether (2:5:8
v/v/v) as described.[23] After removal of organic solvents under vac-
uum, the LOS fraction was precipitated from concentrated phenol
solution with water, the precipitate was washed with aqueous 80 %
phenol and then three times with cold acetone and then lyophilized.
The LOS fractions were analyzed by SDS-polyacrylamide gel elec-
trophoresis on 16 % gels, which were stained with silver nitrate.[24]
MALDI TOF-MS: m/z calcd. for C164H292N2O86P4 3789.74; found
3785.3 [M – H]–. C164H292N2O86P4: calcd. C 51.95, H 7.76, N 0.74;
found C 51.05, H 7.58, N 0.69.
Isolation of Oligosaccharide OS: LOS was treated with anhydrous
hydrazine (2 mL), stirred at 37 °C for 90 min, cooled, poured into
ice-cold acetone (20 mL), and allowed to precipitate. The precipi-
tate was then centrifuged (3000 g, 30 min), washed twice with ice-
cold acetone, dried, dissolved in water, and lyophilized. The OS
product was de-N-acylated with 4 m KOH as described.[25] Salts
were removed by gel permeation chromatography with Sephadex
G-10 (Pharmacia) column (50 ⫻ 1.5 cm) to yield the resulting oli-
gosaccharide OS.[25]
Isolation of Lipid A: Free lipid A was obtained by hydrolysis of
LOS (with 10 mm sodium acetate buffer pH 4.4, 100 °C, 3 h). The
solution was extracted three times with CHCl3/MeOH/H2O
(100:100:30 v/v/v) and centrifuged (4 °C, 5000 g, 15 min). The or-
ganic phase contained lipid A and the aqueous phase contained
the core oligosaccharide.
General and Analytical Methods: For the determination of sugar
residues and their absolute configuration, GLC and GLC-MS were
all carried out as described.[26–27] Monosaccharides were identified
www.eurjoc.org 2663

FULL PAPER
as acetylated O-methyl glycosides derivatives. After methanolysis
(2 m HCl/MeOH, 85°, 24 h) and acetylation with acetic anhydride
in pyridine (85°, 30 min), the sample was analyzed by GLC-MS.
Linkage analysis was carried out by methylation of the complete
core region as described.[26,27] The sample was hydrolyzed with 4 m
trifluoroacetic acid (100 °C, 4 h), carbonyl-reduced with NaBD4,
carboxy-methylated, carboxyl-reduced, acetylated and analyzed by
GLC-MS.
Total fatty acid content was obtained by acid hydrolysis. LOS was
first treated with HCl 4 m (4 h, 100 °C) and then with NaOH 5 M
(30 min, 100 °C). Fatty acids were then extracted in CHCl3, methyl-
ated with diazomethane, and analyzed by GLC-MS. The ester-
bound fatty acids were selectively released by base-catalyzed hy-
drolysis with NaOH 0.5M/MeOH (1:1 v/v, 85°, 2 h), then the prod-
uct was acidified, extracted in CHCl3, methylated with diazometh-
ane, and analyzed by GLC-MS.[28] Elemental analyses were per-
formed with a Carlo Erba 1108 instrument.
NMR Spectroscopy: For structural assignments of OS, 1D and 2D
1
H NMR spectra were recorded in D2O (0.5 mL) at 300 K, pD 7
(uncorrected value) with a Bruker 600 DRX equipped with a cryo
probe. Spectra were calibrated with internal acetone (δH =
2.225 ppm, δC = 31.45 ppm). 31P NMR experiments were carried
out with a Bruker DRX-400 spectrometer; aqueous 85 % phos-
phoric acid was used as external reference (δ = 0.00 ppm). Rotating
frame Overhauser enhancement spectroscopy (ROESY), Total Cor-
relation Spectroscopy (TOCSY), Double quantum-filtered phase-
sensitive correlation spectroscopy (DQF-COSY), Heteronuclear
single quantum coherence (HSQC), and Heteronuclear multiple
bond correlation (HMBC) experiments were performed and pro-
cessed as described.[29–30]
MALDI TOF Mass Spectrometry: MALDI-TOF mass spectra of
the intact LOS were recorded in negative polarity with a Perseptive
(Framingham, MA, USA) Voyager STR equipped with delayed ex-
traction technology. Ions formed by a pulsed UV laser beam (nitro-
gen laser, λ = 337 nm) were accelerated by 24 kV. Reflectron
MALDI TOF MS and MALDI TOF/TOF MS[2] analyses of lipid
A were performed with a 4800 Proteomic analyzer (Applied Biosys-
tems) supplied with a Nd:YAG laser at a wavelength of 355 nm.
