Carbohydrate mediated drug delivery: Synthesis and characterization of new lipid-conjugates

Article abstract of DOI:10.1016/j.chemphyslip.2014.10.003

A new synthetic methodology for cationic glycolipids using p-aminophenyl-α-d-mannopyranoside (PAPM) and p-aminophenyl-α-d-galactopyranoside (PAPG) with spacer in between the quaternary nitrogen atom and the sugar unit is developed. In addition, a new clas

Full text of DOI:10.1016/j.chemphyslip.2014.10.003

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Chemistry and Physics of Lipids xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
1
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Carbohydrate mediated drug delivery: Synthesis and characterization
of new lipid-conjugates
Moghis U. Ahmad a, Shoukath M. Ali a, Ateeq Ahmad a, Saifuddin Sheikh a, Paul Chen b,
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Q1
Q2
Imran Ahmad a,
*
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a
Jina Pharmaceuticals Inc., 28100 N Ashley Circle, Suite 103, Libertyville, IL 60048, USA
b
Nia Life Sciences, 28100 N Ashley Circle, Suite 102, Libertyville, IL 60048, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
A new synthetic methodology for cationic glycolipids using p-aminophenyl-a-D-mannopyranoside
Received 16 September 2014
Received in revised form 16 October 2014
Accepted 19 October 2014
Available online xxx
(PAPM) and p-aminophenyl-a-D-galactopyranoside (PAPG) with spacer in between the quaternary
nitrogen atom and the sugar unit is developed. In addition, a new class of neutral glycolipid conjugates,
such as PAPM–lipids or PAPG–lipids conjugates was also synthesized for targeting drugs to receptors. The
precipitation–inhibition assay showed that conjugate of PAPM inhibited the concanavalin A and
invertase aggregation. This binding inhibition study of a synthesized compound suggests that conjugates
of PAPM can be potentially used to target mannose receptors. In addition, a higher transfection was
Keywords:
Mannopyranoside
Galactopyranoside
Lipids
Lipid-conjugate
Synthesis
obtained by mixing PAPM with pSV-b-gal reporter gene and incubating with mannose binding protein/
receptor expressing A549 cells. The coexistence of both mannose group and a net positive charge may
result in improved transfection efficiency in cells expressing mannose binding proteins/receptors.
ã 2014 Published by Elsevier Ireland Ltd.
Drug delivery
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1. Introduction
carriers. For example, the sulfated polysaccharide, heparin, plays
an essential role in blood coagulation (Linhardt and Toida, 1997).
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Carbohydrates derivatives have been known to be involved in
variety of biological functions. Cell surface carbohydrates are
involved in numerous biological functions, including cellular
recognition, adhesion, cell growth regulation, cancer cell metasta-
sis and inflammation (Dwek, 1996). They also serve as an
attachment sites for infectious bacteria, viruses, toxins, and
hormones that result in pathogenesis (Varki, 1993). Cell surface-
bound receptors represent suitable entry sites for drug delivery
into cells by receptor-mediated endocytosis. Targeted drug
delivery capitalizes on the presence of specific cell surface receptor
mediated endocytosis (Cotton and Wagner, 1993; Perales et al.,
1994; Wu and Wu, 1987). The presence of mannose receptors on a
variety of macrophages such as peritoneal, alveolar and in Kupffer
cells is well documented (Imber et al., 1982; Stahl et al., 1978;
Schlessinger et al., 1978). Synthetic carbohydrate polymers
containing fucosylamine and mannosamine have been targeted
to mouse leukemia L1210 cells, and macrophages, respectively
(Rathi et al., 1991; Duncan et al., 1986). These specific carbohy-
drate-based molecules could be applied as drug or gene delivery
Several researchers are of the opinion that macrophages may
serve as a secondary drug carrier for the delivery of liposomal
drugs (Ahmad et al., 1989; Morgan et al., 1985). The role of
macrophages in the uptake process is quite evident from the
increased deposition of liposomal drug products in cells (Ahmad
et al., 1989). The studies indicated that amphotericin B incorpo-
rated into liposomes composed of egg phosphatidyclcholine and
1,2-dipalmitoyl-3-phosphoethanolamine mannose (EPC/DPPE-
Man) is less toxic as compared to the drug in non-mannosylated
EPC liposomes. This was related to the fact that mannosylated
liposomes are more rapidly taken up by the macrophages
compared with the non-mannosylated ones; thus less time is
available for such liposomes to interact with sensitive red blood
cells, resulting in less toxicity (Ahmad et al.,1991). Liposome-based
drug formulation ligated with mannose successfully demonstrated
targeting of therapeutic drug to the disease site.
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Cationic liposomes bearing covalently grafted receptor specific
ligands, having so called “homing devices”, on their surfaces are also
capable of delivering their genetic payloads to specific cells in the
body. Many reported specific cationic glycolipid based transfection
vectors based on the covalent grafting of the cyclic pyranose form of
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the
D-galactose ligands onto their polar head-group region
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(Kawakami et al., 1998, 2000a,b; Fumoto et al., 2004; Shigeta
et al., 2007; Sun et al., 2005; Mangit et al., 2005; Hwang et al., 2001;
* Corresponding author. Tel.: +1 847 573 0771; fax: +1 847 573 0770.
