This document describes the synthesis and characterization of a series of anionic liposaccharide derivatives intended to act as absorption enhancers and improve oral bioavailability of drugs. The liposaccharides were designed with a lipophilic lipid side chain and a hydrophilic head containing glucose and glutamic acid. Isothermal titration calorimetry was used to determine the critical aggregation concentrations and thermodynamic profiles of the liposaccharides. Two liposaccharides formed nanoparticles below 100 nm and had critical aggregation concentrations below 0.325 mM, indicating favorable aggregation in aqueous solution driven by entropy.

1. Liposaccharide-based nanoparticulate drug delivery system
Adel S. Abdelrahim a
, Pavla Simerska a,*, Istvan Toth a,b
a
The University of Queensland, School of Chemistry and Molecular Biosciences (SCMB), St Lucia, Brisbane, Queensland 4072, Australia
b
The University of Queensland, School of Pharmacy, Woolloongabba, Queensland 4102, Australia
a r t i c l e i n f o
Article history:
Received 3 February 2012
Received in revised form 30 March 2012
Accepted 16 April 2012
Available online 23 April 2012
Keywords:
Charged liposaccharide
Microcalorimetry
Tobramycin
Absorption enhancer
Nanoparticle
Drug delivery
a b s t r a c t
A series of anionic liposaccharide derivatives were synthesized in order to develop a system, which
would have the capacity to act as an absorption enhancer and to improve oral bioavailability of drugs.
The addition of a liposaccharide to a drug enhances drug stability against enzymatic degradation, while
the lipophilicity can be controlled by variation of the lipid side chain. All liposaccharide derivatives were
purified and fully characterized by nuclear magnetic resonance and high-resolution mass spectrometry.
The thermodynamic profiles, critical aggregation concentrations and size of the synthesized lip-
osaccharides were determined by isothermal titration microcalorimetry, transmission electron micros-
copy and dynamic light scattering. These liposaccharides formed nanoparticles with sizes below 100 nm.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
A large number of newly developed drug candidates cannot be
administered orally for various reasons such as poor penetration
through the intestinal mucosa, and/or binding in the gastrointes-
tinal tract due to the highly hydrophilic properties.1
Therefore, the
administration of these drugs is limited to intravenous or in-
tramuscular routes. To overcome these challenges, medicinal and
pharmaceutical research has focused on development of alterna-
tives with enhanced oral bioavailability.2
One of the main strategies
being investigated is increasing the lipophilicity of the constructs,
thereby facilitating their penetration across the intestine. Recently,
many studies have been carried out to study the influence of ab-
sorption enhancers (e.g., bile salts, fatty acids, surfactants) on the
drug’s intestinal absorption and membrane permeability, especially
by passive diffusion.3
The addition of a safe and effective absorption
enhancer into the conventional oral dosage form is considered to be
easier and cheaper than development of a novel drug or pro-drug.4
Also aggregation, surfactant and ion-pairing characteristics of the
formed compounds can increase intestinal uptake.5
We have demonstrated earlier that the co-administration of
liposaccharide-based absorption enhancers with various drugs
(e.g., piperacillin6
and gentamicin7
) improved absorption of the
parent drug in vivo. However, the permeability of those compounds
was still low.8
To further improve the permeability, we describe the
synthesis and characterization of a novel series of anionic lip-
osaccharide derivatives with good absorption enhancing activity.
These derivatives are unique amphiphilic synthetic compounds
with a lipophilic tail (lipoamino acid) and a hydrophilic head con-
taining a carbohydrate (glucose) and a glutamic acid sodium salt.
Sodium salt formation of an acidic drug increases the solubility and
stability during oral administration.9
This structural arrangement
modulates aqueous solubility as well as the lipophilicity of the
drugeliposaccharide complex. The incorporation of a lipoamino
acid (LAA),10
an amino acid with a lipophilic alkyl side chain, into
the molecules was previously reported to increase oral absorption
of drugs with poor bioavailability.11
It has been shown, when LAAs
form amphiphilic ion pairs with macrolide class antibiotics (e.g.,
erythromycin) there was no decrease in its antibacterial activity.12
The incorporation of a carbohydrate into the system not only im-
proves water solubility, but also can utilize active or facilitated
glucose transport systems during absorption.
The amphoteric structural design of the molecules was de-
veloped in order to promote surfactant like properties and further
aggregation and/or micellization of the liposaccharides. Isothermal
titration calorimetry (ITC) was performed to determine the critical
aggregation concentration (CAC) of the synthesized compounds.
Enthalpy of aggregation (DHagg), the Gibbs’ free energy of aggre-
gation (DGagg) and the entropy of aggregation (DSagg) were also
calculated. The size and shape of the liposaccharides were mea-
sured by transmission electron microscopy (TEM) and dynamic
light scattering (DLS).
* Corresponding author. Tel.: þ61 7 33469892; fax: þ61 7 33654273; e-mail
address: p.simerska@uq.edu.au (P. Simerska).
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.04.064
Tetrahedron 68 (2012) 4967e4975

2. 2. Results and discussion
2.1. Synthesis
tert-Butyloxocarbonyl (Boc) protected LAA derivatives 1aed were
synthesized from their bromoalkane precursors and diethyl acet-
amidomalonate followed by Boc protection as described pre-
viously.13
The carboxyl groups of glutamic acid (Glu) were esterified
using thionyl chloride in methanol to yield Glu dimethyl ester hy-
drochloride salt. Following the removal of the excess thionyl chlo-
ride under vacuum and neutralization by aqueous sodium
bicarbonate (NaHCO3), dimethylated Glu 2 was obtained in a quan-
titative yield.14
Boc-LAAs 1aed with different lipid side chain lengths
(C8eC14) were coupled to dimethyl-Glu 2 using O-benzotriazole-
N,N,N0,N0-tetra-methyl-uronium-hexafluoro-phosphate (HBTU)/dii-
sopropylethyl amine (DIPEA) in dry dichloromethane (DCM) to
produce compounds 3aed in approximately 65% yield (Scheme 1).
The Boc protecting group was removed by trifluoroacetic acid (TFA)
in DCM followed by the neutralization of the TFA salt with aqueous
NaHCO3 to give dimethyl-Glu-LAAs 4aed in 90e95% yields.
Peracetylation of D-glucose was performed using acetic anhy-
dride, followed by bromination using hydrogen bromide in acetic
acid.15
Several methodologies were tested to prepare the azide de-
rivative from the bromide including addition of sodium azide in the
mixture of acetone and water16
and the method using tetra-butyl
ammonium hydrogen sulfate in DCM/aqueous NaHCO3 mixture.17
Applying the first method for azide synthesis, we obtained, after
the crude product was re-crystallized from hot ethanol, higher
yields (86% instead of 64%) of the b-D-glucopyranosyl azide.
b-D-Glucopyranosyl azide16
was reduced to amine by hydrogenation
(H2 on Pd/C) and immediately reacted in situ with a 1 mol equiv of
succinic anhydride to overcome the instability of peracetylated
glucosyl amine.18,19
The concentration of succinic anhydride used in
the reaction mixture was optimized to 1 mol equiv due to the ob-
served difficulties during the purification of the formed sugar de-
rivative. An acidebase wash was applied to remove unreacted
peracetylated glucosyl amine from the reaction.20
The peracetylated
glucosylamido propionic acid was coupled to the free amine de-
rivatives 4aed using HBTU/DIPEA in dry DCM and following flash
column chromatography yielded pure liposaccharides 5aed.
Zemplen deacetylation using 1 M NaOCH3 in methanol at pH 13 was
applied, and the reaction mixture was stirred with water for an
additional 12 h to hydrolyse di-methyl esters. Then the reaction
mixture was acidified using acidic resin IR-120 [H]þ
, filtered, evap-
orated under vacuum and lyophilized using acetonitrile/water (1:1)
to form free acids 6aed in 90% yields. The free acids of the lip-
osaccharides 6aed were sonicated with 2 equiv of NaHCO3 in water
to facilitate ion-pairing of the formed liposaccharide with the pos-
itively charged drug and to increase the aqueous solubility of the
final complex. The sodium derivatives of the liposaccharides 7aed
with C8eC14 lipid side chain lengths (Scheme 2) were obtained
after lyophilization in quantitative yields as white powders.
All the structural elucidations were done by 1
H, 13
C nuclear
magnetic resonance (NMR) and mass spectroscopy (MS).