External calibration was performed and mass accuracy was better
than 100 ppm. The mass resolution of the spectra obtained in re-
flector mode was approximately 13000.
LOS and Lipid A Sample Preparation: R-type LPS MALDI prepa-
ration was performed as recently reported in detail.[14] MALDI
preparation of lipid A was performed as described;[31] samples were
dissolved in CHCl3/CH3OH (1:1 v/v), and the matrix solution was
prepared by dissolving 2,4,6-trihydroxyacetophenone (THAP) in
CH3OH/TFA (0.1 %)/CH3CN (7:2:1). A sample/matrix solution
mixture (1:1 v/v, 1 μL) was deposited onto the MALDI plate and
allowed to dry at room temperature.
HEK293-hTLR4/CD14/MD2 Cell Culture, Transfection and Stimu-
lation: Stably transfected cell line HEK293-hTLR4/MD2-CD14
and HEK293-hTLR2 (both by InvivoGen) were cultured in
DMEM (Lonza) with 10 % FBS (Euroclone). Blasticidin-S (10 μg/
mL) and HygroGold (50 μg/mL) (InvivoGen) were added to cell
cultures following the manufacturer’s instructions. Both cell lines
were transiently transfected with PolyFect Transfection Reagent
(Qiagen) according to the manufacturer’s instructions. For NF-κB
studies, the cells were transfected overnight with a reaction mix
composed of PolyFect Transfection Reagent (Qiagen; 1 μL), Firefly
luciferase reporter constructs, pGL3 (150 ng), ELAM tk (contain-
ing NF-κB promoter sequences), and Renilla luciferase reporter
plasmid, pRLTK (15 ng, as an internal control).[32] HEK293-
2664 www.eurjoc.org © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
A. Silipo et al.
hTLR4/MD2/CD14 and HEK293-hTLR2 were exposed to dif-
ferent concentrations of Alkalimonas or E. coli LPS (LPS-EB ultra-
pure, InvivoGen) (1, 10 and 100 ng/mL) and stimulation was pro-
longed for 4 h for HEK293-hTLR4/MD2/CD14 and 6 h for HEK
293-hTLR2, respectively. In the experiments aimed at measuring
TLR2 activation, synthetic triacylated lipoprotein Pam3CSK4
(0.5 μg/mL) (InvivoGen) was used as a positive control.
In the competition assays in HEK293-hTLR4/MD2/CD14, the
cells were primed with Alkalimonas LPS (1, 10 and 100 ng/mL) for
1 h and then re-stimulated with E. coli LPS (10 ng/mL) for 4 h.
After 4 h stimulation, NF-κB-dependent luciferase activity was
measured with a Dual-Luciferase Reporter Assay System (Pro-
mega, Milan, Italy), as reported.[32] CXCL-8 production was quan-
tified by ELISA assays (DuoSet, R&D System) according to manu-
facturer’s instructions.
Bone Marrow-Derived Macrophages (BMDMs) Culture and Stimu-
lation Assay: BMDMs were derived from the bone marrow cells
collected from five-week old wild-type C57BL/6 female mice
(Charles River Laboratories). BMDMs were differentiated during
7 d in RPMI1640 (Lonza, Italy), supplemented with 10 % heat-in-
activated FBS (Euroclone), 2 mm l-Glutamine, 5 % Na pyruvate
(Lonza, Italy), 0.1 mm NEAA (Euroclone), 0.5 % 2-ME (Gibco, It-
aly), and 30 ng/mL macrophage colony-stimulating factor (M-CSF)
(Miltenyi Biotec). At 7 d, BMDMs were characterized by immuno-
staining with F4-80 and MAC-1 monoclonal antibodies (BD
Pharmingen) through flow cytometric analysis with a FACSCalibur
cytometer.
