NMR conformational analysis of biosynthetic precursor-type lipid A: Monomolecular state and supramolecular assembly

Article abstract of DOI:10.1039/b410544c

The detailed conformational analysis of a single molecule of the tetraacyl biosynthetic precursor-type lipid A and its characteristic supramolecular assembly in aqueous SDS-micelles are described. Regular molecular arrangements were observed by detailed a

Full text of DOI:10.1039/b410544c

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A R T I C L E
NMR conformational analysis of biosynthetic precursor-type lipid
A: monomolecular state and supramolecular assembly†
Masato Oikawa, Tetsuya Shintaku, Naohiro Fukuda, Harald Sekljic, Yoshiyuki Fukase,
Hiroaki Yoshizaki, Koichi Fukase* and Shoichi Kusumoto
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama,
Toyonaka, Osaka, 560-0043, Japan. E-mail: koichi@chem.sci.osaka-u.ac.jp
Received 12th July 2004, Accepted 30th September 2004
First published as an Advance Article on the web 8th November 2004
The detailed conformational analysis of a single molecule of the tetraacyl biosynthetic precursor-type lipid A and its
characteristic supramolecular assembly in aqueous SDS-micelles are described. Regular molecular arrangements
were observed by detailed analysis of the NMR spectra of synthetically pure specimens, including regiospecifically
13C-labeled ones. NMR analysis of a biologically inactive precursor-type analogue with four shorter acyl chains
demonstrated its conformational flexibility, indicating the importance of hydrophobic interactions for maintaining
the conformation of such molecules.
transform and attenuated total reflectance infrared spectroscopy
Introduction
to estimate the approximate molecular shapes responsible for
Lipopolysaccharide (LPS) is a characteristic component of the
the activity.5,17–24 Only X-ray crystallographic data of a complex
outer membrane of Gram-negative bacteria and forms the
of FhuA and E. coli K12 LPS provided a precise LPS con-
outermost leaflet of the lipid bilayer of the membrane.1–5 LPS is
formation of the molecular level before the present study.25,26
also known as endotoxin for its toxicity and its potent immunos-
Molecular modeling studies of lipid A and LPS have also been
timulation in higher animals. LPS is a typical inducer of innate
reported.22,23,27–32 The dihedral angles of the glycosidic bonds of
immunity and leads to the production of various mediators such
some models coincide with the X-ray crystallographic data but
as cytokines, prostaglandins, platelet activating factor, oxygen
the inclination of the lipid A backbone to acyl moieties did
free radicals and NO. These mediators activate and modulate
not.27–29 Conformational studies of lipid A in organic solvents
the immune system to protect against infection. When a large
using NMR afforded different results from those obtained by
amount of LPS is released by a severe infection of Gram-negative
the above X-ray analyses.32,33
microbes, overproduced mediators lead to endotoxin-related
We therefore started independent conformational analysis
symptoms such as high fever, serious inflammation, hypotension
of lipid A by NMR in aqueous media to obtain the three-
and, in serious cases, lethal shock.
LPS consists of a glycolipid component termed lipid A and
dimensional structure in relation to its biological functions. In
this paper, we describe direct NMR evidence leading to the
a polysaccharide part. It has been unequivocally proven that
elucidation of 1) the monomolecular conformation and 2) a
lipid A is the chemical entity responsible for the biological
supramolecular assembly of biosynthetic precursor-type lipid A
activity of LPS by our total synthesis of Escherichia coli lipid
2 in aqueous micelles. We also analyzed the monomolecular
A (1) (Fig. 1).6,7 We have also synthesized LPS from Re-mutant
conformation of a biologically inactive analogue 3 in both
bacteria (Re-LPS),8 which contains two 3-deoxy-D-manno-2-
aqueous solutions and micelles. The importance of hydrophobic
octulosonic acid (Kdo) residues linked to lipid A and various
interaction of acyl groups for maintaining the conformation of
structural analogues of lipid A. Biological tests of synthetic
the molecule of lipid A is also discussed.
analogues led us to conclude that hydrophobic acyl groups in
lipid A play a critical role for the expression of activity.9–13
The tetraacyl lipid A 2, designated as precursor Ia14 or lipid
Results
IVA,15 corresponds to a lipid A component from LPS of a
temperature-sensitive mutant of Salmonella typhimurium16 and
was identified as a key biosynthetic precursor of LPS. Inter-
estingly, the tetraacyl biosynthetic precursor 2 shows endotoxic
activity in mice but acts as a potent antagonist in suppressing the
action of LPS in human systems.9–13 By contrast, precursor-type
analogue 3 with shorter acyl chains, which possesses four (R)-3-
hydroxydecanoic acids in place of (R)-3-hydroxytetradecanoic
acids in 2, shows neither endotoxic nor antagonistic activities.11
We assumed that the hydrophobic interaction between the
acyl groups in lipid A should be important in maintaining its
particular molecular conformation and such a conformation
may be recognized by LPS binding proteins such as CD14, toll
like receptor 4 (TLR4)–MD-2 complex and CD55.1–5
Conformations of oligosaccharides in solution have generally
been determined by NMR using information regarding the
atom distances between monosaccharide residues and dihedral
angles around the glycosidic bonds.34 The former information
is obtained from NOE analysis and the latter is determined
from heteronuclear coupling constants, 3JC,H, across glyco-
3
sidic linkages.35 It had been difficult to obtain JC,H values
because of the low natural abundance of the 13C isotope.