2.2. Isothermal titration calorimetry and size measurements
Isothermal titration calorimetry (ITC) was used to monitor the
interactions of anionic liposaccharides in aqueous solution.21
The
synthesised liposaccharides were expected to form aggregates due
to their intra- and/or intermolecular-hydrophobic interactions in
aqueous media as they possessed both hydrophilic and lipophilic
moieties. Determination of the critical aggregation concentration
(CAC) of the liposaccharides was an important step in order to
understand the interactions between the liposaccharide and
a model drug during their complexation. The importance of the CAC
value has been reported elsewhere describing the effect of higher
CAC values on aggregation and permeation through biological
membranes.22
Also thermodynamic profile results of the newly
designed penetration enhancers would be valuable in predicting
potential toxicity of the compounds, especially disruption to bi-
ological membranes.
The liposaccharides 7b,c formed aggregates at their CACs, which
were calculated from the ITC experiments. The CACs of the lip-
osaccharides 7b,c were estimated by the van Os method;23
and the
Scheme 1. Coupling of Boc-LAAs to di-methylated Glu. Reagents and conditions: (a)
HBTU, DIPEA, DCM, 24 h; (b) (i) TFA/DCM (1:1), 1 h; (ii) NaHCO3.
Scheme 2. Synthesis of the liposaccharides 7aed with different lipid side chain lengths. Reagents and conditions: (a) HBTU, DIPEA, DCM, 24 h; (b) (i) 0.1 M NaOCH3, methanol, 2 h;
(ii) H2O addition,12 h; (iii) Amberlite IR-120 (Hþ
); (c) NaHCO3.
A.S. Abdelrahim et al. / Tetrahedron 68 (2012) 4967e49754968

3. cumulative enthalpy was plotted as a function of surfactant con-
centration. The enthalpy was expressed as a function of the com-
pound’s concentration (mM) in the calorimeter cell and it reflected
the contribution of individual interactions occurring between the
liposaccharide molecules and deionized water. It was found that
the liposaccharides GlcC10Glu (7b) and GlcC12Glu (7c) aggregated
in the aqueous state mainly due to their higher lipophilicity than
that of the liposaccharide GlcC8Glu (7a). The CAC of liposaccharide
7a could not be accurately estimated due to the low enthalpy data
( 0.5 kJ molÀ1
). The liposaccharide 7a either did not aggregate or
the aggregates were not stable in water to allow ITC results to be
measured. It is assumed that the liposaccharide GlcC14Glu (7d)
with the longest alkyl side chain was more lipophilic than the op-
timal lipophilicity and so stable and detectable aggregates were not
observed. A higher degree of lipophilicity is known to lead to poor
solubility in aqueous media and may cause large changes in the
titration curve, which may relate to the smaller demicellization
enthalpy for this compound’s aggregates. The calorimetric titration
graphs of the liposaccharides GlcC10Glu (7b) and GlcC12Glu (7c)
are presented in Fig. 1 and their thermodynamic values in Table 1.
The enthalpy changes of aggregation (DHagg) were observed to
be similar for GlcC10Glu (7b)¼3.75 kJ molÀ1
and for GlcC12Glu
(7c)¼2.40 kJ molÀ1
(Fig. 1a). A change of slope of cumulative en-
thalpy was used to calculate the CAC values by selecting data above
and below these concentrations. These data were fitted into a linear
regression with the point of their intersections selected as the CACs
(Fig. 1b).24
CACs were also determined from the maximum of the
first derivative curves (Fig. 1c).25
The CACs for GlcC10Glu (7b) and
GlcC12Glu (7c) were calculated to be 0.275Æ0.008 mM and
0.253Æ0.012 mM, respectively (Table 1). A decrease in peak height
was noticed after a certain number of injections (Fig. 1c). This was
caused by the concentrations in the reaction cell exceeding the CAC
and the aggregates titrated into the reaction cell were no longer
dissociated. Above the CAC the enthalpy change is therefore solely
the result of aggregate dilution effects of the CAC.26
The Gibbs free energy of aggregation (DGagg) was calculated to
determine the binding process of the liposaccharides (DGagg¼RT
lnXagg; R is the gas constant 8.314 J KÀ1
molÀ1
, T is the absolute
temperature 298 K and Xagg is the CAC value in moles). DGagg of
(a)
(b)
(c)
Fig. 1. Determination of the enthalpy of aggregation and critical aggregation concentrations (CACs) of 4 mM liposaccharides GlcC10Glu (7b) and GlcC12Glu (7c) at 298 K; (a) heat of
the reaction versus concentration of 7b or 7c; (b) determination of the CACs through cumulative enthalpy versus concentration of 7b or 7c; (c) first derivative of the enthalpy of
liposaccharides 7b and 7c.
Table 1
Summary of the thermodynamic values of compounds 7b and 7c obtained by iso-
thermal titration calorimetry (ITC) measurements
Liposaccharide CAC
(mM)
DHagg
(kJ molÀ1
)
DGagg
(kJ molÀ1
)
TDSagg
(kJ molÀ1
)
DSagg
(kJ molÀ1
)
GlcC10Glu (7b) 0.275 3.75 À3.19 6.94 0.023
GlcC12Glu (7c) 0.325 2.40 À2.77 5.17 0.017
A.S. Abdelrahim et al. / Tetrahedron 68 (2012) 4967e4975 4969

4. GlcC10Glu 7b was calculated to be À3.19 kJ molÀ1
and of GlcC12Glu
7c À2.77 kJ molÀ1
. These results suggested that favourable changes
during the aggregation process led to the formation of stabilised
entities in the aqueous environment.
The entropy of aggregation (DSagg) of both liposaccharides 7b
and 7c was calculated using the GibbseHelmoltz equation
DSagg¼(DHaggÀDGagg)/T. The endothermic nature of the processes
(DHagg0) (Fig. 1a) indicated that disaggregation led to an increase
in the overall entropy of the system, because aggregate dissociation
was thermodynamically favourable below the CMC (DH0);
therefore, TDSDH. DSagg was calculated for both liposaccharides
7b and 7c to be 0.023 and 0.017 kJ KÀ1
molÀ1
, respectively. This
positive entropy change implied a decrease in the general degree of
order in the system (e.g., desolvation process associated with the
pairing of molecules)27
and was attributed to the release of counter
ions associated with the surfactant head groups when aggregates
broke down to monomers.26
Moreover, the negative value of
ÀTDSagg (Table 1), which contributed to lowering DGagg, also in-
dicated that aggregation was a favourable process.
The size and shape of the liposaccharides 7b and 7c at their CACs
were measured by TEM. It was previously reported by our group
that more lipophilic compounds form larger aggregates. In this
study, different methods of size measurement of peptides, lip-
opeptides and lipoglycopeptides were compared.28
The lip-
osaccharide GlcC10Glu (7b) formed poly-dispersed aggregates
around 60e80 nm in size with smaller individual nanoparticles
around 30 nm (Fig. 2a, b). The liposaccharide GlcC12Glu (7c)
showed similar sized spherical aggregates (Fig. 2c, d). These results
correlated with the liposaccharide sizes determined by DLS (a
highly poly-disperse size distribution with a peak below 100 nm;
data not shown).
3. Conclusion
Anionic liposaccharides 7aed were designed and synthesized
from biocompatible non-toxic precursors such as carbohydrate,
lipoamino and amino acid derivatives and all products were purified
and fully characterised by NMR and High-resolution mass
spectrometry (HRMS). ITC results confirmed the ability of the lip-
osaccharides 7b and 7c (comprising C10 and C12 LAA) to aggregate in
an aqueous environment. The thermodynamic profiles including
CAC, DHagg, DGagg and DSagg of the liposaccharides 7b and 7c were
also determined by ITC and showed formation of aggregates. In-
terestingly C10 and C12 had the optimal lipid side chain length for
the aggregation process to occur. We also found that the lip-
osaccharides GlcC10Glu (7b) and GlcC12Glu (7c) formed poly-
disperse aggregates around 60e80 nm in size as showed by TEM
and DLS. The liposaccharide-based drug delivery system presented
herewillbe further tested invitro andinvivo foritsabilitytoenhance
intestinal absorption of otherwise poorly orally available drugs.