For stimulation assays, BMDMs were seeded into 24-well plates
(5 ⫻ 105 cells), exposed to different concentrations of Alkalimonas
or E. coli LPS (1, 10 and 100 ng/mL) and stimulation was pro-
longed for 6 h. Cell supernatants were recovered and processed for
the measurement of TNF-α and KC production through ELISA
assays (DuoSet, R&D System) according to manufacturer’s in-
structions.
Statistical Analysis: Data are reported as mean ⫾ S. D., and the
numbers of independent experiments were indicated in each legend
of the figures Statistical calculations and tests were performed
using Student’s t-test. A P-value ⬍ 0.05 was considered statistically
significant. A P-value ⬍ 0.0001 was considered extremely signifi-
cant.
Supporting Information (see footnote on the first page of this arti-
cle): Spectra (Figure S1, Figure S2, Figure S3, and Figure S4) are
provided.
Acknowledgments
Ida Paciello is a fellow of the Istituto Pasteur-Fondazione Cenci
Bolognetti.
[1] W. D. Grant, B. E. Jones, in: Encyclopedia of Microbiology
(Ed.: J. Lederberg), Academic Press, London, 2000, 2nd ed., p.
73–80.
[2] W. D. Grant, W. E. Mwatha, B. E. Jones, FEMS Microbiol.
Rev. 1990, 75, 255–270.
[3] Y. Ma, Y. Xue, W. D. Grant, N. C. Collins, A. W. Duckworth,
R. P. van Steenbergen, B. E. Jones, Extremophiles 2004, 8, 193–
200.
[4] O. Takeuchi, S. Akira, Cell 2010, 140, 805–820.
[5] Silipo, A. Molinaro, in: Endotoxins: structure, function and re-
cognition, vol. 53 of the Subcellular Biochemistry series,
Springer, Heidelberg, Germany, 2010, 53, p. 69–99.
Eur. J. Org. Chem. 2013, 2653–2665
Lipopolysaccharide from Alkalimonas delamerensis
[6] A. Silipo, A. Molinaro, in: Bacterial Lipopolysaccharides,
Springer-Verlag, Berlin Heidelberg, Germany, 2011, p. 1–20 .
[7] R. Medzhitov, Nat. Rev. Immunol. 2001, 1, 135–134.
[8] A. Ialenti, P. Di Meglio, G. Grassia, P. Maffia, M. Di Rosa, R.
Lanzetta, A. Molinaro, A. Silipo, W. Grant, A. Ianaro, Eur. J.
Immunol. 2006, 36, 354–360.
[9] G. I. Birnbaum, R. Roy, J. R. Brisson, H. Jennings, J. Car-
bohydr. Chem. 1987, 6, 17–39; O. Holst, J. E. Thomas-Oates,
H. Brade, Eur. J. Biochem. 1994, 222, 183–194.
[10] A. Silipo, A. Molinaro, D. Comegna, L. Sturiale, P. Cescutti,
D. Garozzo, M. Parrilli, R. Lanzetta, Eur. J. Org. Chem. 2006,
4874–4883.
[11] P. J. Garegg, P. E. Jansson, B. Lindberg, F. Lindh, J. Lonngren,
I. Kvarnström, W. Nimmich, Carbohydr. Res. 1980, 78, 127–
132.
[12] A. G. Goncalves, D. R. B. Ducatti, M. E. R. Duarte, M. D.
Noseda, Carbohydr. Res. 2002, 337, 2443–2453.
[13] S. Leone, V. Izzo, A. Silipo, L. Sturiale, D. Garozzo, R. Lan-
zetta, M. Parrilli, A. Molinaro, A. Di Donato, Eur. J. Biochem.
2004, 271, 2691–704.