E-mail address: Imran@jinaphama.com (I. Ahmad).
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http://dx.doi.org/10.1016/j.chemphyslip.2014.10.003
0009-3084/ã 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Ahmad, M.U., et al., Carbohydrate mediated drug delivery: Synthesis and characterization of new lipid-
conjugates. Chem. Phys. Lipids (2014), http://dx.doi.org/10.1016/j.chemphyslip.2014.10.003

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Hashida et al., 2001; Gaucheron et al., 2001; Plank et al., 1992).
Following the above concept, we have synthesized novel cationic
nitrogen atmosphere and monitored by thin layer chromatography
(TLC) on Merck silica gel F254 plates (250 m) with visualization
m
p-aminophenyl-
a
-D-mannopyranoside (PAPM) 1a and p-amino
using UV light or by heating plates sprayed with a solution of
phosphomolybdic acid (5% ethanol solution). Flash column
chromatography was carried out on silica gel 60 Å (230–400 mesh).
Organic solvent extracts in the isolation procedures were dried
over anhydrous sodium sulfate. Melting points were obtained in
open capillary tubes in a Mel-Temp1 melting point apparatus and
are uncorrected.
phenyl- -galactopyranoside (PAPG) 1b (Fig. 1) containing spacer
a-D
in betweenthe sugarheadandpositivelychargednitrogenatom,for
gene or drug delivery. A different chemistry is used to conjugate
lipids inviewof targetingtospecific cells. Cell specific receptorsuch
as mannose or galactose can be effectively utilized s for selective
delivery of drugs by employing synthetic sugar–lipid conjugates,
such as p-(tetradecanoylamido) phenyl
and p-(tetradecanoylamido) phenyl -galactopyranoside 2b and
analogs; and p-(3-cholesterylimido) phenyl- -mannopyrano-
side 3a and p-(30cholesterylimido) phenyl-
-galactopyranoside
a-
D-mannopyranoside 2a
1H NMR spectra were recorded using Varian Inova spectrome-
ter. Unless otherwise stated, 1H NMR spectra were recorded at 25 ꢀ
C using an internal tetramethylsilane standard at 0 ppm. IR spectra
were recorded on a Nicolet Nexus 470 FT-IR spectrometer. Samples
were prepared by attenuated total reflectance (ATR) method. High
resolution mass spectra were recorded on BioTOF II ESI mass
spectrometer. Optical rotations were obtained on PerkinElmer
Polarimeter 341.
a-D
a-D
a-D
conjugate 3b (Fig. 1). Here we describe the complete synthesis and
structural characterization by 1H NMR and high resolution mass
spectroscopy (HRMS) along with precipitation–inhibition assay of
synthesized compound as an example with Concanavalin A.
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2. Materials and methods
2.1. Synthesis of 2,3,4, 5-tetraacetyl-p-nitrophenyl-a-D-
mannopyranoside (5a)
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p-Nitrophenyl
a-D-mannopyranoside and p-nitrophenyl a-D-
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galactopyranoside were purchased from Toronto Research Chemi-
cal, Inc., Toronto, ON, Canada. Anhydrous solvents were purchased
from Sigma–Aldrich and used without further drying. Reagents of
the highest commercial quality were purchased and used without
further purification. All reactions were carried out under a dry
To a solution of p-nitrophenyl mannopyranoside (2.0 g,
6.64 mmol) in anhydrous pyridine (32 mL) was added acetic
anhydride (312 mL) and 4-dimethylaminopyridine (DMAP)
(20 mg), and the reaction mixture was stirred at room temperature
for 4 h. Progress of reaction was checked by TLC (CHCl3:MeOH; 9:1,
Fig. 1. Lipid conjugates of p-aminophenyl-a-D-galactopyranoside and p-aminophenyl-a-D-mannopyranoside.