4. Experimental section
4.1. General
Dichloromethane (DCM), trifluoroacetic acid (TFA) and diiso-
propylethyl amine (DIPEA) were purchased from Auspep (Mel-
bourne, VIC, Australia). O-Benzotriazole-N,N,N0,N0-tetra-methyl-
uronium-hexafluoro-phosphate (HBTU) and di-tert-butyldicar-
bonate (Boc2O) were obtained from GL Biochem Ltd. (Shanghai,
China). Na-Boc-protected amino acids were supplied by Nova-
biochem (Laufelfingen, Switzerland). Palladium (10 wt % on carbon)
was purchased from Lancaster Synthesis (Lancashire, England).
Amberlite ion exchange resin (IR-120) [HÀ
] was provided by British
Drug Houses (BDH) Ltd. (England). Gases (nitrogen, hydrogen and
argon) were supplied by BOC Gases (Brisbane, QLD, Australia). Silica
(silica gel 60, 230e400 mesh) for flash chromatography was
obtained from Lomb Scientific (Taren Point, NSW, Australia). Deu-
terated solvents DCl3-d1 and DMSO-d6 were manufactured by
Cambridge Isotope Laboratories Inc. (Andover, MA, USA). All com-
mercial reagents were purchased in analytical grade or higher pu-
rity from Sigma-Aldrich (Castle Hill, NSW, Australia) or Merck Pty.
Ltd. (Kilsyth, VIC, Australia) and were used without further purifi-
cation. Solvents were freshly distilled prior to use and all moisture-
sensitive reactions were carried out in an inert atmosphere under
Fig. 2. Transmission electron microscopy (TEM) images of liposaccharide GlcC10Glu (7b) (a, b) and GlcC12Glu (7c) (c, d) at their critical aggregation concentrations (CACs).
A.S. Abdelrahim et al. / Tetrahedron 68 (2012) 4967e49754970

5. nitrogen or argon using oven-dried glassware. Reactions were
carried out at room temperature unless otherwise specified. Thin-
layer chromatography (TLC) was performed on silica gel 60 F254
aluminium sheets (Merck, Darmstadt, Germany), and compounds
were visualized by either ninhydrin dip (0.1% ninhydrin in ethanol)
or ceric sulfate dip (15% aqueous H2SO4 saturated with ceric sul-
fate). All TLC plates were developed by heating after treatment with
the developing agent. Purification of the synthesized compounds
was achieved by flash column chromatography that was performed
on silica gel 60, 230e400 mesh ASTM (Scharlau, Barcelona, Spain).
Melting points were measured with a capillary apparatus.
Infrared measurements were performed on an IR spectrometer
Spectrum 2000 (Perkin Elmer Pty Ltd, Glen Waverley, VIC, Aus-
tralia), at a resolution of 4 cmÀ1
ATR. Nuclear Magnetic Resonance
(NMR) spectra (1
H and 13
C NMR) were recorded at room temper-
ature in deuterated chloroform (CDCl3) solutions (unless otherwise
indicated). A Bruker AM 500 instrument operating at 500 MHz was
used. Chemical shifts are listed in parts per million (ppm) down
field from internal tetramethylsilane (TMS). Signal multiplicities
are represented as singlet (s), doublet (d), double doublet (dd),
triplet (t), quartet (q), quintet (quint), multiplet (m), broad (br) and
broad singlet (br s).
Mass spectra (MS) were recorded on a PerkineElmer Sciex API
3000 mass spectrometer (Applied Biosystems/MDS Sciex, Toronto,
Canada) operating in positive ion electrospray mode (ESI-MS). Liquid
chromatography mass spectroscopy (LCeMS/MS) data were mea-
sured on a Waters 2790 instrument using positive mode electrospray
ionization. The mobile phase used for the measurement was a mix-
ture of solvent A (0.1% acetic acid in water) and solvent B (0.1% acetic
acid in 90% acetonitrile and 10% water). Results were analysed by
Analyst 1.4 software. High-resolution mass spectrometry (HRMS)
data were obtained on a Qstar Pulsar mass spectrometer (Applied
Biosystems) operating in positive ion electrospray mode. Analytical
results were within Æ0.4% of the theoretical values for the formula
given unless otherwise indicated.
4.2. Synthesis
4.2.1. Dimethyl N-(2-(N-tert-butyloxycarbonyl)amino-D,L-octanoyl)-
L-glutamate (3a). 2-(Na-Boc)amino-D,L-heptanoic acid 1a7
(1.11 g,
4.28 mmol), HBTU (0.97 g, 5.14 mmol) and DIPEA (1.49 ml,
8.52 mmol) were dissolved in dry DCM (50 ml) followed by the
addition of dimethyl glutamic acid 214
(0.75 g, 4.28 mmol). The
reaction mixture was stirred at room temperature for 12 h, then
washed with 5% HCl (2Â50 mL) and a saturated solution of NaHCO3
(2Â50 mL), and dried over MgSO4. The residual solvent was evap-
orated under vacuum and the crude product was purified by col-
umn chromatography (Rf¼0.3 ethyl acetate/hexane, 1:2 (v/v)) to
produce pure compound 3a (1.15 g, 2.76 mmol, 65%) as a colourless
oil (1:1 mixture of diastereomers). 1
H NMR (500 MHz, CDCl3)
d 7.78e7.73 (1H, m, amide NH), 6.09e6.08 (1H, m, amide NH),
4.65e4.59 (1H, m, CH (lipid)), 4.30e4.24 (1H, m, CH (glutamic)),
3.72 (3H, s, OCH3), 3.64 (3H, s, OCH3), 2.451 (2H, t, J¼11.7 Hz, CH2
(glutamic)), 2.24e2.21 (2H, m, b-CH2 (glutamic)), 1.39 (9H, s, Boc),
1.79e1.74 (2H, m, b-CH2 (lipid)), 1.28e1.23 (8H, m, 4CH2 (lipid)),
0.87 (3H, t, J¼4.5 Hz, CH3 (lipid)); 13
C NMR (500 MHz, CDCl3)
d 172.65, 172.59, 172.45, 172.35, 171.51, 171.49, 170.28, 155.36,
155.33, 79.65, 78.54, 67.10, 59.69, 56.19, 54.07, 54.02, 51.92, 51.61,
51.56, 51.21, 50.97, 50.94, 50.90, 50.42, 45.74, 32.28, 32.16, 31.36,
31.36, 31.24, 31.10, 30.05, 29.44, 29.41, 29.09, 28.80, 28.56, 27.75,
26.54, 25.14, 25.05, 22.17, 22.13, 22.09, 22.04, 20.23, 13.60, 13.48,
13.44, 11.10; HRMS calculated for [C20H36N2NaO7]þ
[MþNa]þ
439.2420, found 439.2415.
4.2.2. Dimethyl N-(2-(N-tert-butyloxycarbonyl)amino-D,L-decanoyl)-
L-glutamate (3b). Following the procedure described for compound
3a, except 2-(Na-Boc) amino-D,L-decanoic acid 1b7
(1.22 g,
4.28 mmol) was used instead of 1a to synthesise 3b. Crude product
3b was purified by flash column chromatography (Rf¼0.3 ethyl
acetate/hexane,1:2 (v/v)) to give pure 3b (1.20 g, 2.70 mmol) in 63%
yield as a colourless oil (1:1 mixture of diastereomers). ESI-MS, MS,
m/z: 467 [MþNa]þ
. 1
H NMR (500 MHz, CDCl3) d 7.37e7.30 (1H, m,
amide NH), 7.27e7.25 (1H, m, amide NH), 4.55 (1H, t, J¼5.7 Hz, CH
(lipid)), 4.07e4.00 (1H, m, CH (glutamic)), 3.67 (3H, s, OCH3), 3.58
(3H, s, OCH3), 2.37 (2H, t, J¼13.1 Hz, CH2 (glutamic)), 1.96e1.95 (2H,
m, b-CH2 (glutamic)), 1.36 (9H, s, Boc), 1.18e1.17 (14H, m, 7CH2
(lipid)), 0.81 (3H, t, J¼10.9 Hz, CH3 (lipid)); 13
C NMR (500 MHz,
CDCl3) d 172.66, 172.55, 172.35, 171.68, 171.60, 170.60, 155.33, 79.04,
59.88, 54.10, 53.13, 51.86, 51.82, 51.19, 51.17, 51.02, 32.21, 31.40,
29.50, 29.02, 28.94, 28.78, 27.83, 26.69, 25.18, 25.07, 22.18, 20.47,
13.71, 13.60; HRMS calculated for [C22H40N2NaO7] [MþNa]þ
467.2733, found 467.2731.
