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Tetrahedron 68 (2012) 4967e4975

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Tetrahedron

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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, Australiab 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 2012Received in revised form 30 March 2012Accepted 16 April 2012Available online 23 April 2012

Keywords:Charged liposaccharideMicrocalorimetryTobramycinAbsorption enhancerNanoparticleDrug delivery

* Corresponding author. Tel.: þ61 7 33469892; faaddress: p.simerska@uq.edu.au (P. Simerska).

0040-4020/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.tet.2012.04.064

a b s t r a c t

A series of anionic liposaccharide derivatives were synthesized in order to develop a system, whichwould 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, whilethe lipophilicity can be controlled by variation of the lipid side chain. All liposaccharide derivatives werepurified 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.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

A large number of newly developed drug candidates cannot beadministered orally for various reasons such as poor penetrationthrough the intestinal mucosa, and/or binding in the gastrointes-tinal tract due to the highly hydrophilic properties.1 Therefore, theadministration of these drugs is limited to intravenous or in-tramuscular routes. To overcome these challenges, medicinal andpharmaceutical research has focused on development of alterna-tives with enhanced oral bioavailability.2 One of the main strategiesbeing 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 thedrug’s intestinal absorption andmembrane permeability, especiallyby passive diffusion.3 The addition of a safe and effective absorptionenhancer into the conventional oral dosage form is considered to beeasier and cheaper than development of a novel drug or pro-drug.4

Also aggregation, surfactant and ion-pairing characteristics of theformed compounds can increase intestinal uptake.5

We have demonstrated earlier that the co-administration ofliposaccharide-based absorption enhancers with various drugs(e.g., piperacillin6 and gentamicin7) improved absorption of theparent drug in vivo. However, the permeability of those compounds

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was still low.8 To further improve the permeability, we describe thesynthesis and characterization of a novel series of anionic lip-osaccharide derivatives with good absorption enhancing activity.These derivatives are unique amphiphilic synthetic compoundswith 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 andstability during oral administration.9 This structural arrangementmodulates aqueous solubility as well as the lipophilicity of thedrugeliposaccharide complex. The incorporation of a lipoaminoacid (LAA),10 an amino acid with a lipophilic alkyl side chain, intothe molecules was previously reported to increase oral absorptionof drugs with poor bioavailability.11 It has been shown, when LAAsform 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 facilitatedglucose transport systems during absorption.

The amphoteric structural design of the molecules was de-veloped in order to promote surfactant like properties and furtheraggregation and/or micellization of the liposaccharides. Isothermaltitration calorimetry (ITC) was performed to determine the criticalaggregation 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 alsocalculated. The size and shape of the liposaccharides were mea-sured by transmission electron microscopy (TEM) and dynamiclight scattering (DLS).

A.S. Abdelrahim et al. / Tetrahedron 68 (2012) 4967e49754968

2. Results and discussion

2.1. Synthesis

tert-Butyloxocarbonyl (Boc) protected LAA derivatives 1aedweresynthesized 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 esterifiedusing 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 sodiumbicarbonate (NaHCO3), dimethylated Glu 2 was obtained in a quan-titative yield.14 Boc-LAAs 1aedwith 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) toproduce 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 aqueousNaHCO3 to give dimethyl-Glu-LAAs 4aed in 90e95% yields.

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.

Peracetylation of D-glucose was performed using acetic anhy-dride, followed by bromination using hydrogen bromide in aceticacid.15 Several methodologies were tested to prepare the azide de-rivative from the bromide including addition of sodium azide in themixture of acetone and water16 and the method using tetra-butylammonium hydrogen sulfate in DCM/aqueous NaHCO3 mixture.17

Applying the first method for azide synthesis, we obtained, afterthe crude product was re-crystallized from hot ethanol, higheryields (86% instead of 64%) of the b-D-glucopyranosyl azide.