Please cite this article in press as: Ahmad, M.U., et al., Carbohydrate mediated drug delivery: Synthesis and characterization of new lipid-
conjugates. Chem. Phys. Lipids (2014), http://dx.doi.org/10.1016/j.chemphyslip.2014.10.003

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v/v). After completion of reaction the solvent was removed under
vacuum. The residue was dissolved in chloroform (200 mL) and
washed with cold aq. HCl (2%) followed by water and brine, dried
over anhydrous sodium sulfate, and concentrated under reduced
pressure to afford 5a (2.97 g, 95.5%) as a faint yellow solid. mp 144–
4). The acidified product was concentrated at reduced pressure and
the residue was triturated with methanol (2 ꢂ 20 mL) and filtered
through Whatman #54 paper, concentrated under vacuum and
desiccated over P2O5 to give cationic product 1a (790 mg, 94.3%) as
white solid glassy material. mp 49–50 ꢀC; IR (KBr):3276, 2931,
1663, 1604, 1542, 1508, 1412, 1218, 1008 cmꢁ1; 1H NMR (500 MHz,
145 ꢀC, [
a]
25 + 83.7 (c 1.0, DMSO); IR (KBr): 2995, 1744, 1610, 1592,
D
1493, 1376, 1300 cmꢁ1
;
1H NMR(500 MHz, CDCl3):
d
2.03 (s, 3H),
DMSO-d6): d 1.56–1.8 (m, 2H), 1.91–2.1 (m, 2H), 2.41 (t, 2H,
2.05 (s, 3H), 2.06 (s, 3H), 2.22 (s, 3H), 4.02 (m, 1H), 4.07 (dd, 1H, J
1 = 2.0 Hz, J2 = 2.5 Hz), 4.27 (dd, 1H, J1 = 5.5 Hz, J2 = 5.5 Hz), 5.39 (t,
1H, J = 10 Hz), 5.46 (dd, 1H, J1 = 2 Hz, J2 = 2 Hz), 5.54 (dd, 1H, J
1 = 3.5 Hz, J2 = 3.5 Hz), 5.63 (d, 1H, J = 2.0 Hz), 7.21 (d, 2H, J = 9.0 Hz),
J = 7.0 Hz), 2.73 (s, 3H), 2.74 (s, 3H), 3.05 (m, 2H), 3.17 (s, 2H), 3.39
(m, 1H), 3.47 (m, 2H), 3.59 (m, 1H), 3.66 (dd, 1H, J1 = 3 Hz, J2 = 3 Hz),
3.81 (m, 1H), 7.01 (d, 2H, J = 9 Hz), 7.52 (d, 2H, 9 Hz), 10.10 (s, 1H),
10.62 (br, 1H); 13C NMR (100 MHz, DMSO-d6): 19.84, 32.86, 41.9,
55.94, 60.99, 66.74, 70.06, 70.70, 74.81, 99.25, 106.95, 117.06,
120.42, 133.67, 138.91, 152.10, 169.58; HRMS (ESI) Calcd. for
C18H28N2O7 (M + H+) 385.1969, found: 385.1969.
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111
8.22 (d, 2H, J = 9 Hz); 13C NMR(100 MHz, CDCl3):
d 20.58, 20.74,
112
113
114
115
61.93, 65.60, 68.48, 68.91, 69.82, 95.09, 116.45, 125.78, 143,16,
160.11, 169.54, 169.83, 170.28; HRMS (ESI) Calcd. for C20H23NO12
(M + Na+) 492.1112, found: 492.1097.
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2.4. Synthesis of p-(tetradecanoylamido) phenyl-
a-D-
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2.2. Synthesis of 2,3,4,5-tetraacetyl-p-aminophenyl-
mannopyranoside (6a)
a-D-
mannopyranoside (2a)
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To a solution of compound 6a (1.0 g, 2.27 mmol) in anhydrous
dichloromethane (50 mL) were added myristic acid (796 mg,
3.48 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodimide hy-
drochloride (EDC) (660 mg, 3.44 mmol), and DMAP (30 mg), and
the reaction mixture was stirred overnight at room temperature.
The completion of reaction was checked by TLC (CHCl3: MeOH; 9:1,
v/v). The reaction mixture was diluted with dichloromethane
(50 mL) and washed with ice-cold 2% HCl, water, and brine, dried
over sodium sulfate, and concentrated under diminished pressure.
Purification of the crude product (1.67 g) by silica gel chromatog-
raphy (eluted with CHCl3 only) gave 8a (1.22 g, 85.8%) as off-white
foam like product which solidify on cooling. The crude product was
carried over to next step without further purification.
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The compound 2,3,4,5-tetraacetyl-p-nitrophenyl-a-D-manno-
pyranoside (5a) (2.64 g; 5.62 mmol) was dissolved in warm
methanol (150 mL) and ethyl acetate (75 mL). To this solution,
10% palladium on carbon (720 mg) in methanol (10 mL) was added
in small portions, followed by ammonium formate (7.5 g,
118.9 mmol), and the reaction mixture was stirred at room
temperature for 30 min. The progress of reaction was checked
by TLC (CHCl3:MeOH; 9.5:0.5, v/v) and filtered through celite bed
immediately after completion of reaction. The celite bed was
washed with a mixture of ethyl acetate (75 mL) and methanol
(25 mL). The filtrate and washing were combined and the solvents
were removed under reduced pressure. The residue was again
dissolved in ethyl acetate (200 mL) and washed with water and
brine, dried over sodium sulfate and concentrated under vacuum.
Compound 6a was dried in desiccator overnight to provide a faint
p-Aminophenyl-a-D-mannopyranoside myristate (2a) was
obtained by hydrolysis of compound 8a (1.1 g, 1.92 mmol) in
anhydrous methanol (30 mL), and using 0.5 M sodium methoxide
in anhydrous methanol (8 mL) at room temperature stirring for 1 h.