4.2.3. Dimethyl N-(2-(N-tert-butyloxycarbonyl)amino-D,L-dodeca-
noyl)-L-glutamate (3c). Compound 3c was prepared by the pro-
cedure described above for compound 3a, except 2-(Na-Boc)
amino-D,L-dodecanoic acid 1c (1.34 g, 4.28 mmol) was used instead
of 1a. The crude product was purified by flash chromatography
(Rf¼0.3 ethyl acetate/hexane, 1:2 (v/v)) to give pure compound 3c
(1.54 g, 3.26 mmol) in 76% yield as a colourless oil (1:1 mixture of
diastereomers). 1
H NMR (500 MHz, CDCl3) d 7.56e7.46 (1H, m,
amide NH), 7.38e7.31 (1H, m, amide NH), 5.65 (1H, t, J¼9.85 Hz, CH
(lipid)), 4.57e4.52 (1H, m, CH (glutamic)), 3.65 (3H, s, OCH3), 3.58
(3H, s, OCH3), 2.33 (2H, t, J¼11.7 Hz, CH2 (glutamic)), 1.96e1.94 (2H,
m, b-CH2 (glutamic)), 1.73e1.69 (2H, m, b-CH2 (lipid)), 1.36 (9H, s,
Boc), 1.20e1.18 (16H, m, 8CH2 (lipid)), 0.81 (3H, t, J¼6.80 Hz, CH3
(lipid)); 13
C NMR (500 MHz, CDCl3) d 172.78, 172.66, 172.56, 171.81,
171.72, 155.48, 143.18, 127.73, 126.45, 124.39, 119.88, 108.29, 79.09,
67.27, 59.99, 54.22, 54.16, 51.96, 51.91, 51.28, 51.26, 51.14, 32.40,
32.34, 31.57, 29.62, 29.27, 29.21, 29.08, 28.99, 27.96, 26.78, 25.32,
25.20, 22.33, 13.83, 13.73; HRMS calculated for [C24H44N2NaO7]
[MþNa]þ
495.3046, found 495.3041.
4.2.4. Dimethyl N-(2-(N-tert-butyloxycarbonyl)amino-D,L-tetradeca-
noyl)-L-glutamate (3d). Compound 3d was prepared by the procedure
described above for compound 3a, except 2-(Na-Boc) amino-D,L-tet-
radecanoic acid 1d7
(1.46 g, 4.28 mmol) was used instead of 1a. The
crude product was purified by flash chromatography (Rf¼0.3 ethyl
acetate/hexane, 1:2 (v/v)) to give pure compound 3d (1.23 g,
2.46 mmol) in 57% yield as a colourless oil (1:1 mixture of di-
astereomers). 1
H NMR (500 MHz, CDCl3) d 77.53e7.51 (1H, m, amide
NH), 7.05e7.03 (1H, m, amide NH), 5.65e4.52 (1H, t, J¼9.85 Hz, CH
(lipid)), 4.08e4.03 (1H, m, CH (glutamic)), 3.65 (3H, s, OCH3), 3.58
(3H, s, OCH3), 2.33 (2H, t, J¼11.7 Hz, CH2 (glutamic)),1.93e1.90 (2H, m,
b-CH2 (glutamic)), 1.53e1.49 (2H, m, b-CH2 (lipid)), 1.36e1.35 (9H, m,
Boc), 1.20e1.17 (20H, m, 10CH2 (lipid)), 0.80 (3H, t, J¼6.85 Hz, CH3
(lipid)); 13
C NMR (500 MHz, CDCl3) d 1722.99, 172.88, 172.32, 171.93,
171.83, 155.52, 128.58, 128.50, 124.87, 119.98, 109.23, 84.99, 79.62,
54.43, 52.24, 52.21, 51.55, 51.29, 32.32, 31.72, 29.73, 29.47, 29.38,
29.30, 29.16, 26.99, 25.41, 22.49, 13.91; HRMS calculated for
[C26H48N2NaO7] [MþNa]þ
523.3359, found 523.3354.
4.2.5. Dimethyl N-(2-amino-D,L-octanoyl)-L-glutamate (4a). Com-
pound 3a (1.50 g, 3.60 mmol) was dissolved in TFA/DCM (1:1;
20 ml) and stirred for 1 h. The mixture was diluted in DCM (50 ml),
evaporated and washed with NaHCO3 solution. The organic layer
was separated, dried over MgSO4, filtered and evaporated under
vacuum to produce compound 4a (1.08 g, 3.42 mmol) in 95% yield
as a colourless oil (1:1 mixture of diastereomers). 1
H NMR
(500 MHz, CDCl3) d 7.98e7.87 (1H, m, amide NH), 7.84e7.56 (2H, m,
amine NH2), 4.59e4.57 (1H, m, CH (lipid)), 4.25e4.20 (1H, m, CH
(glutamic)), 3.75 (3H, s, OCH3), 3.68 (3H, s, OCH3), 2.45 (2H, t,
A.S. Abdelrahim et al. / Tetrahedron 68 (2012) 4967e4975 4971

6. J¼11.7 Hz, CH2 (glutamic)), 2.09e2.05 (2H, m, b-CH2 (glutamic)),
1.88e1.79 (2H, m, b-CH2 (lipid)), 1.25e1.24 (8H, m, 4CH2 (lipid)),
0.84 (3H, t, J¼10.95 Hz, CH3 (lipid)); 13
C NMR (500 MHz, CDCl3)
d 175.48,174.89,172.17,171.58,169.80,169.57,161.44,160.89,160.35,
159.81, 120.69, 116.89, 113.10, 109.31, 54.58, 53.31, 53.10, 53.04,
52.57, 52.51, 52.47, 54.58, 53.31, 53.10, 53.04, 52.52, 52.51, 52.47,
38.97, 31.36, 31.17, 29.91, 29.83, 28.49, 27.27, 26.25, 24.60, 24.33,
22.23, 13.50; HRMS calculated for [C15H28N2NaO5]þ
[MþNa]þ
339.1900, found 339.1886.
4.2.6. Dimethyl N-(2-amino-D,L-decanoyl)-L-glutamate (4b). Com-
pound 4b was prepared by the procedure described for compound
4a, except 3b (1.50 g, 3.37 mmol) was used instead of 3a to produce
compound 4b (1.10 g, 3.20 mmol) in 95% yield as a colourless oil
(1:1 mixture of diastereomers). 1
H NMR (500 MHz, CDCl3)
d 78.00e7.98 (1H, m, amide NH), 4.94 (2H, br s, amine NH2),
4.60e4.55 (1H, m, CH (lipid)), 4.26e4.25 (1H, m, CH (glutamic)),
3.74 (3H, s, OCH3), 3.69 (3H, s, OCH3), 2.44 (2H, t, J¼11.7 Hz, CH2
(glutamic)),1.90e1.89 (2H, m, b-CH2 (glutamic)),1.24e1.21 (14H, m,
7CH2 (lipid)), 0.83 (3H, t, J¼6.5 Hz, CH3 (lipid)); 13
C NMR (500 MHz,
CDCl3) d 175.62, 175.06, 172.15, 171.65, 169.86, 169.66, 160.53,
160.20, 159.87, 159.55, 118.314, 116.04, 113.76, 111.49, 62.12, 54.66,
53.13, 53.07, 52.56, 31.62, 31.35, 30.44, 29.96, 29.86, 28.99, 28.93,
28.83, 26.23, 24.63, 24.36, 22.44, 13.63; HRMS calculated for
[C17H33N2O5]þ
[MþH]þ
345.24, found 345.2381.
4.2.7. Dimethyl N-(2-amino-D,L-dodecanoyl)-L-glutamate (4c). Com-
pound 4c was prepared by the procedure described for compound
4a, except 3c (1.50 g, 3.17 mmol) was used instead of 3a to produce
compound 4c (1.06 g, 2.85 mmol) in 90% yield as a colourless oil
(1:1 mixture of diastereomers). 1
H NMR (500 MHz, CDCl3)
d 7.54e7.82 (1H, m, amide NH), 4.98 (2H, br s, amine NH2),
4.57e4.55 (1H, m, CH (glutamic)), 4.23e4.20 (1H, m, CH (lipid)),
3.75 (3H, s, OCH3), 3.69 (3H, s, OCH3), 2.44 (2H, t, J¼7.5 Hz, CH2
(glutamic)), 2.23e2.17 (2H, m, b-CH2 (glutamic)), 1.87e1.84 (2H, m,
b-CH2 (lipid)),1.25e1.24 (16H, m, 8CH2 (lipid)), 0.86 (3H, t, J¼6.7 Hz,
CH3 (lipid)); 13
C NMR (500 MHz, CDCl3) d 175.21, 174.74, 174.59,
171.90, 171.31, 169.62, 169.39, 160.77, 160.45, 160.12, 159.80, 118.20,
115.94, 113.67, 111.39, 109.85, 54.36, 52.85, 52.79, 52.35, 52.28,
52.23, 31.61, 31.19, 30.34, 29.70, 29.62, 29.23, 29.14, 29.00, 28.90,
28.73, 28.71, 26.10, 26.05, 24.49, 24.21, 22.37, 20.54, 13.59, 13.39;
HRMS calculated for [C19H37N2O5]þ
[MþH]þ
373.27, found
373.2697.