Scheme 2. Synthesis of the liposaccharides 7aed with different lipid side chain lengths. Rea(ii) H2O addition,12 h; (iii) Amberlite IR-120 (Hþ); (c) NaHCO3.

b-D-Glucopyranosyl azide16 was reduced to amine by hydrogenation(H2 on Pd/C) and immediately reacted in situ with a 1 mol equiv ofsuccinic anhydride to overcome the instability of peracetylatedglucosyl amine.18,19 The concentration of succinic anhydride used inthe 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 unreactedperacetylated glucosyl amine from the reaction.20 The peracetylatedglucosylamido propionic acid was coupled to the free amine de-rivatives 4aed using HBTU/DIPEA in dry DCM and following flashcolumn chromatography yielded pure liposaccharides 5aed.Zempl�en deacetylation using 1 MNaOCH3 inmethanol at pH 13 wasapplied, and the reaction mixture was stirred with water for anadditional 12 h to hydrolyse di-methyl esters. Then the reactionmixture 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 6aedwere sonicated with 2 equiv of NaHCO3 in waterto facilitate ion-pairing of the formed liposaccharide with the pos-itively charged drug and to increase the aqueous solubility of thefinal complex. The sodium derivatives of the liposaccharides 7aedwith C8eC14 lipid side chain lengths (Scheme 2) were obtainedafter lyophilization in quantitative yields as white powders.

All the structural elucidations were done by 1H, 13C nuclearmagnetic resonance (NMR) and mass spectroscopy (MS).

2.2. Isothermal titration calorimetry and size measurements

Isothermal titration calorimetry (ITC) was used to monitor theinteractions of anionic liposaccharides in aqueous solution.21 Thesynthesised liposaccharides were expected to form aggregates dueto their intra- and/or intermolecular-hydrophobic interactions inaqueous media as they possessed both hydrophilic and lipophilicmoieties. Determination of the critical aggregation concentration(CAC) of the liposaccharides was an important step in order tounderstand the interactions between the liposaccharide andamodel drug during their complexation. The importance of the CACvalue has been reported elsewhere describing the effect of higherCAC values on aggregation and permeation through biologicalmembranes.22 Also thermodynamic profile results of the newlydesigned penetration enhancers would be valuable in predictingpotential toxicity of the compounds, especially disruption to bi-ological membranes.

The liposaccharides 7b,c formed aggregates at their CACs, whichwere calculated from the ITC experiments. The CACs of the lip-osaccharides 7b,cwere estimated by the van Os method;23 and the

gents and conditions: (a) HBTU, DIPEA, DCM, 24 h; (b) (i) 0.1 M NaOCH3, methanol, 2 h;

Table 1Summary of the thermodynamic values of compounds 7b and 7c obtained by iso-thermal titration calorimetry (ITC) measurements

Liposaccharide CAC(mM)

DHagg

(kJmol�1)DGagg

(kJmol�1)TDSagg(kJmol�1)

DSagg(kJmol�1)

GlcC10Glu (7b) 0.275 3.75 �3.19 6.94 0.023GlcC12Glu (7c) 0.325 2.40 �2.77 5.17 0.017

A.S. Abdelrahim et al. / Tetrahedron 68 (2012) 4967e4975 4969

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 reflectedthe contribution of individual interactions occurring between theliposaccharide molecules and deionized water. It was found thatthe liposaccharides GlcC10Glu (7b) and GlcC12Glu (7c) aggregatedin the aqueous state mainly due to their higher lipophilicity thanthat of the liposaccharide GlcC8Glu (7a). The CAC of liposaccharide7a could not be accurately estimated due to the low enthalpy data(�0.5 kJmol�1). The liposaccharide 7a either did not aggregate orthe aggregates were not stable in water to allow ITC results to bemeasured. 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 notobserved. A higher degree of lipophilicity is known to lead to poorsolubility in aqueous media and may cause large changes in thetitration curve, which may relate to the smaller demicellizationenthalpy for this compound’s aggregates. The calorimetric titrationgraphs 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 tobe similar for GlcC10Glu (7b)¼3.75 kJmol�1 and for GlcC12Glu(7c)¼2.40 kJmol�1 (Fig. 1a). A change of slope of cumulative en-thalpy was used to calculate the CAC values by selecting data aboveand below these concentrations. These datawere fitted into a linear

(a)

(b)

(c)

Fig. 1. Determination of the enthalpy of aggregation and critical aggregation concentrationsthe reaction versus concentration of 7b or 7c; (b) determination of the CACs through cumuliposaccharides 7b and 7c.

regressionwith the point of their intersections selected as the CACs(Fig. 1b).24 CACs were also determined from the maximum of thefirst derivative curves (Fig. 1c).25 The CACs for GlcC10Glu (7b) andGlcC12Glu (7c) were calculated to be 0.275�0.008 mM and0.253�0.012 mM, respectively (Table 1). A decrease in peak heightwas noticed after a certain number of injections (Fig. 1c). This wascaused by the concentrations in the reaction cell exceeding the CACand the aggregates titrated into the reaction cell were no longerdissociated. Above the CAC the enthalpy change is therefore solelythe result of aggregate dilution effects of the CAC.26