After completion of reaction, the solution was made neutral by
adding Amberlite1 IR-120H+ resin. The solution was diluted by
adding chloroform and the resin was filtered. The filtrate was
concentrated and the product was triturated with acetone
(2 ꢂ 25 mL) to remove traces of salt and impurities and to give
the desired product 2a (0.83 g, 90.2%) as a white powder. mp 158–
yellow solid. Yield 2.43 g (98.3%) mp 52–54 ꢀC, [
DMSO); IR (KBr): 3446, 3369, 3229, 2959, 1741, 1511, 1368, 1210,
1036 cmꢁ1; 1H NMR (500 MHz, CDCl3):
2.03 (s, 3H), 2.05 (s, 6H),
a]
25 + 64.7 (c 1.0,
D
d
2.18 (s, 3H), 4.09 (dd, 1H, J1 = 2.5 Hz, J2 = 2.0 Hz), 4.15 (m, 1H), 4.28
(dd, 1H, J1 = 5.5 Hz, J2 = 5.5 Hz), 5.36 (m, 4H), 5.42 (dd, 1H, J1 = 2 Hz,
J2 = 1.5 Hz), 5.54 (dd, 1H, J1 = 3.5 Hz, J2 = 3.0 Hz), 6.61 (d, 2H, J
1 = 8.5 Hz), 6.89 (d, 2H, J = 8.5 Hz); 13C NMR (100 MHz CDCl3): 20.4,
20.60, 61.82, 65.38, 68.46, 68.54, 68.63, 96.71, 114.51, 118.63,
144.64, 146.03, 169.51, 169.63, 169.88; HRMS (ESI) Calcd. for
C20H25NO10 (M + Na+) 462.1371, found: 462.1377.
59 ꢀC; [
a]
25 + 68.4 (c 1.0, DMSO); IR (KBr): 3324, 2916, 2848, 1655,
140
141
142
D
1535, 1511, 1220 cmꢁ1; 1H NMR (500 MHz, DMSO-d6):
d
0.85 (t, 3H,
J = 6.5 Hz), 1.23 (br, m, 20H), 1.56 (m, 2H), 2.25 (t, 2H, J = 7.5 Hz),
3.38–3.49 (m, 3H), 3.57–3.66 (m, 2H), 3.80 (br, 1H), 4.41 (t, 1H,
J = 6.0 Hz), 4.70 (br, OH), 4.78 (br, OH), 4.95 (br, OH), 5.26 (d, 1H,
J = 1.5 Hz), 7.00 (d, 2H. J = 9.0 Hz), 7.47 (d, 2H, J = 9.5 Hz); 9.72 (s,1H);
13C NMR (100 MHz, DMSO-d6): 113.93, 22.05, 25.16, 28.67, 28.77,
28.91, 28.98, 31.27, 36.27, 61.02, 66.72, 70.09, 70.67, 74.78, 99.30,
117.05, 120.27, 133.81, 151.99, 170.78; HRMS (ESI) Calcd. for
C26H43NO7 (M + Na+) 504.2932, found: 504.2915.
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2.3. Synthesis of p-(dimethylamino butylamido) phenyl-
mannopyranoside (1a)
a-
D-
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To a solution of 2,3,4,5-tetraacetyl-p-aminophenyl-
a
-D-man-
nopyranoside (6a) (2.02 g, 4.60 mmol) in anhydrous dichloro-
methane (100 mL) were added 4-dimethylaminobutyric acid (1.6 g,
9.54 mmol),
N,N0-dicyclohexylcarbodiimide
(DCC)
(2.5 g,
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12.1 mmol), and DMAP (66 mg), and the reaction mixture was
stirred at room temperature for 48 h. The mixture was filtered and
the filtrate was kept in refrigerator (2–8 ꢀC) for 4 h. The precipitated
by product, dicyclohexylurea (DCU) was filtered and the filtrate
was concentrated under reduced pressure. The residue was
purified by chromatography (silica gel, 0–10% methanol in
chloroform) to give compound 7a (2.41 g, 94.8%) as off white
solid. To a solution of compound 7a (1.1 g, 1.99 mmol) in anhydrous
methanol (30 mL) was added 0.5 M sodium methoxide in anhy-
drous methanol (8 mL) and stirred at room temperature for 1.5 h.
The completion of reaction checked by TLC (CHCl3:MeOH; 8:2, v/v).
The solution was neutralized by adding Amberlite1 IR-120H+ resin
and the suspension was filtered. The filtrate was concentrated to
small volume and acidified with ice-cold 2% HCl in methanol (pH
2.5. Synthesis of p-(30-cholesterylimido) phenyl-
mannopyranoside (3a)
a-D-
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To
a solution of compound 6a (250 mg, 0.569 mmol) in
anhydrous dichloromethane (5 mL) were added triethylamine
(Et3N) (0.25 mL) and cholesteryl chloroformate (289 mg,
0.643 mmol), and the reaction mixture was stirred overnight at
room temperature. The completion of reaction checked by TLC
(CHCl3:MeOH; 9.5:0.5, v/v). After completion, the reaction mixture
was diluted with CH2Cl2 (25 mL) and washed with ice cold 2% HCl,
water, and brine, dried over sodium sulfate, and concentrated
under diminished pressure. The residue on purification by silica gel
column chromatography gives 9a (290 mg, 60%) as a white solid. To
a solution of 9a (280 mg, 0.33 mmol) in a mixture of anhydrous
Please cite this article in press as: Ahmad, M.U., et al., Carbohydrate mediated drug delivery: Synthesis and characterization of new lipid-
conjugates. Chem. Phys. Lipids (2014), http://dx.doi.org/10.1016/j.chemphyslip.2014.10.003

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methanol (5 mL) and anhydrous tetrahydrofuran (8 mL) was added
0.5 M sodium methoxide in anhydrous methanol (1.1 mL), and the
reaction mixture was stirred at room temperature for 30 min. The
progress of reaction was checked by TLC (CHCl3:MeOH; 8:2, v/v).