Recent development of J-HMBC 2D experiments overcame this
disadvantage.36–38 Combinations of hetero half-filtered total cor-
relation spectroscopy (HETLOC) and phase-sensitive HMBC
3
have also proved to be useful in determining JC,H values.39,40
These methods, however, failed to give a reliable result with the
natural abundance of 13C owing to the low solubility of the lipid
A analogue 2. Hence, we decided to synthesize two biosynthetic
precursor derivatives, 13C-labeled at the 1ꢀ- and 6-positions, to
overcome the solubility problem. Having already completed the
synthesis of 6-13C-labeled 2 and its short chain analogue 3,41 in
the present study we synthesized the 1ꢀ-13C-labeled derivatives of
2 and 3.
Conformations of lipid A and LPS have so far been in-
vestigated mainly using X-ray powder-diffraction and Fourier-
† Electronic supplementary information (ESI) available: other NMR
data, synthetic data and rotatable 3D crystal structure diagrams of con-
formation i, conformation ii, conformation short, supramolecule I and
supramolecule II. See http://www.rsc.org/suppdata/ob/b4/b410544c/
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 5 7 – 3 5 6 5
3 5 5 7
©

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Fig. 1 The chemical structures of Escherichia coli lipid A, the biosynthetic precursor-type lipid A and a short chain analogue of the biosynthetic
precursor.
Synthesis of 13C-labeled compounds
The 1ꢀ-13C-labeled derivative of 2 was de novo synthesized as
shown in Schemes 1 and 2. The 1,6:2,3-dianhydro-(1-13C)
Scheme 2 Synthesis of 1ꢀ-13C-labeled biosynthetic precursor.
mannopyranose 4 was prepared from commercially available
D-(1-13C)glucose as described previously for the 6ꢀ-labeled
compound.41 The benzyl group of 4 was changed to an
acetyl group. An azido group was then introduced at the 2-
position and the resulting 2-azido anhydroglucose 7 was treated
with phenyl trimethylsilyl sulfide and zinc iodide to give the
thioglycoside 8 (a : b = 5 : 1). Removal of the acetyl groups of
8 followed by benzylidenation afforded 10. The azido group
of 10 was reduced and then a 2,2,2-trichloroethoxycarbonyl
(Troc) group was introduced to the resulting 2-amino group as a
stereochemical auxiliary for b-selective glycosylation to give 11.
(R)-3-Benzyloxytetradecanoic acid11 20 was introduced to the 3-
position of the a-anomer of 11, which was chromatographically
isolated from the anomeric mixture. Regioselective reductive
opening of the benzylidene group of 12 and subsequent cleavage
of the thioglycoside gave the 1,4-dihydroxy compound 14. We
have traditionally used the trichloroacetimidate method for
glycosylation with N-Troc glucosaminyl donors. In the present
study however, we used a glycosyl phosphate as a donor42–46
in order to reduce the number of reaction steps. In this way,
compound 14 was phosphorylated using Watanabe’s reagent47
to give the bisphosphate 15, which was to be used as the glycosyl
donor (Scheme 1). The glycosylation of the acceptor 16 with
Scheme 1 Synthesis of 13C-labeled glycosyl donor.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 5 7 – 3 5 6 5
15 proceeded smoothly, using trimethylsilyltrifluoromethane
3 5 5 8

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1
ꢀ
Fig. 2 One-dimensional NMR spectra of unlabeled 2. A: H NMR spectrum (500 MHz). B: GOESY spectrum irradiated at 4.67 ppm (H1 ) with a
mixing time of 100 ms.
sulfonate (TMSOTf), to give the disaccharide 17 in a good yield.
Cleavage of the N-Troc group was followed by N-acylation
to give fully the acylated disaccharide 18. The allyl group
of 18 was then removed and the resulting 1-hydroxy group
was phosphorylated. Final deprotection of all the benzyl-type
protective groups by catalytic hydrogenation gave the desired
1ꢀ-13C-labeled 2. The 1ꢀ-13C-labeled derivative of the short chain
analogue 3 was synthesized in a similar manner.
Conformation of the biosynthetic precursor 2
Compound 2 gave well-resolved 1H NMR spectra in DMSO-d6
33
but broad signals in D2O, owing to the short spin–spin relaxation
time (T2) due to self-aggregation. Addition of SDS to aqueous
solutions of amphiphilic glycolipids has been shown to give
sharp NMR signals of their hydrophilic parts.48–50 Well-resolved
sharp spectra were obtained of 2 in SDS-micelles (Fig. 2, A)
and the following precise analysis thus became possible. Micelle
conditions under a large excess of SDS employed in this work
are regarded as a simulation of the amphiphilic environment on
the cell surface membrane of both bacteria and host animals.
Under such amphiphilic conditions, the conformation of lipid
A must be regulated by the packing of acyl chains in lipid layers
or micelles owing to the lipophilic aggregation.
The labeled and unlabeled 2 gave identical 1H NMR spectra,
except for the splitting of the proton(s) on the labeled position in
the former (1JC6,H6 = 150 and 143.5 Hz for 6-13C-2, and 1JC1 ,H1
=
ꢀ
ꢀ
163.9 Hz for 1ꢀ-13C-2), respectively. The concentration of SDS, at
88 mM, was far higher than its critical micelle point (8.2 mM),51
hence thesolution was considered to be under micelleconditions.