4.2.8. Dimethyl N-(2-amino-D,L-tetradecanoyl)-L-glutamate(4d). Com-
pound 4d was prepared by the procedure described for compound 3a,
except 3d (1.50 g, 3.00 mmol) was used instead of 3a to produce
compound 4d (1.11 g, 2.78 mmol) in 92% yield as a colourless oil (1:1
mixture of diastereomers). 1
H NMR (500 MHz, CDCl3) d 77.53e7.51
(1H, m, amide NH), 5.65e4.52 (1H, t, J¼9.85 Hz, CH (glutamic)), 4.98
(2H, br s, amine NH2), 4.08e4.03 (1H, m, CH (lipid)), 3.65 (3H, s,
OCH3), 3.58 (3H, s, OCH3), 2.33 (2H, t, J¼11.7 Hz, CH2 (glutamic)),
1.93e1.90 (2H, m, b-CH2 (glutamic)), 1.53e1.49 (2H, m, b-CH2 (lipid)),
1.20e1.17 (20H, m, 10CH2 (lipid)), 0.80 (3H, t, J¼6.85 Hz, CH3 (lipid));
13
C NMR (500 MHz, CDCl3) d 175.21, 174.74, 174.59, 171.90, 171.31,
169.62, 169.39, 160.77, 160.45, 160.12, 159.80, 118.20, 115.94, 113.67,
111.39, 109.85, 54.36, 52.85, 52.79, 52.35, 52.28, 52.23, 31.61, 31.19,
30.34, 29.70, 29.62, 29.23, 29.14, 29.00, 28.90, 28.73, 28.71, 26.10,
26.05, 24.49, 24.21, 22.37, 20.54, 13.59, 13.39; HRMS calculated for
[C21H41N2O5]þ
[MþH]þ
401.3000, found 401.3010.
4.2.9. Dimethyl N-(2-(N-(4-(2,3,4,6-tetra-O-acetyl-b-D-glucopyr-
anosylamino)succinyl))amino-D,L-octanoyl)-L-glutamate (5a). N-(4-
(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylamino)succinic) acid29
(1.00 g, 2.23 mmol), HBTU (0.50 g, 2.68 mmol) and DIPEA
(0.77 ml, 4.47 mmol) were dissolved in dry DCM (50 ml).
Compound 4a (0.70 g, 2.23 mmol) was added to the reaction
mixture and stirred at room temperature for 12 h. Then it was
washed with 5% HCl solution, 10% NaHCO3 solution, dried over
MgSO4, filtered and evaporated to give an oily product, 5a. The
crude product was purified by column chromatography (Rf¼0.6
methanol/DCM, 1:9 (v/v)) to give pure 5a (1.16 g, 1.56 mmol) in
70% yield as a colourless oil (1:1 mixture of diastereomers). 1
H
NMR (500 MHz, CDCl3) d 7.45e7.43 (1H, m, amide NH), 7.38e7.36
(1H, m, amide NH), 7.14e7.00 (1H, m, amide NH), 6.54e6.52 (1H, t,
J¼8.05 Hz, H-1 (glucose)), 5.37e5.23 (2H, m, H-2 and H-3 (glu-
cose)), 5.02e4.98 (1H, m, H-5 (glucose)), 4.90e4.84 (1H, m, H-4
(glucose)), 4.51e4.38 (2H, m, H-6a,b (glucose)), 4.24 (1H, t,
J¼4.1 Hz, CH (glutamic)), 4.01 (1H, t, J¼12.3 Hz, CH (lipid)), 3.67
(3H, s, OCH3), 3.60 (3H, s, OCH3), 2.52e2.44 (4H, m, 2CH2 (glu-
cose)), 2.38e2.33 (2H, m, CH2 (glutamic)), 2.00, 1.97, 1.95, 1.93
(12H, 4s, 4CH3CO (glucose)), 1.78 (2H, m, b-CH2 (glutamic)),
1.56e1.52 (2H, m, b-CH2 (lipid)), 1.23e1.19 (8H, m, 4CH2 (lipid)),
0.82 (3H, t,J¼5.45 Hz, CH3 (lipid)); 13
C NMR (500 MHz, CDCl3)
d 173.24, 172.79, 172.57, 172.23, 171.92, 171.82, 171.79, 171.73,
171.59, 170.91, 170.50, 169.80, 169.77, 169.42, 77.84, 73.45, 73.32,
73.13, 72.89, 72.55, 70.59, 70.43, 68.14, 67.98, 61.63, 61.54, 53.09,
52.89, 52.32, 52.24, 51.78, 51.74, 51.52, 38.50, 32.16, 31.76, 31.48,
31.16, 31.00, 30.68, 30.61, 30.00, 29.93, 28.88, 28.80, 26.81, 26.54,
25.36, 25.18, 22.40, 20.59, 20.57, 20.51, 20.44, 13.89; HRMS calcu-
lated for [C33H51N3NaO16]þ
[MþNa]þ
768.3167, found 768.3162.
4.2.10. Dimethyl N-(2-(N-(4-(2,3,4,6-tetra-O-acetyl-b-D-glucopyrano-
sylamino)succinyl))amino-D,L-decanoyl)-L-glutamate (5b). Compound
5b was prepared by following the procedure described for compound
5a, except compound 4b (0.76 g, 2.23 mmol) was used to produce
compound 5b (Rf¼0.6 methanol/DCM, 1:9 (v/v)) (1.36 g, 1.76 mmol)
in 79% yield as a colourless oil (1:1 mixture of diastereomers). 1
H
NMR (500 MHz, CDCl3) d 7.66e7.65 (1H, m, amide NH), 7.52e7.50
(1H, m, amide NH), 7.38e7.28 (1H, m, amide NH), 6.87e6.80 (1H, t,
J¼8.1 Hz, H-1 (glucose)), 5.35e5.23 (2H, m, H-2 and H-3 (glucose)),
5.02e4.98 (1H, m, H-5 (glucose)), 4.88e4.85 (1H, m, H-4 (glucose)),
4.49e4.21 (2H, m, H-6a,b (glucose)), 4.01 (1H, t, J¼4.1 Hz, CH (gluta-
mic)), 3.90 (1H, t, J¼12.3 Hz, CH (lipid)), 3.66 (3H, s, OCH3), 3.64 (3H,
s, OCH3), 2.47e2.43 (4H, m, 2CH2 (succinic)), 2.36e2.33 (2H, m, CH2
(glutamic)), 2.16e2.13 (2H, m, bCH2 (glutamic)), 2.00, 1.97, 1.95, 1.93
(12H, 4s, 4CH3CO (glucose)), 1.55e1.51 (2H, m, b-CH2 (lipid)),
1.19e1.17 (12H, m, 6CH2 (lipid)), 0.80 (3H, t, J¼6.8 Hz, CH3 (lipid)); 13
C
NMR (500 MHz, CDCl3) d 173.17, 173.15, 172.86, 172.74, 172.23, 172.05,
171.88, 171.82, 171.66, 170.62, 170.47, 170.46, 170.26, 169.77, 169.75,
169.38, 162.53, 73.22, 73.08, 72.99, 72.69, 70.54, 70.43, 68.12, 67.98,
61.67, 61.58, 52.97, 52.62, 52.21, 52.17, 51.69, 61.66, 51.4552.97, 52.82,
52.21, 52.17, 51.69, 51.66, 51.45, 36.35, 32.27, 31.83, 31.63, 31.29, 31.09,
30.95, 30.56, 30.52, 29.94, 29.88, 29.25, 29.16, 29.05, 26.73, 26.51,
25.41, 25.21, 22.43, 20.52, 20.50, 20.43, 20.41, 20.37, 13.88; HRMS
calculated for [C35H55N3NaO16]þ
[MþNa]þ
796.3480, found
796.3475.