After completion of reaction the solution was neutralized by
adding Amberlite1 IR-120H+ resin. The resin was filtered,
concentrated under vacuum. The product was dissolved in hot
mixture of acetone (20 mL)–methanol (5 mL) and filtered through
glass filter. The filtrate was stored at ꢁ20 ꢀC overnight. The
precipitated final product (3a) was filtered, washed with cold
mixture of acetone–methanol, and desiccated under high vacuum.
2.7. Synthesis of p-(dimethylamino butylamido) phenyl-
galactopyranoside (1b)
a
-
D
-
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To a solution of 2,3,4, 5-tetraacetyl-p-aminophenyl-
a
-D
-gal-
actopyranoside (6b) (1.0 g, 2.27 mmol) in anhydrous dichloro-
methane (50 mL) were added 4-dimethylaminobutyric acid
(0.808 g, 4.82 mmol), DCC (1.5 g, 7.27 mmol), and DMAP (30 mg),
and the reaction mixture was stirred overnight at room tempera-
ture. The progress of reaction was monitored by TLC (CHCl3:MeOH;
9:1, v/v). The reaction mixture was filtered and the filtrate was kept
in refrigerator (2–8 ꢀC) for 4 h. The precipitated DCU was filtered
and the filtrate was concentrated under diminished pressure. The
residue was purified by chromatography (silica gel, eluted with
0–10% MeOH in CHCl3) to give compound 7b (1.41 g,100%) as an off
white solid. To a solution of compound 7b (1.4 g, 2.53 mmol) in
anhydrous methanol (30 mL) was added 0.5 M sodium methoxide
in anhydrous methanol (8 mL) and stirred at room temperature for
1.5 h. The completion of reaction checked by TLC (CHCl3:MeOH;
8:2, v/v). The solution was neutralized by adding Amberlite1 IR-
120H+ resin (pH 6.0) and the suspension was filtered. The filtrate
was concentrated to small volume and acidified with ice-cold 2%
HCl in methanol (pH 4.0). The acidified product was concentrated
at reduced pressure and the residue was triturated with methanol
(2 ꢂ 20 mL) to remove any salt from the final product. The resulting
suspension was filtered through Whatman #54 filter paper,
concentrated under vacuum and dried in desiccator over P2O5 to
Yield 230 mg (100%). Mp 185–187 ꢀC; [
(KBr): 3369, 2935, 2867, 1731, 1703, 1513, 1216, 1054, 1013 cmꢁ1; 1H
NMR (500 MHz, DMSO-d6): 0.66 (s, 3H), 0.84 (d, 6H, J = 6.03 Hz),
a]
D
25 + 65.5(c 0.5 DMSO); IR
d
0.89 (d, 3H, J = 5.3 Hz), 0.99 (s, 3H),1.09–1.22 (m,10H),1.31–1.53 (m,
12H), 1.79–1.95 (m, 6H), 2.29–2.39 (m, 2H), 3.42–3.47 (m, 3H),
3.57–3.65 (m, 2H), 3.79 (s, 1H), 4.43 (s, br, 2H), 4.76–4.83 (m, br,
2H), 4.97 (s, 1H), 5.24 (s, 1H), 5.37 (s, 1H), 6.98 (d, 2H, J = 8.12 Hz),
7.34 (d, 2H, J = 7.43), 9.44 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
11.61, 18.51, 18.94, 20.62, 22.35, 22.58, 23.47, 23.87, 27.42, 27.81,
28.88, 31.39, 35.33, 35.78, 36.06, 36.64, 41.85, 49.53, 55.74, 56.16,
61.02, 66.74, 67.00, 70.15, 70.70, 73.32, 74.71, 99.41, 117.24, 119.38,
121.93, 133.65, 139.55, 151.66, 152.91; HRMS (ESI) Calcd. for
C40H61NO8 (M + Na+) 706.4289, found 706.4453.