[14] a) L. Sturiale, A. Palmigiano, A. Silipo, Y. Knirel, A. P. Anisi-
mov, R. Lanzetta, M. Parrilli, A. Molinaro, D. Garozzo, J.
Mass Spectrom. 2011, 46, 1135–42; b) L. Sturiale, D. Garozzo,
A. Silipo, R. Lanzetta, M. Parrilli, A. Molinaro, Rapid Com-
mun. Mass Spectrom. 2005, 19, 1829.
[15] A. Domon, C. E. Costello, Glycoconjugate J. 1988, 5, 397.
[16] L. D. Hawkins, W. J. Christ, D. P. Rossignol, Curr. Top. Med.
Chem. 2004, 4, 1147–71.
[17] D. S. Zamboni, M. A. Campos, A. C. Torrecilhas, K. Kiss, J. E.
Samuel, D. T. Golenbock, F. N. Lauw, C. R. Roy, I. C. Alme-
ida, R. T. Gazzinelli, J. Biol. Chem. 2004, 52, 54405–15.
[18] R. P. Darveau, T. T. Pham, K. Lemley, R. A. Reife, B. W. Bain-
bridge, S. R. Coats, W. N. Howald, S. S. Way, A. M. Hajjar,
Infect. Immun. 2004, 9, 5041–51.
Eur. J. Org. Chem. 2013, 2653–2665 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
[19] P. Cescutti, R. Toffanin, P. Polesello, I. W. Sutherland, Car-
bohydr. Res. 1999, 315, 159–168.
[20] Y. Knirel, in: Bacterial Lipopolysaccharides Structure, Chemical
Synthesis Biogenesis and Interaction with Host Cells (Eds.: A. Y.
Knirel, M. A. Valvano), Springer, Wien, Austria, 2011.
[21] G. Zhao, A. V. Perepelov, S. N. Senchenkova, A. S. Shashkov,
L. Feng, X. Li, Y. A. Knirel, L. Wang, Carbohydr. Res. 2007,
342, 1275–1279.
[22] E. V. Vinogradov, R. Pantophlet, S. R. Haseley, L. Brade, O.
Holst, H. Brade, Eur. J. Biochem. 1997, 243, 167–173.
[23] A. Galanos, O. Luderitz, O. Westphal, Eur. J. Biochem. 1969,
9, 245–249.
[24] F. Kittelberger, J. Hilbink, J. Biochem. Biophys. Methods 1993,
26, 81–86.
[25] A. Silipo, L. Sturiale, C. De Castro, R. Lanzetta, M. Parrilli,
D. Garozzo, A. Molinaro, Carbohydr. Res. 2012, 1, 75–82.
[26] K. Leontein, J. Lönngren, Methods Carbohydr. Chem. 1978, 62,
359–362.
[27] S. Hakomori, J. Biochem. 1964, 55, 205–208.
[28] E. T. Rietschel, Eur. J. Biochem. 1976, 64, 423–428.
[29] A. Silipo, M. R. Leone, G. Erbs, R. Lanzetta, M. Parrilli, W. S.
Chang, M. A. Newman, A. Molinaro, Angew. Chem. 2011, 123,
12818; Angew. Chem. Int. Ed. 2011, 50, 12610–12612.
[30] T. Ieranò, A. Silipo, P. Cescutti, M. R. Leone, R. Rizzo, R.
Lanzetta, M. Parrilli, A. Molinaro, Chem. Eur. J. 2009, 15,
7156–7166.
[31] A. Silipo, L. Sturiale, D. Garozzo, C. de Castro, R. Lanzetta,
M. Parrilli, W. Grant, A. Molinaro, Eur. J. Org. Chem. 2004,
2263–2271.
[32] G. Nigro, L. L. Fazio, M. C. Martino, G. Rossi, I. Tattoli, V.
Liparoti, C. De Castro, A. Molinaro, D. J. Philpott, M. L. Ber-
nardini, Cell Microbiol. 2008, 3, 682–95.
Received: December 17, 2012
Published Online: March 19, 2013
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