Proton signals of 2 in SDS-d25–D2O were assigned by COSY and
NOESY. The distinct signal of 13C-labeled C6 at d 68.5 made it
easy to assign the signals of H4 and H5 by the use of a 13C–1H
Fig. 3 NOESY (600 MHz) spectra of 13C-labeled 2. A: The correlated
signals between the lower half of H6a/H5 (X) and the upper half of
H6b/H5 (Y) of 6-13C-2 with a mixing time of 300 ms. Both signals are
2
split by JC6,H5 in the T2 dimension. B: The correlated signals between
the left half of H6a/H1 (X) and the right half of H6b/H1 (Y) of 6-13C-2
ꢀ
ꢀ
with a mixing time of 500 ms. Both signals are split by 3JC6,H1 in the T1
ꢀ
ꢀ
HMBC spectrum. Protons at C6 and C6 were assigned on the
dimension. C: The correlated signals of H6a/H1 and H6b/H1 of 1ꢀ-13C-2
ꢀ
ꢀ
basis of Ohrui’s empirical rule.52 The clear assignment for all
with a mixing time of 300 ms. Both signals are split by 3JC1 ,H6a or 3JC1 ,H6b
in the T2 dimension. The slope of the linked line between the split cross
peaks against the slope of diagonal peaks represents the relative sign of
the coupling constants.53
ꢀ
ꢀ
ꢀ
protons, C1 (d 102.7) and C6 (d 68.5) of 2, shows that there are
no conformational changes in SDS-d25–D2O which are slower
than the time scale of NMR. Important nJX,H-coupling constants
which were determined are as follows. The 3JH5,H6a (1.4 Hz), 3JP1,H1
3
1
ꢀ
ꢀ
(1.0 Hz) and JP4 ,H4 (7.2 Hz) values were obtained from H J-
3
2
resolutional spectroscopy. JC6,H1 (6.6 Hz) and JC6,H5 (1.7 Hz)
peak structure in the NOESY spectra of 6-13C-2 (Fig. 3, A and
ꢀ
were from the splitting of the respective signals (H6/H1 and
Fig. 3, B). The 3JC1 ,H6a (0 Hz) and 3JC1 ,H6b (4.2 Hz) values were
evaluated from the NOESY spectrum of 1ꢀ-13C-2 (Fig. 3, C) in
ꢀ
ꢀ
ꢀ
H5/H6), both appearing as an “exclusive COSY”-like53 cross-
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 5 7 – 3 5 6 5
3 5 5 9

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a similar manner. These selected NMR data are summarized in
Fig. 4.
4
Since the two glucosamine residues in 2 share normal C1
chair conformations, as concluded from the 3JH,H values of the
ring protons (Table 1), the rotational angles of the three bonds
which determine the overall shape of the molecule, i.e., C5–C6,
ꢀ
C6–O, and O–C1 between the two pyranosyl residues, had to be
estimated.
Fig. 4 Important nJX,H-cou◦pling co◦nstants of 2 for the conformational
◦
ꢀ
analysis. {q, w, φ} = {−120 , −150 , −60 } [q = (O–C1 –Og–C6), w =
ꢀ
(C1 –Og–C6–C5), φ = (Og–C6–C5–O)].
Fig. 5 T-ROESY (500 MHz) spectrum of unlabeled 2 obtained with a
mixing time of 75 ms. Negative cross peaks are omitted for clarity.
The dihedral angle of the H5–C5–C6–Og bond was determined
possible dihedral angles for H6a–C6–Og–C1 ( 90◦) and H6b–
2
3
2
ꢀ
from JC6,H5 and JH5,H6a values: the JC6,H5 value of 1.7 Hz
indicates H5–C5(OR)–C6–OgR to be in a trans-conformation,54
C6–Og–C1 ( 30◦ or 140◦) were calculated by putting these
ꢀ
which is also supported by the small 3JH5,H6a value (1.4 Hz).
two JC,H values into the above Karplus equation. From the
stereochemical assignment for H6a (pro-S) and H6b (pro-R) and
3
3
ꢀ
The JC6,H1 value (6.6 Hz) was used for the determination
ꢀ
ꢀ
ꢀ
ꢀ
of the dihedral angle of C6–Og–C1 –H1 . The large coupling
constant observed shows that the conformation is either cis
or trans according to the Karplus equation with Tvaroska’s
the observed NOEs at H6a/H1 and H6b/H1 , two dihedral angles
for the C6–Og bond were concluded to be +90◦ and −30◦ for
ꢀ
ꢀ
H6a–C6–Og–C1 and H6b–C6–Og–C1 , respectively.
coefficients, JC,H = 5.7·cos2h − 0.6·cosh + 0.5, where h is
The orientation of the phosphates can be likewise determined
by the 3JP,H value using the Karplus equation, 3JP,H = A·cos2h −
B·cosh, where A (e.g., 18.1) and B (e.g., 4.8) are the coefficients
reported in several reports56–59 and h is the dihedral angle of
3
55
ꢀ
ꢀ
the dihedral angle of C6–Og–C1 –H1 . Taking into account the
ꢀ
observed strong NOE between H6b and H1 (Fig. 2, B and Fig. 5),
the conformation should be cis but never trans.