give 1b (820 mg, 84.2%) as white solid mp 62–63 ꢀC; [
(c 1.0, DMSO); IR (KBr): 3255, 2933, 2700, 1647, 1543, 1508, 1412,
1216, 1076, 1026 cmꢁ1 1H NMR (400 MHz, DMSO-d6):
1.97 (m,
a]
25 + 103.0
249
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2.6. Synthesis of 2,3,4,5-tetraacetyl-p-aminophenyl-
galactopyranoside (6b)
a-D-
D
;
d
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To a solution of p-nitrophenyl galactopyranoside 4b (5.0 g,
16.6 mmol) in anhydrous pyridine (50 mL) was added acetic
anhydride (25 mL) and 4-dimethylaminopyridine (DMAP)
(50 mg), and the reaction mixture was stirred at room
temperature for 4 h. The progress of reaction was checked by
TLC (CHCl3:MeOH; 9:1, v/v). After completion of reaction, the
solvent was removed under vacuum. The residue was dissolved
in chloroform (200 mL) and washed with cold aq. HCl (2%),
water and brine, and dried over sodium sulfate, concentrated
under reduced pressure to afford the intermediate 2,3,4,5-
2H), 2.42 (t, 2H, J = 7.1 Hz), 2.73 (s, 6H), 3.05 (m, 2H), 3.17 (d, 4H,
J = 2.04), 3.38 (m, 3H), 3.55 (m, 1H), 3.75 (m, 2H), 5.31 (d, 1H,
J = 2.65), 7.01 (d, 2H, J = 7.58), 7.53 (d, 2H, J = 8.91), 10.16 (s, 1H); 13
C
NMR (100 MHz, DMSO-d6): 19.83, 32.84, 41.89, 55.93, 60.12, 68.07,
68.39, 69.44, 72.05, 98.64, 106.93, 117.25, 120.36, 133.50, 139.12,
153.01, 169.54, HRMS (ESI) Calcd. for C18H28N2O7 (M + H+)
385.1969, found: 385.1977.
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2.8. Synthesis of p-(tetradecanoylamido) phenyl-
galactopyranoside (2b)
a-D-
tetracetyl-p-nitrophenyl-
an off-white solid. The peracetylated nitro compound (7.98 g,
17.0 mmol) was dissolved in mixture of warm methanol
a-D-galactopyranoside (7.98 g. 100%) as
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A solution of compound 6b (1.04 g, 2.27 mmol) in anhydrous
dichloromethane (50 mL) was stirred with myristic acid (787 mg,
3.41 mmol), EDC (663 mg, 3.44 mmol), and DMAP (30 mg) over-
night at room temperature. The progress of reaction was
monitored by TLC (CHCl3:MeOH, 9:1, v/v). The reaction mixture
was diluted with dichloromethane (50 mL) and washed with ice
cold 2% HCl (20 mL), water and brine, dried over sodium sulfate,
and concentrated under reduced pressure. The resulting white
foamy solid was loaded on silica gel column and eluted with CHCl3
a
(200 mL) and ethyl acetate (150 mL). The solution was treated
with 10% palladium on carbon (2.2 g) in ethyl acetate (10 mL) in
small portions, followed by ammonium formate (23 g, 0.36 mol)
and the reaction mixture was stirred at room temperature for
30 min. The progress of reaction was checked by TLC (CHCl3:
MeOH; 9.5:0.5, v/v). The reaction mixture was filtered through
celite bed immediately after completion of reaction and the bed
was washed with
a mixture of ethyl acetate (75 mL) and
to yield 2,3,4,5-tetraacetyl-p-aminophenyl-a-D-galactopyranoside
methanol (25 mL). The filtrate and the washings were combined
and concentrated under vacuum. The residue was again
dissolved in ethyl acetate (300 mL) and washed with water
(3 ꢂ100 mL) and brine, dried over sodium sulfate and concen-
trated under vacuum. The compound 6b was desiccated
overnight and used in next step without purification (6.7 g;
myristate (8b) (1.36 g, 88.3%) as off white foam like product which
solidified on cooling. The crude product was carried over to next
step without further purification.
A solution of 0.5 M sodium methoxide in methanol (8 ml) was
added to a flask containing compound 8b (1.25 g, 1.92 mmol) in
anhydrous methanol (30 mL) at room temperature and the
solution was stirred for 30 min. The solution was made neutral
by adding Amberlite1 IR-120H+ resin (pH 6.0). The thin suspension
was diluted by adding ethanol (150 mL) and the resin was filtered.
The filtrate was concentrated and the product was triturated with
acetone (2 ꢂ 25 mL) to remove traces of salt and impurities and to
give the desired product 2b (0.9 g, 97.2%) as a white solid. mp
89.7%) as off white solid. Mp 131–132 ꢀC; [
CHCl3); IR (KBr): 3325, 2925, 2854, 1749, 1660, 1511, 1373, 1231,
1070 cmꢁ1 1H NMR (400 MHZ, CDCl3):
1,98 (s, 3H), 2.02 (s,
a]
25 + 176.4(c 1.0,
D
;
d
3H), 2.08 (s, 3H), 2.16 (s, 3H), 4.12 (m, 2H), 4.40 (t, 1H,
J = 6.85 Hz), 5.25 (dd, 1H, J1 = 3.79 Hz, J2 = 2 = 3.59 Hz), 5.53–5.56
(m, 2H), 5.59 (d, 1H, J = 3.83 Hz), 6.61 (d, 2H, J = 8.91 Hz), 6.86 (d,
2H, J = 8.63); 13C NMR (100 MHZ, CDCl3):
d 20.55, 20.59, 20.68,
198 ꢀC; [
1652, 1603, 1541, 1511, 1467, 1230, 1081 cmꢁ1
DMSO-d6): 0.85 (t, 3H, J = 6.5), 1.24 (br, m, 20H), 1.57 (m, 3H), 2.20
(m, 2H), 3.23–3.45 (m, 3H,), 3.74 (m,1H), 4.27 (br,1H), 4.57 (br, 3H),
a]
25 + 114.3 (c 1.0, DMSO); IR (KBr): 3291, 1918, 2850,
D
;
1H NMR (400 MHz,
61.55, 66.87, 67.55, 67.92, 67.98, 76.68, 76.99, 77.32, 99.99, 115.92,
118.33, 142.18, 149.16, 169.93, 170.12, 170.25, 170.30; HRMS (ESI)
Calcd. for C20H25NO10 (M + H+) 440.1551, found: 440.1520.