4.2.11. Dimethyl N-(2-(N-(4-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosy-
lamino)succinyl))amino-D,L-dodecanoyl)-L-glutamate (5c). Compound
5c was prepared by following the procedure described for compound
5a, except compound 4c (0.82 g, 2.23 mmol) was used to produce
compound 5c (Rf¼0.6 methanol/DCM,1:9 (v/v)) (1.16 g,1.45 mmol) in
65% yield as a colourless oil (1:1 mixture of diastereomers). 1
H NMR
(500 MHz, CDCl3) d 7.63e7.62 (1H, m, amide NH), 7.53e7.52 (1H, m,
amide NH), 7.37e7.30 (1H, m, amide NH), 6.87 (1H, t, H-1 (glucose)),
5.37e5.32 (2H, m, H-2 and H-3 (glucose)), 5.04e4.99 (1H, m, H-5
(glucose)), 4.91 (1H, m, H-4 (glucose)), 4.50e4.42 (2H, m, H-6a,b
(glucose)), 4.25 (1H, t, J¼4.35 Hz, CH (glucose)), 4.06 (1H, t, J¼7.1 Hz,
CH (lipid)), 3.66 (3H, s, OCH3), 3.64 (3H, s, OCH3), 2.50e2.46 (4H, m,
2CH2 (succinic)), 2.37 (2H, t, J¼8.0 Hz, CH2 (glutamic)), 2.18e2.14 (2H,
m, bCH2 (glutamic)), 2.02, 1.98, 1.97, 1.95 (12H, 4s, 4CH3CO (glucose)),
A.S. Abdelrahim et al. / Tetrahedron 68 (2012) 4967e49754972

7. 1.57e1.53 (2H, m, b-CH2 (lipid)),1.20e1.19 (16H, m, 8CH2 (lipid)), 0.82
(3H, t, J¼6.8 Hz, CH3 (lipid)); 13
C NMR (500 MHz, CDCl3) d 173.13,
172.81, 172.13, 172.03, 171.84, 170.56, 170.45, 169.73, 169.36, 73.06,
72.71, 70.55, 68.11, 61.66, 52.83, 52.13, 51.62, 31.86, 30.96, 30.54, 29.91,
29.31, 29.19, 29.11, 26.51, 25.43, 20.51, 20.41, 20.36; HRMS calculated
for [C37H59N3NaO16]þ
[MþNa]þ
824.3793, found 824.3788.
4.2.12. Dimethyl N-(2-(N-(4-(2,3,4,6-tetra-O-acetyl-b-D-glucopyr-
anosylamino)succinyl))amino-D,L-tetradecanoyl)-L-glutamate
(5d). Compound 5d was prepared by following the procedure de-
scribed for compound 5a, except compound 4d (1.07 g, 2.23 mmol)
was used to produce compound 5d (Rf¼0.6 methanol/DCM, 1:9
(v/v)) (0.70 g, 0.84 mmol) in 38% yield as a colourless oil (1:1
mixture of diastereomers). 1
H NMR (500 MHz, CDCl3) d 7.45e7.44
(1H, m, amide NH), 7.34e7.32 (1H, m, amide NH), 7.25e7.23 (1H, m,
amide NH), 6.80e6.78 (1H, t, J¼7.85 Hz, H-1 (glucose)), 5.43e5.29
(2H, m, H-2 and H-3 (glucose)), 5.08e5.04 (1H, m, H-5 (glucose)),
4.96e4.92 (1H, m, H-4 (glucose)), 4.55e4.46 (2H, m, H-6a,b (glu-
cose)), 4.31e4.28 (1H, m, CH (glutamic)), 4.10e4.07 (1H, m, CH
(lipid)), 3.66 (3H, s, OCH3), 3.62 (3H, s, OCH3), 2.52e2.39 (4H, m,
2CH2 (succinic)), 2.35e2.32 (2H, m, CH2 (glutamic)), 2.23e2.19 (2H,
m, bCH2 (glutamic)), 2.00, 1.97, 1.95, 1.93 (12H,4s, 4CH3CO (glu-
cose)), 1.54e1.51 (2H, m, b-CH2 (lipid)), 1.19e1.17 (20H, m, 10CH2
(lipid)), 0.80 (3H, t, J¼6.85 Hz, CH3 (lipid)); 13
C NMR (500 MHz,
CDCl3) d 173.78, 173.30,173.18,172.87, 172.78, 172.66,172.46, 172.22,
172.06, 172.01, 171.87, 171.83, 171.81, 171.65, 170.74, 170.46, 170.35,
169.76, 169.74, 169.39, 165.55, 162.53, 73.27, 73.10, 72.95, 72.62,
70.56, 70.43, 68.14, 61.64, 61.55, 53.41, 53.07, 52.89, 52.24, 52.17,
51.71, 51.67, 51.63, 51.48, 38.44, 32.15, 31.73, 31.13, 30.96, 30.61,
30.57, 29.97, 29.91, 29.51, 29.49, 29.46, 29.35, 29.33, 29.29, 29.17,
28.56, 28.41, 27.83, 26.74, 26.49, 25.45, 25.28, 22.50, 20.52, 20.46,
20.40, 18.38, 17.27, 13.93; HRMS calculated for [C39H63N3NaO16]þ
[MþNa]þ
852.4106, found 852.4101.
4.2.13. N-(2-(N-(4-(b-D-Glucopyranosylamino)succinyl))amino-D,L-
octanoyl)-L-glutamic acid (6a). Compound 5a (1.2 g, 1.61 mmol)
was dissolved in methanol (30 ml) and the pH was adjusted to 12
using 1 M NaOCH3 for 2 h. Water (10 ml) was added to the reaction
mixture and the pH was readjusted to 13. The solution was stirred
at room temperature for an additional 12 h. Upon completion, the
reaction mixture was acidified using Amberlite resin IR-120 [Hþ
]
until an acidic pH was obtained. The reaction mixture was filtered
and the filtrate was evaporated under vacuum. The residue was
lyophilised in acetonitrile/water (1:1) to give compound 6a (0.79 g,
1.44 mmol) in 89% yield as a white powder; mp 172 C (1:1 mixture
of diastereomers). 1
H NMR (500 MHz, MeOD) d 7.65e7.64 (1H, m,
amide NH), 7.51e7.50 (1H, m, amide NH), 7.39e7.36 (1H, m, amide
NH), 4.39e4.33 (1H, m, CH (glutamic)), 4.23e4.19 (1H, m, CH
(lipid)), 3.78e3.72 (1H, m, H-2 (glucose)), 3.58e3.55 (1H, m, H-3
(glucose)), 3.34e3.31 (2H, m, H-4 and H-5 (glucose)), 3.18e3.14
(2H, m, H-6a,b (glucose)), 2.55e2.35 (4H, m, 2CH2 (succinic)),
2.17e2.13 (2H, m, CH2 (glutamic)), 1.99e1.95 (2H, m, bCH2 (gluta-
mic)), 1.82e1.75 (2H, m, b-CH2 (lipid)), 1.37e1.27 (8H, m, 4CH2
(lipid)), 0.80 (3H, t, J¼6.75 Hz, CH3 (lipid)); 13
C NMR (500 MHz,
MeOD) d 174.00, 173.82, 173.79, 173.59, 173.50, 173.46, 173.40,
173.29, 173.18, 172.01, 171.9079.54, 79.48, 78.04, 78.01, 77.99, 77.33,
72.56, 72.49, 69.89, 69.84, 61.18, 61.10, 53.41, 53.32, 51.52, 51.37,
31.31, 31.21, 31.18, 30.60, 30.43, 30.13, 30.10, 29.62, 29.51, 29.49,
29.36, 28.55, 28.51, 26.10, 25.99, 25.83, 25.79, 25.35, 25.32, 22.11,
12.89; HRMS calculated for [C23H38N3O12]À
[MÀH]À
548.2455,
found 548.2461.