The Gibbs free energy of aggregation (DGagg) was calculated todetermine the binding process of the liposaccharides (DGagg¼RTlnXagg; R is the gas constant 8.314 J K�1mol�1, T is the absolutetemperature 298 K and Xagg is the CAC value in moles). DGagg of

(CACs) of 4 mM liposaccharides GlcC10Glu (7b) and GlcC12Glu (7c) at 298 K; (a) heat oflative enthalpy versus concentration of 7b or 7c; (c) first derivative of the enthalpy of

A.S. Abdelrahim et al. / Tetrahedron 68 (2012) 4967e49754970

GlcC10Glu 7bwas calculated to be �3.19 kJmol�1 and of GlcC12Glu7c �2.77 kJmol�1. These results suggested that favourable changesduring the aggregation process led to the formation of stabilisedentities in the aqueous environment.

The entropy of aggregation (DSagg) of both liposaccharides 7band 7c was calculated using the GibbseHelmoltz equationDSagg¼(DHagg�DGagg)/T. The endothermic nature of the processes(DHagg>0) (Fig. 1a) indicated that disaggregation led to an increasein the overall entropy of the system, because aggregate dissociationwas thermodynamically favourable below the CMC (DH>0);therefore, TDS>DH. DSagg was calculated for both liposaccharides7b and 7c to be 0.023 and 0.017 kJ K�1mol�1, respectively. Thispositive entropy change implied a decrease in the general degree oforder in the system (e.g., desolvation process associated with thepairing of molecules)27 and was attributed to the release of counterions associated with the surfactant head groups when aggregatesbroke down to monomers.26 Moreover, the negative value of�TDSagg (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 CACswere measured by TEM. It was previously reported by our groupthat more lipophilic compounds form larger aggregates. In thisstudy, different methods of size measurement of peptides, lip-opeptides and lipoglycopeptides were compared.28 The lip-osaccharide GlcC10Glu (7b) formed poly-dispersed aggregatesaround 60e80 nm in size with smaller individual nanoparticlesaround 30 nm (Fig. 2a, b). The liposaccharide GlcC12Glu (7c)showed similar sized spherical aggregates (Fig. 2c, d). These resultscorrelated with the liposaccharide sizes determined by DLS (ahighly poly-disperse size distribution with a peak below 100 nm;data not shown).

Fig. 2. Transmission electron microscopy (TEM) images of liposaccharide GlcC10Glu (7b) (a, b) and GlcC12Glu (7c) (c, d) at their critical aggregation concentrations (CACs).

3. Conclusion

Anionic liposaccharides 7aed were designed and synthesizedfrom biocompatible non-toxic precursors such as carbohydrate,lipoamino and amino acid derivatives and all productswere purifiedand fully characterised by NMR and High-resolution mass

spectrometry (HRMS). ITC results confirmed the ability of the lip-osaccharides7band7c (comprisingC10andC12 LAA) to aggregate inan aqueous environment. The thermodynamic profiles includingCAC, DHagg, DGagg and DSagg of the liposaccharides 7b and 7c werealso determined by ITC and showed formation of aggregates. In-terestingly C10 and C12 had the optimal lipid side chain length forthe 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 TEMand DLS. The liposaccharide-based drug delivery system presentedherewill be further tested invitro and invivo for its ability toenhanceintestinal 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) [H�] was provided by BritishDrug Houses (BDH) Ltd. (England). Gases (nitrogen, hydrogen andargon) were supplied by BOC Gases (Brisbane, QLD, Australia). Silica(silica gel 60, 230e400 mesh) for flash chromatography wasobtained 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

A.S. Abdelrahim et al. / Tetrahedron 68 (2012) 4967e4975 4971

nitrogen or argon using oven-dried glassware. Reactions werecarried out at room temperature unless otherwise specified. Thin-layer chromatography (TLC) was performed on silica gel 60 F254aluminium sheets (Merck, Darmstadt, Germany), and compoundswere 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 withthe developing agent. Purification of the synthesized compoundswas achieved by flash column chromatography that was performedon silica gel 60, 230e400 mesh ASTM (Scharlau, Barcelona, Spain).Melting points were measured with a capillary apparatus.