d
Please cite this article in press as: Ahmad, M.U., et al., Carbohydrate mediated drug delivery: Synthesis and characterization of new lipid-
conjugates. Chem. Phys. Lipids (2014), http://dx.doi.org/10.1016/j.chemphyslip.2014.10.003

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4.91 (br 1H), 5.30 (s, 1H), 7.01 (d, 2H, J = 8.67 Hz), 7.46 (d, 2H,
J = 8.81 Hz), 9.78 (s, 1H); 13C NMR (100 MHz, DMSO-d6): 13.92,
22.09, 25.19, 28.72, 28.85, 28.96, 29.05, 31.30, 36.30, 60.28, 68.07,
68.55, 69.46, 72.07, 98.76, 117.28, 120.28, 133.70, 152.96, 170.76,
HRMS (ESI) Calcd. for C26H43N2O7 (M + Na+) 504.2932, found:
504.2936.
3H, J = 6.04), 0.94 (s, 3H), 1.11–1.22 (m, 9H), 1.31–1.40 (m, 6H), 1.42–
1.53 (m, 6H), 1.78–1.95 (m, 6H), 2.29–2.40 (m, 2H), 3.17–3.43 (m,
1H), 3.46–3.57 (m, 1H), 3.62–3.78 (m, 5H), 4.40–4.45 (m, 1H), 5.27
(s, 1H), 5.37 (s, 1H), 6.98 (d, 2H, J = 8.78), 7.33 (d, 2H, J = 8.3 Hz), 9.42
(s, 1H); 13C NMR (100 MHz, DMSO-d6): 11.63, 18.51, 18.96, 20.60,
22.35, 22.61, 22.34, 23.86, 27.40, 27.80, 31.37, 35.28, 25.72, 36.07,
36.60, 38.17, 41.84, 49.51, 55.65, 56.14, 60.25, 68.05, 68.53, 69.43,
72.02, 73.33, 98.82, 117.22, 119.35, 122.00, 133.46, 139.55, 152.58,
152.96; HRMS (ESI) Calcd. for C40H61NO8 (M + Na+) 706.4289,
found: 706.4279.
2.9. Synthesis of p-(30-cholesterylimido) phenyl-
galactopyranoside (3b)
a-D-
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A solution of compound 6b (1.07 g, 2.28 mmol) in anhydrous
dichloromethane (5 mL) was treated with triethylamine (Et3N)
(1.0 mL) and cholesteryl chloroformate (1.20 g, 2.67 mmol), and the
reaction mixture was stirred overnight at room temperature. After
completion of reaction, (TLC; CHCl3:MeOH; 9.5:0.5, v/v), the
reaction mixture was diluted with dichloromethane (25 mL) and
washed with ice cold 2% HCl, water, and brine, dried over sodium
sulfate, and concentrated under diminished pressure. The residue
was purified by column chromatography (silica gel; eluted with
chloroform only) to yield 2,3,4,5-tetracetyl-p-aminophenyl-(cho-
398
2.10. Binding inhibition study
399
400
Concanavalin A (Con A), invertase, and
from Sigma–Aldrich. Solutions of compound 1a (2.5 mM, 1.25 mM,
0.625 mM), -MM (2.5 mM, 1.25 mM, 0.625 mM), Con A (20 M)
and invertase (2 M) were prepared in phosphate buffered saline
(PBS pH 7.4). Solutions of 1a and -MM, at various concentrations
a-MM were purchased
401
a
m
402
m
403
a
404
were mixed with equal volumes of Con A or PBS in triplicate in
microtiter plate. Equal volumes of invertase were added to each
well resulting in the final concentration of 1a and
0.833 mM, 0.416 mM, and 0.208 mM. The final concentration of
Con A, and invertase in test wells were 6.6 mM and 0.66 mM,
respectively. An increase in turbidity at 320 nm (OD320) was
monitored at room temperature using SpectraMax M2e (Molecular
Devices, CA, USA). Binding of Con A to invertase was used as control
(100%) aggregation and PBS was used as blank.
405
406
lesteryl)-
a
-D-galactopyranoside (9b) (1.2 g, 58%) as a reaction
a-MM
407
intermediate.