4.2.14. N-(2-(N-(4-(b-D-Glucopyranosylamino)succinyl))amino-D,L-
decanoyl)-L-glutamic acid (6b). Compound 6b was prepared by the
procedure described for compound 6a, except compound 5b
(1.35 g, 1.75 mmol) was used to give compound 6b (0.92 g,
1.59 mmol) in 91% yield as a white powder; mp 182 C (1:1 mixture
of diastereomers). 1
H NMR (500 MHz, MeOD) d 7.70e7.69 (1H, m,
amide NH), 7.63e7.62 (1H, m, amide NH), 7.52e7.50 (1H, m, amide
NH), 4.35e4.28 (1H, m, CH (glutamic)), 4.23e4.15 (1H, m, CH
(lipid)), 3.72e3.69 (1H, m, H-2 (glucose)), 3.55e3.52 (1H, m, H-3
(glucose)), 3.32e3.28 (2H, m, H-4 and H-5 (glucose)), 3.16e3.11
(2H, m, H-6a,b (glucose)), 2.55e2.35 (4H, m, 2CH2 (succinic)),
2.17e2.13 (2H, m, CH2 (glutamic)), 1.99e1.95 (2H, m, bCH2 (gluta-
mic)), 1.82e1.75 (2H, m, b-CH2 (lipid)), 1.37e1.27 (12H, m, 6CH2
(lipid)), 0.80 (3H, t, J¼6.75 Hz, CH3 (lipid)); 13
C NMR (500 MHz,
MeOD) d 174.01, 173.79, 173.59, 173.50, 173.46, 173.32, 173.29,
173.19, 173.03, 79.54, 79.48, 78.00, 77.96, 77.34, 77.34, 72.56, 72.49,
69.89, 61.18, 61.10, 53.42, 53.33, 31.51, 31.34, 31.22, 30.63, 30.46,
30.14, 29.63, 29.58, 29.53, 29.50, 29.37, 29.04, 28.91, 28.68, 26.11,
26.00, 25.42, 25.38, 22.20, 12.94; HRMS calculated for
[C25H42N3O12]À
[MÀH]À
576.2768, found 576.2774.
4.2.15. N-(2-(N-(4-(b-D-Glucopyranosylamino)succinyl))amino-D,L-
dodecanoyl)-L-glutamic acid (6c). Compound 6c was prepared by
the procedure described for compound 6a, except compound 5c
(1.10 g, 1.37 mmol) was used to give compound 6c (0.79 g,
1.31 mmol) in 95% yield as a white powder; mp 190 C (1:1 mixture
of diastereomers). 1
H NMR (500 MHz, MeOD) d 7.70e7.69 (1H, m,
amide NH), 7.60e7.57 (1H, m, amide NH), 7.50e7.49 (1H, m, amide
NH), 4.41e4.38 (1H, m, CH (glutamic)), 4.30e4.27(1H, m, CH
(lipid)), 3.81e3.79 (1H, m, H-2 (glucose)), 3.63e3.60 (1H, m, H-3
(glucose)), 3.40e3.32 (2H, m, H-4 and H-5 (glucose)), 3.24e3.20
(2H, m, H-6a,b (glucose)), 2.55e2.35 (4H, m, 2CH2 (succinic)),
2.17e2.13 (2H, m, CH2 (glutamic)), 1.99e1.95 (2H, m, bCH2 (gluta-
mic)), 1.82e1.75 (2H, m, b-CH2 (lipid)), 1.37e1.27 (16H, m, 8CH2
(lipid)), 0.80 (3H, t, J¼6.75 Hz, CH3 (lipid)); 13
C NMR (500 MHz,
MeOD) d 173.72,173.64,172.70,172.46,172.15,172.15,172.00,171.96,
171.91, 171.80, 78.17, 78.14, 76.64, 76.62, 76.00, 71.24, 71.20, 68.57,
59.85, 52.11, 52.00, 50.23, 50.16, 30.23, 29.99, 29.30, 29.12, 28.80,
28.28, 27.87, 27.76, 27.61, 27.58, 27.53, 24.83, 24.67, 24.10, 24.06,
20.89, 11.60; HRMS calculated for [C27H46N3O12]À
[MÀH]þ
604.3081, found 604.3087.
4.2.16. N-(2-(N-(4-(b-D-Glucopyranosylamino)succinyl))amino-D,L-
tetradecanoyl)-L-glutamic acid (6d). Compound 6d was prepared by
the procedure described for compound 6a, except compound 5d
(1.25 g, 1.50 mmol) was used to give compound 6d (0.85 g,
1.34 mmol) in 89% yield as a white powder; mp 110 C (1:1 mixture
of diastereomers). 1
H NMR (500 MHz, MeOD) d 7.63e7.60 (1H, m,
amide NH), 7.55e7.53 (1H, m, amide NH), 7.40e7.38 (1H, m, amide
NH), 4.35e4.30 (1H, m, CH (glutamic)), 4.23e4.20(1H, m, CH
(lipid)), 3.73e3.71 (1H, m, H-2 (glucose)), 3.57e3.53 (1H, m, H-3
(glucose)), 3.33e3.28 (2H, m, H-4 and H-5 (glucose)), 3.17e3.13
(2H, m, H-6a,b (glucose)), 2.52e2.35 (4H, m, 2CH2 (glucose)),
2.10e2.07 (2H, m, CH2 (glutamic)), 1.92e1.88 (2H, m, bCH2 (gluta-
mic)), 1.75e1.72 (2H, m, b-CH2 (lipid)), 1.37e1.27 (20H, m, 10CH2
(lipid)), 0.80 (3H, t, J¼6.8 Hz, CH3 (lipid)); 13
C NMR (500 MHz,
MeOD) d 174.23,174.11,173.89,173.56,173.48,173.40,173.26,173.03,
172.00, 80.02, 79.60, 78.07, 77.29, 72.44, 69.90, 61.19, 55.59, 53.58,
53.46, 52.44, 52.34, 51.52, 50.32, 50.14, 49.96, 37.72, 31.55, 31.22,
31.11, 31.00, 30.65, 30.51, 30.15, 30.02, 29.97, 29.55, 29.43, 29.25,
29.19, 29.16, 29.10, 29.00, 28.96, 28.85, 28.73, 28.35, 25.81, 25.34,
24.32, 22.22, 19.10, 12.99; HRMS calculated for [C29H51N3NaO12]þ
[MþNa]þ
656.3370, found 656.3365.
4.2.17. Disodium N-(2-(N-(4-(b-D-glucopyranosylamino)succinyl))
amino-D,L-octanoyl)-L-glutamate (7a). The free acid 6a (0.54 g,
1.00 mmol) was suspended in water (50 mL), NaHCO3 (0.16 g,
2.00 mmol) was added and the mixture was sonicated. The reaction
mixture was lyophilized to give liposaccharide 7a (0.59 g,
1.00 mmol) in a quantitative yield as a white powder; mp 200 C
A.S. Abdelrahim et al. / Tetrahedron 68 (2012) 4967e4975 4973

8. (1:1 mixture of diastereomers). IR (powder) nmax¼3264, 2926,
2859, 1639, 1549, 1396, 1113, 1076, 1020, 893 cmÀ1
. 1
H NMR
(500 MHz, MeOD) d 7.70e7.68 (1H, m, amide NH), 7.60e7.59 (1H,
m, amide NH), 7.50e7.49 (1H, m, amide NH), 4.39e4.33 (1H, m, CH
(glutamic)), 4.23e4.19 (1H, m, CH (lipid)), 3.78e3.72 (1H, m, H-2
(glucose)), 3.58e3.55 (1H, m, H-3 (glucose)), 3.34e3.31 (2H, m, H-4
and H-5 (glucose)), 3.18e3.14 (2H, m, H-6a,b (glucose)), 2.55e2.35
(4H, m, 2CH2 (succinic)), 2.17e2.13 (2H, m, CH2 (glutamic)),
1.99e1.95 (2H, m, bCH2 (glutamic)), 1.82e1.75 (2H, m, b-CH2
(lipid)), 1.37e1.27 (8H, m, 4CH2 (lipid)), 0.80 (3H, t, J¼6.75 Hz, CH3
(lipid)); 13
C NMR (500 MHz, MeOD) d 174.00, 173.82, 173.79, 173.59,
173.50, 173.46, 173.40, 173.29, 173.18, 172.01, 171.9079.54, 79.48,
78.04, 78.01, 77.99, 77.33, 72.56, 72.49, 69.89, 69.84, 61.18, 61.10,
53.41, 53.32, 51.52, 51.37, 31.31, 31.21, 31.18, 30.60, 30.43, 30.13,
30.10, 29.62, 29.51, 29.49, 29.36, 28.55, 28.51, 26.10, 25.99, 25.83,
25.79, 25.35, 25.32, 22.11, 12.89; HRMS calculated for
[C23H38N3O12]À
[MÀ2NaþH]À
548.2455, found 548.2461.