Rotational conformation for C6–Og can be drawn by two
3
P–O–C–H. The coupling constants for JP1,H1 (1.0 Hz) and
3
ꢀ
ꢀ
ꢀ
ꢀ
dihedral angles for H6a–C6–Og–C1 and H6b–C6–Og–C1 , and was
uniquely determined on the basis of two values, i.e., 0 Hz for
JP4 ,H4 (7.2 Hz) show the relationships between these nuclei
to be exclusively gauche ( 60◦ or 100◦) and non-restricted,
respectively. It should be noted here that Batley et al. reported
the corresponding value of a deacylated LPS in D2O to be
7.2 Hz which differs from our result.49 This discrepancy is
attributable to the different mode of aggregation and molecular
3
3
3
ꢀ
ꢀ
JC1 ,H6a and 4.2 Hz for JC1 ,H6b as follows. Since JC,H generally
3
ꢀ
assumes positive values, 0 Hz for JC1 ,H6a should indicate the
occurrence of a single conformation but not an equilibrium
mixture of various conformers around the C6–O bond. The
Table 1 Assignments of 1H NMR (500 MHz) signals of 6-13C-2 in SDS-d25–D2O
Protons
Chemical shifts d
Multiplicity
Coupling constants J/Hz
H1
H2
H3
H4
5.41
4.12
5.16
3.84
3.94
4.09
3.85
4.67
4.01
5.18
4.09
3.57
3.84
3.80
dd
dd
dd
m
m
ddd
m
dd
dd
dd
ddd
m
m
m
1.0, 1.0
9.8, 1.0
10.4, 9.8
10.4c
H5
1.7, 1.4c
150, 10.2, 1.4
143.5, 10.2c
11.5, 6.6
11.5, 7.9
9.2, 7.9
9.2, 9.2, 7.2
9.2c
H6a(pro-S)a
H6b(pro-R)a
H1ꢀ
H2ꢀ
H3ꢀ
H4ꢀ
H5ꢀ
H6’a(pro-S)a
H6’b(pro-R)a
acyl groupsa
oxymethines (4H)
methylenes (8H)
other methylenes (80H)
methyl (12H)
—
—
—
—
—
—
—
3.95, 3.94, 3.91, 3.91
2.57, 2,47, 2.45, 2.39, 2.34, 2.31, 2.29, 2.25
1.50–1.14
0.86–0.75
m
m
m
m
a The low-field protons on C6 and C6 designated ‘a’ were assigned to be pro-S according to the literature.52 b Protons on the acyl groups were not
ꢀ
completely assigned. c These protons have other undetermined coupling constants.
3 5 6 0
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 5 7 – 3 5 6 5

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conformation (see below) owing to the different acylation
patterns.
Further important conformational information was obtained
from NOE data. Because there are negative correlations caused
by Hartmann–Hahn effects or spin diffusions in T-ROESY
spectra (Fig. 5), some positive ROESYs are not clearly observed
ꢀ
from H1 , such as NOEs shown in the GOESY spectrum (Fig. 2,
B). Both GOESY and T-ROESY spectra were, therefore, con-
sidered complementary to the conformational analysis. From
these spectra, a number of NOE signals can be seen between
ꢀ
the protons of interglycosidic relationships, such as H6a/H1
ꢀ
(medium) and H6b/H1 (strong) as well as the normal intragly-
ꢀ
ꢀ
ꢀ
ꢀ
cosidic correlations of H1 /H3 (strong), H3 /H5 (strong) and
ꢀ
ꢀ
ꢀ
H1 /H5 (strong). Though the H1 signal (d 4.67) is overlapped
1
by HDO in the H NMR spectrum, the GOESY correlations
(Fig. 2, B) are apparently not those between HDO and 2 for
the following reasons; 1) irradiation at HDO (d 4.65) itself
showed no GOESY signals in the region of d 3.5–4.2; and 2)
reverse irradiation at d 3.5–4.2 gave no GOESY with HDO (d
ꢀ
4.65) but with H1 (d 4.67). Two interglycosidic NOEs agree
ꢀ
with the dihedral angles around the C6–Og and Og–C1 bonds
Fig. 7 Supramolecular and molecular conformation of 2 (conformer I).
Elements are distinguished by color: carbon (white), hydrogen (white),
oxygen (red), nitrogen (blue) and phosphorus (green). The CPK model
is used for visualization in C and D. A: single molecular conformation i.
B: front view of a supramolecule, consisting of 5 molecules of 2. C: top
view of the supramolecule. A central single molecule is shown in yellow.
D: side view of the supramolecule.
assigned above. From these NMR data, the monomolecular
conformation of 2 can be roughly drawn as illustrated in Fig. 6.
C1 –H1 (+52◦)] was found by pure Low mode conformational
search.
ꢀ
ꢀ
The monomolecular conformation i was then used to con-
struct supramolecular structures. Since intermolecular NOEs
ꢀ
ꢀ
at H1/H3 and H2 /H5 cannot occur simultaneously, the NOE
data may possibly account for the fluctuation of supramolecular
aggregation or the presence of two types of aggregations.
In the present study, the geometry of the supramolecular
assembly was optimized independently for two intermolecular
relationships, assuming that one particular assembly exhibits
an intermolecular NOE at H1/H3 (conformer I) and the other
ꢀ
ꢀ
shows that of H2 /H5 (conformer II).