Infrared measurements were performed on an IR spectrometerSpectrum 2000 (Perkin Elmer Pty Ltd, Glen Waverley, VIC, Aus-tralia), at a resolution of 4 cm�1 ATR. Nuclear Magnetic Resonance(NMR) spectra (1H and 13C NMR) were recorded at room temper-ature in deuterated chloroform (CDCl3) solutions (unless otherwiseindicated). A Bruker AM 500 instrument operating at 500 MHz wasused. Chemical shifts are listed in parts per million (ppm) downfield from internal tetramethylsilane (TMS). Signal multiplicitiesare represented as singlet (s), doublet (d), double doublet (dd),triplet (t), quartet (q), quintet (quint), multiplet (m), broad (br) andbroad singlet (br s).

Mass spectra (MS) were recorded on a PerkineElmer Sciex API3000 mass spectrometer (Applied Biosystems/MDS Sciex, Toronto,Canada) operating inpositive ion electrospraymode (ESI-MS). Liquidchromatography mass spectroscopy (LCeMS/MS) data were mea-sured onaWaters 2790 instrument using positivemode electrosprayionization. The mobile phase used for the measurement was a mix-ture of solvent A (0.1% acetic acid inwater) and solvent B (0.1% aceticacid in 90% acetonitrile and 10% water). Results were analysed byAnalyst 1.4 software. High-resolution mass spectrometry (HRMS)data were obtained on a Qstar Pulsar mass spectrometer (AppliedBiosystems) operating in positive ion electrospray mode. Analyticalresults were within �0.4% of the theoretical values for the formulagiven 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 theaddition of dimethyl glutamic acid 214 (0.75 g, 4.28 mmol). Thereaction mixture was stirred at room temperature for 12 h, thenwashed with 5% HCl (2�50 mL) and a saturated solution of NaHCO3(2�50 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)) toproduce pure compound 3a (1.15 g, 2.76 mmol, 65%) as a colourlessoil (1:1 mixture of diastereomers). 1H 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)); 13C 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 product3b was purified by flash column chromatography (Rf¼0.3 ethylacetate/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. 1H 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)); 13C 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 insteadof 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 ofdiastereomers). 1H 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)); 13C 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 3dwas prepared by the proceduredescribed 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. Thecrude product was purified by flash chromatography (Rf¼0.3 ethylacetate/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). 1H NMR (500MHz, CDCl3) d 77.53e7.51 (1H, m, amideNH), 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)); 13C NMR (500MHz, 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 layerwas separated, dried over MgSO4, filtered and evaporated undervacuum to produce compound 4a (1.08 g, 3.42 mmol) in 95% yieldas a colourless oil (1:1 mixture of diastereomers). 1H 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) 4967e49754972

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)); 13C 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 compound4a, except 3b (1.50 g, 3.37 mmol) was used instead of 3a to producecompound 4b (1.10 g, 3.20 mmol) in 95% yield as a colourless oil(1:1 mixture of diastereomers). 1H 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)); 13C 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 compound4a, except 3c (1.50 g, 3.17 mmol) was used instead of 3a to producecompound 4c (1.06 g, 2.85 mmol) in 90% yield as a colourless oil(1:1 mixture of diastereomers). 1H 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)); 13C 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, found373.2697.

4.2.8. DimethylN-(2-amino-D,L-tetradecanoyl)-L-glutamate (4d). Com-pound 4dwas prepared by the procedure described for compound 3a,except 3d (1.50 g, 3.00 mmol) was used instead of 3a to producecompound 4d (1.11 g, 2.78 mmol) in 92% yield as a colourless oil (1:1mixture of diastereomers). 1H 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));13C NMR (500MHz, 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 reactionmixture and stirred at room temperature for 12 h. Then it waswashed with 5% HCl solution, 10% NaHCO3 solution, dried overMgSO4, filtered and evaporated to give an oily product, 5a. Thecrude product was purified by column chromatography (Rf¼0.6methanol/DCM, 1:9 (v/v)) to give pure 5a (1.16 g, 1.56 mmol) in70% yield as a colourless oil (1:1 mixture of diastereomers). 1HNMR (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)); 13C 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). Compound5bwas prepared by following the procedure described for compound5a, except compound 4b (0.76 g, 2.23 mmol) was used to producecompound 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). 1HNMR (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)); 13CNMR (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; HRMScalculated for [C35H55N3NaO16]þ [MþNa]þ 796.3480, found796.3475.