4.2.18. Disodium N-(2-(N-(4-(b-D-glucopyranosylamino)succinyl))
amino-D,L-decanoyl)-L-glutamate (7b). Compound 7b was pre-
pared by the procedure described for compound 7a, except com-
pound 6b (0.57 g, 1.00 mmol) was used to produce compound 7b
in a quantitative yield as a white powder; mp 209 C (1:1 mixture
of diastereomers). IR (powder) nmax¼3266, 2925, 2855, 1643, 1548,
1399, 1112, 1077, 1022, 892 cmÀ1
. 1
H NMR (500 MHz, MeOD)
d 7.75e7.73 (1H, m, amide NH), 7.62e7.60 (1H, m, amide NH),
7.52e7.51 (1H, m, amide NH), 6.52 (1H, m, H-1 (glucose)), 5.28 (2H,
m, H-2 and H-3 (glucose)), 5.02 (1H, m, H-5 (glucose)), 4.88 (1H,
m, H-4 (glucose)), 4.46 (2H, m, H-6a,b (glucose)), 4.24 (1H, t,
J¼4.1 Hz, CH (lipid)), 4.01 (1H, t, J¼12.35 Hz, CH (glutamic)), 2.49
(4H, m, 2CH2 (succinic)), 2.37 (2H, m, CH2 (glutamic)), 2.30 (2H, m,
bCH2 (glutamic)), 1.75 (2H, m, b-CH2 (lipid)), 1.23 (12H, m, 6CH2
(LAA)), 0.80 (3H, t, J¼6.75 Hz, CH3 (LAA)); 13
C NMR (500 MHz,
MeOD) d 174.01, 173.79, 173.59, 173.50, 173.46, 173.32, 173.29,
173.19, 173.03, 79.54, 79.48, 78.00, 77.96, 77.34, 77.34, 72.56, 72.49,
69.89, 61.18, 61.10, 53.42, 53.33, 31.51, 31.34, 31.22, 30.63, 30.46,
30.14, 29.63, 29.58, 29.53, 29.50, 29.37, 29.04, 28.91, 28.68, 26.11,
26.00, 25.42, 25.38, 22.20, 12.94; HRMS calculated for
[C25H42N3O12]À
[MÀ2NaþH]À
576.2768, found 576.2774.
4.2.19. Disodium N-(2-(N-(4-(b-D-glucopyranosylamino)succinyl))
amino-D,L-dodecanoyl)-L-glutamate (7c). Compound 7c was pre-
pared by the procedure described for compound 7a, except com-
pound 6c (0.60 g,1.00 mmol) was used to produce compound 7c in
a quantitative yield as a white powder; mp 221 C (1:1 mixture of
diastereomers). IR (powder) nmax¼3267, 2923, 2854, 1644, 1549,
1397, 1114, 1077, 1022, 895 cmÀ1
. 1
H NMR (500 MHz, MeOD)
d 7.75e7.72 (1H, m, amide NH), 7.61e7.59 (1H, m, amide NH),
7.53e7.52 (1H, m, amide NH), 6.52 (1H, m, H-1 (glucose)), 5.28
(2H, m, H-2 and H-3 (glucose)), 5.02 (1H, m, H-5 (glucose)), 4.88
(1H, m, H-4 (glucose)), 4.46 (2H, m, H-6a,b (glucose)), 4.24 (1H, t,
J¼4.1 Hz, CH (lipid)), 4.01 (1H, t, J¼12.35 Hz, CH (glutamic)), 2.53
(4H, m, 2CH2 (succinic)), 2.37 (2H, m, CH2 (glutamic)), 2.17 (2H, m,
bCH2 (glutamic)), 1.75 (2H, m, b-CH2 (lipid)), 1.27 (16H, m, 8CH2
(lipid)), 0.80 (3H, t, J¼6.75 Hz, CH3 (lipid)); 13
C NMR (500 MHz,
MeOD) d 173.72, 173.64, 172.70, 172.46, 172.15, 172.15, 172.00,
171.96, 171.91, 171.80, 78.17, 78.14, 76.64, 76.62, 76.00, 71.24, 71.20,
68.57, 59.85, 52.11, 52.00, 50.23, 50.16, 30.23, 29.99, 29.30, 29.12,
28.80, 28.28, 27.87, 27.76, 27.61, 27.58, 27.53, 24.83, 24.67, 24.10,
24.06, 20.89, 11.60; HRMS calculated for [C27H46N3O12]À
[MÀ2NaþH]À
604.3081, found 604.3087.
4.2.20. Disodium N-(2-(N-(4-(b-D-glucopyranosylamino)succinyl))
amino-D,L-tetradecanoyl)-L-glutamate (7d). Compound 7d was pre-
pared by the procedure described for compound 7a, except com-
pound 6d (0.63 g, 1.00 mmol) was used to produce compound 7d in
a quantitative yield as a white powder; mp 229 C (1:1 mixture of
diastereomers). IR (powder) nmax¼3268, 2923, 2853, 1645, 1549,
1401, 1112, 1076, 1022, 893 cmÀ1
. 1
H NMR (500 MHz, MeOD)
d 7.70e7.69 (1H, m, amide NH), 7.63e7.62 (1H, m, amide NH),
7.52e7.51 (1H, m, amide NH), 6.52 (1H, m, H-1 (glucose)), 5.28 (2H,
m, H-2 and H-3 (glucose)), 5.02 (1H, m, H-5 (glucose)), 4.88 (1H, m,
H-4 (glucose)), 4.46 (2H, m, H-6a,b (glucose)), 4.24 (1H, t, J¼4.1 Hz,
CH (lipid)), 4.01 (1H, t, J¼12.35 Hz, CH (glutamic)), 2.52 (4H, m,
2CH2 (succinic)), 2.37 (2H, m, CH2 (glutamic)), 2.30 (2H, m, bCH2
(glutamic)), 1.56 (2H, m, b-CH2 (lipid)), 1.21 (20H, m, 10CH2 (lipid)),
0.80 (3H, t, J¼6.65 Hz, CH3 (lipid)); 13
C NMR (500 MHz, MeOD)
d 174.23,174.11,173.89,173.56,173.48,173.40,173.26,173.03,172.00,
80.02, 79.60, 78.07, 77.29, 72.44, 69.90, 61.19, 55.59, 53.58, 53.46,
52.44, 52.34, 51.52, 50.32, 50.14, 49.96, 37.72, 31.55, 31.22, 31.11,
31.00, 30.65, 30.51, 30.15, 30.02, 29.97, 29.55, 29.43, 29.25, 29.19,
29.16, 29.10, 29.00, 28.96, 28.85, 28.73, 28.35, 25.81, 25.34, 24.32,
22.22, 19.10, 12.99; HRMS calculated for [C29H51N3NaO12]þ
[MÀNaþ2H]þ
656.3370, found 656.3365.
4.3. Isothermal titration calorimetry
ITC measurements were carried out using a VP-ITC MicroCalo-
rimeter (MicroCal, Northampton, MA, USA). Solutions (4 mM) of
liposaccharides 7b and 7c were degassed for 15 min prior to each
experiment and the sample cell (1.5 mL) was filled with deionized
water. The titrating solution was automatically added in aliquots
(total 30Â) of 10 mL from a 300 mL modified gas-tight Hamilton
syringe through a thin stainless steel capillary under continuous
stirring at 300 rpm, 298 K and 4 min intervals. The resulting data
were integrated using Origin software (MicroCal) to give the en-
thalpy of each liposaccharide injection. The heat exchanges gen-
erated by liposaccharide/water interactions were obtained from
titrations of liposaccharide solutions into deionized water. The
enthalpy of aggregation of liposaccharides 7b and 7c was obtained
from the difference between the initial and the final asymptotes of
the sigmoidal curves. The CACs were obtained from the transition
point of the enthalpy concentration profiles. All experiments were
repeated three times to check the reproducibility of the results.30
Acknowledgements
We wish to thank Ichun Lin for his help with the TEM mea-
surements. Also we would like to acknowledge the Egyptian Gov-
ernment for providing a PhD scholarship for A.S.A.. We thank the
Australian Research Council for a Professorial Research Fellowship
to I.T. (DP110100212) and an Australian Postdoctoral Fellowship to
P.S. (DP1092829).
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