A supramolecule with five lipid A molecules was chosen
Fig. 6 Important NOE data of 2 for the conformational analysis. Bold
(black), bold (gray), plain and dashed lines indicate the intensity of NOE
correlations: strong, medium, weak and very weak, respectively.
as a model. The geometrical restraints H5–C5–C6–Og (180◦),
◦
H6a–C6–Og–C1 (+90 ), and C6–Og–C1 –H1 (0◦), as well as the
ꢀ
ꢀ
ꢀ
˚
ꢀ
ꢀ
intermolecular distance restraints, H1/H3 (2.5 A) and H1 /H2
˚
Besides these intramolecular correlations used for the con-
formational consideration of the single molecule, the other
signals observed in Fig. 2, B and Fig. 5 include the correlations
(4.0 A), were applied to generate supramolecular structure I. An
additional dihedral restraint was also applied for P1–O–C1–H1
to be −60◦. The geometry of I was then minimized without any
constraints under Amber* force field (Fig. 7).
ꢀ
ꢀ
ꢀ
ꢀ
of H1/H3 (strong), H1 /H2 (very weak), and H2 /H5 (weak),
which cannot be explained by intramolecular interactions from
the stereochemical point of view (Fig. 6). These positive cross
peaks in T-ROESY can be regarded as direct NOEs60 and are,
hence, rationally assigned to be intermolecular ROESY signals.
These intermolecular ROESYs provide direct evidence of the
supramolecular formation of lipid A in aqueous micelles.
Based on the conformational information obtained so far
from NMR, molecular modeling was studied next. The geom-
etry of monomolecular lipid A was optimized by the molecu-
lar mechanics calculations under AMBER* force field61 with
a generalized Born/surface area (GB/SA) continuum water
solvation model62 using MacroModel version 7.0 (Schro¨dinger
Inc.). Since NMR provided only the data for the conformation
of the saccharide part, the geometry of the acyl moieties was
determined as the energy minimized conformation fulfilling the
structural information shown in Fig. 4 and Fig. 6. For the ester
and amide groups the s-trans conformations were employed. The
monomolecular conformation i [H5–C5–C6–Og (172◦), H6a–C6–
The two phosphate groups occupy gauche and syn positions
against the respective◦vicinal protons◦(averaged dihedral angles
ꢀ
of P–O–C–H are −49 for P1 and −8 for P4 ) and the averaged
◦
◦
ꢀ
dihedral angles are H5–C5–C6–Og (169 ), H6a–C6–Og–C1 (+88 ),
◦
◦
ꢀ
ꢀ
H6b–C6–Og–C1 (−32 ) and C6–Og–C1 –H1 (+15 ). The averaged
ꢀ
ꢀ
distances of H6a/H1 showing medium NOE, and H6b/H1
˚
˚
showing strong NOE are 2.9 A and 2.1 A, respectively. The
ꢀ
ꢀ
ꢀ
averaged distances of H1/H3, H1 /H2 and H2 /H5 in the most
˚
˚
˚
stable supramolecule I were 3.0 A, 4.0 A and 5.8 A, respectively.
One further supramolecular structure II was constructed by
a similar calculation protocol using another monomolecular
◦
geometry ii [H5–C5–C6–Og (170 ), H6a–C6–Og–C1 (+90◦) and
ꢀ
C6–Og–C1 –H1 (+17◦)] (Fig. 8, A), whose conformation in
ꢀ
ꢀ
acyl moieties and the information for intermolecular distances
∼
ꢀ
ꢀ
ꢀ ꢀ
H2 /H5
H1 /H2 < H1/H3 differs slightly from that of I.
=
Minimization of the supramolecule generated using i and these
intermolecular distances, afforded the disordered structure. The
optimized supramolecular assembly II is shown in Fig. 8. The
◦
◦
ꢀ
ꢀ
ꢀ
ꢀ
Og–C1 (+84 ) and C6–Og–C1 –H1 (+18 )] was thus obtained as
a stable conformer (Fig. 7, A) without any constraints, though i
was not a global energy◦minimum. The more stable conformer
conformations of phosphate group P1 and P4 were found to be
a mixture of gauche and syn [P1: two gauche (−34.0◦) and three
◦
◦
◦
syn (+8.2 ),63 P4 : four gauche (−50.0 ) and one syn (−6.0 )].
ꢀ
of 2 [H5–C5–C6–Og (178 ), H6a–C6–Og–C1 (+112◦) and C6–Og–
The average dihedral angles for bonds between pyranose rings
ꢀ
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 5 7 – 3 5 6 5
3 5 6 1

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of overlapping NOE (H1ꢀ-H6a) and TOCSY signals (H1ꢀ-H4ꢀ).
ꢀ
ꢀ
Strong NOE signals were observed on H6a/H1 and H6b/H1 .
ꢀ
The possible dihedral angles for H6b–C6–Og–C1 were, therefore,
−40◦ or −130◦. The coupling constants for JP1,H1 (3.2 Hz)
3
3
ꢀ
ꢀ
and JP4 ,H4 (7.0 Hz) show the relationships between these
nuclei to be exclusively gauche and non-restricted, respectively.
In conclusion, several conformers of 3 exist in D2O and the
molecule freely rotates at least around the C5–C6 bond.
The coupling constants in SDS-d25–D2O were different from
those in D2O. Important nJX,H-coupling constants of 3 in SDS-
d25–D2O are as follows: 3JH5,H6a (0 Hz), 3JH5,H6b (5.9 Hz), 3JC6,H1
ꢀ
(2.9 Hz), 3JC1 ,H6a (0.7 Hz), 3JC1 ,H6b (1.8 Hz), 3JP1,H1 (2.0 Hz) and
ꢀ
ꢀ
3
ꢀ
ꢀ
JP4 ,H4 (7.1 Hz). A number of intramolecular NOE signals in-
cluding H6a/H1 (medium) and H6b/H1 (strong) were observed.