4.2.11. DimethylN-(2-(N-(4-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosy-lamino)succinyl))amino-D,L-dodecanoyl)-L-glutamate (5c). Compound5cwas prepared by following the procedure described for compound5a, except compound 4c (0.82 g, 2.23 mmol) was used to producecompound 5c (Rf¼0.6methanol/DCM,1:9 (v/v)) (1.16 g,1.45 mmol) in65% yield as a colourless oil (1:1 mixture of diastereomers). 1H 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) 4967e4975 4973

1.57e1.53 (2H, m, b-CH2 (lipid)), 1.20e1.19 (16H, m, 8CH2 (lipid)), 0.82(3H, t, J¼6.8 Hz, CH3 (lipid)); 13C NMR (500MHz, 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 calculatedfor [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 (1:1mixture of diastereomers). 1H 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)); 13C 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 12using 1 M NaOCH3 for 2 h. Water (10 ml) was added to the reactionmixture and the pH was readjusted to 13. The solution was stirredat room temperature for an additional 12 h. Upon completion, thereaction mixture was acidified using Amberlite resin IR-120 [Hþ]until an acidic pH was obtained. The reaction mixture was filteredand the filtrate was evaporated under vacuum. The residue waslyophilised in acetonitrile/water (1:1) to give compound 6a (0.79 g,1.44 mmol) in 89% yield as awhite powder; mp 172 �C (1:1 mixtureof diastereomers). 1H NMR (500 MHz, MeOD) d 7.65e7.64 (1H, m,amide NH), 7.51e7.50 (1H, m, amide NH), 7.39e7.36 (1H, m, amideNH), 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)); 13C 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]� [M�H]� 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 theprocedure 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 awhite powder; mp 182 �C (1:1 mixtureof diastereomers). 1H NMR (500 MHz, MeOD) d 7.70e7.69 (1H, m,amide NH), 7.63e7.62 (1H, m, amide NH), 7.52e7.50 (1H, m, amideNH), 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)); 13C 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]� [M�H]� 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 bythe 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 mixtureof diastereomers). 1H NMR (500 MHz, MeOD) d 7.70e7.69 (1H, m,amide NH), 7.60e7.57 (1H, m, amide NH), 7.50e7.49 (1H, m, amideNH), 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)); 13C 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]� [M�H]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 6dwas prepared bythe 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 mixtureof diastereomers). 1H NMR (500 MHz, MeOD) d 7.63e7.60 (1H, m,amide NH), 7.55e7.53 (1H, m, amide NH), 7.40e7.38 (1H, m, amideNH), 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)); 13C 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 themixturewas sonicated. The reactionmixture 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) 4967e49754974

(1:1 mixture of diastereomers). IR (powder) nmax¼3264, 2926,2859, 1639, 1549, 1396, 1113, 1076, 1020, 893 cm�1. 1H 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-4and 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)); 13C 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]� [M�2NaþH]� 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 7bin a quantitative yield as a white powder; mp 209 �C (1:1 mixtureof diastereomers). IR (powder) nmax¼3266, 2925, 2855, 1643, 1548,1399, 1112, 1077, 1022, 892 cm�1. 1H 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)); 13C 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]� [M�2NaþH]� 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 ina quantitative yield as a white powder; mp 221 �C (1:1 mixture ofdiastereomers). IR (powder) nmax¼3267, 2923, 2854, 1644, 1549,1397, 1114, 1077, 1022, 895 cm�1. 1H 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)); 13C 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]�

[M�2NaþH]� 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 ofdiastereomers). IR (powder) nmax¼3268, 2923, 2853, 1645, 1549,1401, 1112, 1076, 1022, 893 cm�1. 1H 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)); 13C 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

[M�Naþ2H]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) ofliposaccharides 7b and 7c were degassed for 15 min prior to eachexperiment and the sample cell (1.5 mL) was filled with deionizedwater. The titrating solution was automatically added in aliquots(total 30�) of 10 mL from a 300 mL modified gas-tight Hamiltonsyringe through a thin stainless steel capillary under continuousstirring at 300 rpm, 298 K and 4 min intervals. The resulting datawere 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 fromtitrations of liposaccharide solutions into deionized water. Theenthalpy of aggregation of liposaccharides 7b and 7c was obtainedfrom the difference between the initial and the final asymptotes ofthe sigmoidal curves. The CACs were obtained from the transitionpoint of the enthalpy concentration profiles. All experiments wererepeated 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 theAustralian Research Council for a Professorial Research Fellowshipto I.T. (DP110100212) and an Australian Postdoctoral Fellowship toP.S. (DP1092829).

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