ꢀ
ꢀ
From these values and NOE data, the conformation of 3 in
◦
ꢀ
ꢀ
SDS-d25–D2O was deduced to be C6–Og–C1 –H1 : +45 , H6a–C6–
◦
◦
ꢀ
ꢀ
Og–C1 : +100 , H6b–C6–Og–C1 : −25 , H5–C5(OR)–C6–OgR:
trans (53% population) and gauche (−60 ◦: 47% population).
Molecular mechanics calculation of monomolecular 3 afforded
◦
ꢀ
ꢀ
ꢀ
the stable conformer [C6–Og–C1 –H1 (+54 ), H6a–C6–Og–C1
(+104 ◦), H5–C5–C6–Og (179 ◦)] without any constraints (Fig. 9).
The calculated conformer is hence identical with the observed
major one.
Fig. 8 Supramolecular and molecular conformation of 2 (conformer
II). Elements are distinguished by color: carbon (white), hydrogen
(white), oxygen (red), nitrogen (blue) and phosphorus (green). The
CPK model is used for visualisation in C and D. A: single molecular
conformation ii. B: front view of the supramolecule, consisting of 5
molecules of 2. C: top view of the supramolecule. Centered molecule is
shown in yellow. D: side view of the supramolecule.
◦
are H5–C5–C6–Og (+161.4 ), H6a–C6–Og–C1 (+97.3◦), H6b–C6–
ꢀ
Og–C1 (−22.3 ) and C6–Og–C1 –H1 (+38.8◦).
◦
ꢀ
ꢀ
The average internuclear distances around the glycosidic
˚
˚
ꢀ
ꢀ
linkage are 3.2 A for H6a/H1 and 2.1 A for H6b/H1 . The
ꢀ
˚
ꢀ
ꢀ
averaged distances of H1/H3, H1 /H2 and H2 /H5 in the most
˚
˚
stable supramolecule II were 3.6 A, 2.3 A and 2.6 A, respectively.
On the basis of two independent intermolecular NOE and
other NMR data, two types of aggregation I and II were
illustrated. It is likely that supramolecule I is the major one, as
estimated from the relative intensity of the two intermolecular
Fig. 9 Stable conformer of the short-chain analogue 3.
Contrary to the observation for 2, compound 3 does not form
a supramolecular assembly in SDS-micelles, as judged from the
fact that no intermolecular NOE signals were observed.
ꢀ
ꢀ
NOEs of H1/H3 and H2 /H5 , though this value cannot be
directly related to the population of I and II. The supramolecular
models obtained by the calculation were in good agreement with
the observed data. At present no conclusion can be made as to
whether supramolecular forms I and II are present close together
in each single aggregate or separately from each other in different
aggregates. In addition, it is not known whether 2 forms mixed
aggregates with SDS or forms separate aggregates. In any case,
the supramolecules shown in Fig. 7 and Fig. 8 are assumed to be
present in an aqueous media in not such fragmentary forms, but
as parts of higher aggregates such as micelles or liposome-like
double-layered particles.
Discussions
The present study provides the first NMR evidence of lipid A
conformation in aqueous media. The biosynthetic precursor
lipid A 2 forms two types of supramolecular assembly, which
may be in a kind of dynamic equilibrium. It should be noted here
that the observed monomolecular conformation of 2, especially
ꢀ
ꢀ
the geometry around the glycosidic bond C6–Og–C1 –H1 , is not
energetically the most favored as indicated by the molecular
mechanistic calculation of the single molecule. A set of the
torsion angles of 2 derived from the observed conformation
in the SDS-micelles was {q, w, φ} = {−120◦, −150◦, −60◦}
Conformation of short chain analogue of the biosynthetic
precursor
1
ꢀ
ꢀ
The short chain analogue 3 in D2O gave analysable H-NMR
[q = (O–C1 –Og–C6), w = (C1 –Og–C6–C5), φ = (Og–C6–C5–
O)]. The observed dihedral angle C6–Og–C1 –H1 (ca. 0◦) (q =
−120◦) was significantly low in view of the exoanomeric effect,
which favors the dihedral angle of the glycosidic bond to be
3
3
ꢀ
ꢀ
spectra. The JH5,H6a (2.5 Hz) and JH5,H6b (3.1 Hz) values
were obtained from 1H J-resolutional spectroscopy of non-
labeled 3 in D2O. These results indicated the presence of trans-
conformation (77% population) and two gauche-conformations
(−60◦: 13% population, 60◦: 10% population) in H5–C5(OR)–
ca. 40–60◦ (C6–Og–C1 –H1 ). A set of dihedral angles of the
most stable conformer of monomeric 2 calculated here was {q,
ꢀ
ꢀ
◦
◦
◦
ꢀ
C6–OgR. The populations of conformers were determined by
w, φ} = {−67 , −128 , −63 }. The distortion at C6–Og–C1 –
Ohrui’s equation52 The JC6,H1 (1.5 Hz) was obtained from the
H1 in the micelle should be compensated by the formation
3
ꢀ
ꢀ
ꢀ
splitting of the signal (H6/H1 ) in the NOESY spectra of 6-
of a supramolecular structure; the distorted monomolecular
conformation may become a favorable one by making the
intermolecular hydrophobic interaction largest. The alignment
of both phosphate moieties of the assembling molecules is
another characteristic of this arrangement (Fig. 7C and Fig. 8C),
13C-3. Taking into account the exoanomeric effect, the dihedral
◦
3
ꢀ
ꢀ
ꢀ
angle of C6–Og–C1 –H1 was deduced to be +60 . The JC1 ,H6b
value (3.1 Hz) was evaluated from the NOESY spectrum of
1ꢀ-13C-3, whereas the JC1 ,H6a could not be determined because
3
ꢀ
3 5 6 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 5 7 – 3 5 6 5

View Article Online
which may have a further function to stabilize the supramolecule,
particularly in the presence of multivalent cations.
indicating its lower fluidity compared to that of 2. A similar
arrangement was observed for Re-LPS, which is composed of a
hexaacyl lipid A and two additional sugar units, by using X-ray
diffraction and atomic force microscopic studies.66 These results
suggest that LPS in the bacterial outer membrane forms a similar
assembly, which may be further stabilized by the multivalent
cations such as Ca2+ and Mg2+. The strong tendency of lipid A
molecules to form such a supramolecular structure is expected
to be the origin of the mechanical stability of the bacterial outer
membrane.
Recently, Seydel et al. revealed that monomeric lipid A
and LPS prepared by a dialysis procedure showed no activity,
whereas aggregates at the same concentrations were biologically
active.67 These results suggest that monomeric lipid A and LPS
might be conformationally flexible due to the lack of intermolec-
ular hydrophobic interactions and hence is not recognised by the
LPS receptor system. The particular conformation of lipid A in
the supramolecular assembly should be recognized by the LPS
receptor system.
Triantafilou reported that LPS triggers the recruitment of
receptors in the LPS receptor system, e.g., TLR4-MD2, CD14,
CD55, CD11b/CD18, CD81 and CXCR4, within membrane
microdomains on the cell membrane.68 The driving force for
the cluster formation is not yet known. Seydel et al. reported
lipopolysaccharide-binding protein-mediated intercalation of
lipid A to phospholipid membranes.19 The present study suggests
that lipid A incorporated in the membrane still forms a
supramolecular assembly, which may accumulate in membrane
microdomains such as lipid rafts, since lipid A has many
saturated fatty acids. Lipid A binding proteins, therefore, may
gather on membrane microdomains and trigger LPS signaling.
Before the present study, only X-ray crystallographic data
of the complex of FhuA and E. coli K12 LPS provided the
empirical information of lipid A conformation.25,26 The dihedral
angles around the glycosidic bonds in the lipid A part were {q,
w, φ} = {−75◦, −150◦, −108◦}. Energy minimization of the
hexaacylated lipid A part derived from the reported X-ray data
under Amber* force field led to similar torsion angles, except φ
{q, w, φ} = {−72◦, −132◦, −77◦}. This result suggests that the
complex formation of E. coli K12 LPS with FhuA probably
distorted the φ angle in its lipid A part. The conformation
around the glycosidic bonds of the biosynthetic precursor 2 in
aqueous SDS-micelles obtained as above is essentially the same
as that of the lipid A part in K12 LPS observed in X-ray analysis,
since the differences of all dihedral angles between the two works
are less than 60◦.
Seydel, Brandenburg and coworkers have studied the
supramolecular structures and intrinsic conformations of lipid
A derivatives in relation to their biological activities.5,17–21,24 The
present supramolecular models of 2 are in good accordance
with their observations, as follow. They reported an aggregated
lamellar structure of the biosynthetic precursor lipid A 2 in
water, indicating the cross-sectional areas of hydrophilic and
hydrophobic parts are almost the same. The small tilt angle
(ca. 10◦) of the backbone with respect to the direction of acyl
chains was observed in the same aggregates of 2.21 They also
reported a highly hydrated 1-phosphate and a weakly hydrated
4ꢀ-phosphate in lipid A and LPS.18 These lead to a molecular
shape in which 1-phosphate is more accessible to the aqueous
phase than the 4ꢀ-phosphate.
Several molecular mechanics and molecular dynamics studies
have also been reported for conformational study of lipid A and
LPS.22,23,27–31 As for the torsion angles of the glycosidic bonds,
the models of both Labischinski et al. and Obst and coworkers
are consistent with the X-ray crystallographic data of the FhuA-
E. coli K12 LPS complex.23,27–29 Those models, however, have a
reverse inclination (the 1-phosphate being close to the bilayer
and the 4ꢀ-phosphate being directed to the aqueous phase),
which is inconsistent with the X-ray crystallographic data and
the observations of Seydel et al.
The importance of hydrophobic interactions in the formation
of lipid A conformation was also proven by our NMR study of
the short-chain analogue 3. The short-chain analogue 3 takes
several conformations in water and SDS-d25–D2O, as indicated
by NMR. The hydrophobic interaction between the acyl groups
of 3 may not be strong enough to maintain any particular
conformation, though the major conformation in the SDS-
micelles [{q, w, φ} = {−75◦, −150◦, −60◦}] is similar to the
stable conformations of 2 and E. coli lipid A 1, as described
above.
Experimental
Materials
The 6-13C- labeled 2 and 3 were previously synthesized starting
from commercially available D-(6-13C)-glucose.41 The 1ꢀ-13C-
labeled derivatives of 2 and 3 were newly synthesized. The details
of the synthesis are described in the Electronic Supplementary
Information†. Deuterated sodium dodecyl sulfate (SDS-d25) was
purchased from Aldrich.
NMR spectroscopy
The sample solution of 2 was prepared by addition of 8.27 mg
(0.026 mmol) of SDS-d25 to 0.33 lmol of 2 in 0.30 mL of D2O
(99.96% D). The solution of 3 was prepared by a similar method
with or without SDS-d25. All NMR spectra were measured at
303 K with JEOL JNM-Lambda 500 or Varian Unity-plus
R
600 spectrometers and analyzed using the Felixꢀ program
(version 97.0, Molecular Simulations). Proton chemical shifts
were referenced to the proton of HDO (4.65 ppm). COSY and
heteronuclear multiple-bond correlation (HMBC) spectra were
obtained by standard measurement procedures. NOESY spectra
were collected with a mixing time ranging from 300–1500 ms,
while transverse rotating-frame Overhauser effect spectroscopy
(T-ROESY)60 and gradient enhanced nuclear Overhauser effect
spectroscopy (GOESY)69 were performed with 50–175 ms and
100–300 ms mixing time, respectively. The water signal was
suppressed by the DANTE (delays alternating with nutation for
tailored excitation) pulse.70 Each spectrum size was 1024–4096
complex points in the T2 dimension and 256–512 complex points
in T1. Two-fold zero-filling was carried out in both dimensions,
and the data were processed using exponential or shifted sine-
bell window functions before transformation.
The lack of both endotoxic and antagonistic activities in 3
can be explained by its low tendency to bind to LPS receptors
owing to its conformational flexibility, where a large entropic
loss would arise for the binding of 3 to the LPS receptors. This
explanation is also supported by a TLC blotting assay using a
lipid A monoclonal antibody (mAb A6), which has been proven
to recognise the hydrophilic part of lipid A.64 The antibody
recognises the E. coli lipid A 1 and the biosynthetic precursor
2 but does not bind to 3.65 The importance of hydrophobic
interaction in maintaining the conformation of lipid A is also
well understood, by comparing the present result with our
previous NMR study of 2 in DMSO.33 In this solvent, where
no hydrophobic affinity operates, two flexible conformers of 2
were detected, both of which were similar to two conformers of
3 in SDS-micelle, respectively.
As described, the conformations of tetraacyl lipid A 2 and
its regular supramolecular assembly in SDS were determined by
NMR analysis. NMR spectra of hexaacyl E. coli lipid A (1) in
aqueous media remained broad even after the addition of SDS,
Calculations
The molecular mechanics calculations were carried out un-
der the all-atom AMBER* force field61 with generalized
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 5 7 – 3 5 6 5
3 5 6 3

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Born/surface area (GB/SA) continuum water solvation model62
using MacroModel version 7.0 (Schro¨dinger Inc.). The energy
minimization was performed by truncated Newton conju-
26 A. D. Ferguson, W. Welte, E. Hofmann, B. Lindner, O. Holst,
J. W. Coulton and K. Diederichs, Structure Fold. Des., 2000, 8,
585–592: we found that the stereochemistry of the 3ꢀ-o-acyloxyacyl
group of K12 LPS in the X-ray crystallographic analysis was
incorrect. The X-ray data showed the 3ꢀ-o-acyloxyacyl group has
an S-configuration, whereas other acyloxyacyl and hydroxyacyl
groups have an R-configuration. We hence checked the chirality
of 3-hydroxytetradecanoic acid obtained from K12 LPS and did
not detect the S-isomer at all (O. Holst, K. Fukase, unpublished
results). The S-configuration of the 3ꢀ-o-acyloxyacyl group in the
X-ray analysis should be corrected as R-configuration.
27 H. Labischinski, G. Barnickel, H. Bradaczek, D. Naumann, E. T.
Rietschel and P. Giesbrecht, J. Bacteriol., 1985, 162, 9–20.
28 M. Kastowsky, A. Sabisch, T. Gutberlet and H. Bradaczek,
Eur. J. Biochem., 1991, 197, 707–716.
gate gradient minimization (the convergence threshold was
−1
0.05 kJ−1 A mol ).
˚
Acknowledgements
The authors thank Mr S. Adachi and Dr K. Li of the NMR
laboratory of the department for their skillful measurements and
Dr E. Fukushi of the GC-MS & NMR laboratory, Hokkaido
University, for helpful discussions. S. K. is grateful to Prof.
R. R. Schmidt at Universita¨t Konstanz, Germany, for his
valuable suggestions about the use of SDS-micelles in the NMR
study of lipid A. This work was partly supported by a Grant-
in-Aid for Scientific Research on Priority Areas No. 09273102
from the Ministry of Education, Science, Sports and Culture,
Japan. We also appreciate the financial support of “Research for
the Future” Program No. 97L00502 from the Japan Society for
the Promotion of Science.
29 S. Obst, M. Kastowsky and H. Bradaczek, Biophys. J., 1997, 72,
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30 P. Pristovsek and J. Kidric, J. Med. Chem., 1999, 42, 4604–4613.
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34 T. B. Grindley, in Glycoscience, eds. B. Fraser-Reid, K. Tatsuta and
J. Thiem, Springer, Berlin, 2001, vol 1, p. 3 and references cited
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