Q6 408
409
Following the procedure used for 3a, a solution of 0.5 M sodium
methoxide in methanol (6 mL) was added to flask containing
solution of 2,3,4,5-tetracetyl-p-aminophenyl-a-D-galactopyrano-
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411
side (9b) (1.1 g, 1.29 mmol) in a mixture of anhydrous methanol
(10 mL) and anhydrous tetrahydrofuran (30 mL). After 30 min
stirring at room temperature, progress of reaction was checked by
TLC (CHCl3:MeOH; 8:2, v/v). After completion of reaction the
solution was neutralized by adding Amberlite1 IR-120H+ resin. The
product starts to precipitate which was dissolved by adding CHCl3.
The resin was filtered, concentrated under vacuum. The product
was dissolved in hot mixture of acetone (20 mL)–methanol (5 mL)
and filtered through glass filter. The filtrate was stored at ꢁ20 ꢀC
overnight. The precipitated product (3b) was filtered, washed with
cold mixture of acetone–methanol, and desiccated under high
vacuum. Yield 910 mg (100%). Mp 152–154 ꢀC; IR (KBr): 3392, 2936,
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413
2.11. In vitro transfection
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A549 cells (human lung cancer) were obtained from the
National Cancer Institute (Frederick, MD) and maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% heat-inactivated fetal bovine serum (FBS). DMEM, FBS and
other cell culture reagents were purchased from Life Technologies
(Carlsbad, CA).
Log-phase A549 cells were seeded in 96-well plates
(20,000 cells/well) and cultured overnight in a standard 5% CO2
incubator. The culture medium was removed and cells were
2868, 1727, 1704, 1605, 1514, 1467, 1216, 1057 cmꢁ1
;
1H NMR
(500 MHz, DMSO-d6): 0.66 (s, 3H), 0.84 (d, 6H, J = 5.72), 0.89 (d,
d
Fig. 2. Reagents: (a) Ac2O, pyridine, rt (b) amm. formate, Pd-C, MeOH, EtOAc (c) (CH3)2N(CH2)3CO2H, DCC, DMAP, CH2Cl2 (d) NaOMe/MeOH.
Please cite this article in press as: Ahmad, M.U., et al., Carbohydrate mediated drug delivery: Synthesis and characterization of new lipid-
conjugates. Chem. Phys. Lipids (2014), http://dx.doi.org/10.1016/j.chemphyslip.2014.10.003

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M.U. Ahmad et al. / Chemistry and Physics of Lipids xxx (2014) xxx–xxx
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washed with sterile phosphate-buffered saline (PBS) before adding
the transfection mixture. The transfection mixture was prepared
by adding reporter gene, pSV-b-gal control vector (Promega,
Madison, WI) into equal volume of different concentrations of (1a)
to yield a series of mixtures with (ꢃ) charge ratios of 2:1; 4:1; 8:1;
16:1. The mixture was added to and incubated with the cells for 6 h
before replaced with regular culture medium. The cells were
cultured for a total of 24 h. The total amount of DNA plasmid was
derivatives 6a and b were obtained in average 90% isolated yield
and used as starting material for the preparation of different
compounds of interest (Fig. 1). Compounds (6a) (Fig. 2) and (6b)
(Fig. 3) upon treatment with 4-dimethylaminobutyric acid in the
presence of N,N0-dicyclohexylcarbodiimide (DCC) and DMAP in
methylene chloride provided intermediates (7a and b) which on
subsequent deacetylation with sodium methoxide in methanol
and neutralization over ion exchange Amberlite1 IR 120H+ resin
0.2
diluted with OptiMEM I medium (Life Technologies (Carlsbad, CA).
The transfection efficiency was determined using -galactosidase
m
g/well in 96-well plates. Both DNA plasmid and 1a were
afforded p-aminophenyl-(dimethylamino butyrate)-
pyranoside (1a) and p-aminophenyl-(dimethylamino butyrate)-
a-D-galactopyranoside (1b) respectively. Structures of all the
a-D-manno-
b
enzyme assay system (Promega, Madison, WI) according to the
manufacturer’s protocol.
synthetic intermediates and final products (1a and b) were
confirmed by NMR and HRMS.
This the first report on neutral glycolipids as excipients and
their role in targeting drugs to specific body cells. We have
436
3. Results and discussion
designed and synthesized p-(tetradecanoylamido) phenyl-
a
-
D
-
-
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448
449
450
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Towards probing an hitherto unexplored structure–activity of
cationic sugar derivatives, we have designed and synthesized two
novel cationic sugar derivatives (1a) (Fig. 2) and (1b) (Fig. 3)
containing spacer between sugar heads and the positively charged
nitrogen atoms. The common intermediates 2,3,4,5-tetra-O-
mannopyranoside (2a) and p-(tetradecanoylamido) phenyl-
a
-D
galactopyranoside (2b), and their analogs. The solution of 2,3,4,5-
tetraacetyl-p-aminophenyl- -mannopyranoside (6a) in anhy-
a-D
drous methylene chloride was added myristic acid, 1-ethyl-3-(3-
dimethyl aminopropyl) carbodimide hydrochloride (EDC), and
DMAP at room temperature, and stirring overnight afforded
intermediate 8a (Fig. 4). The intermediate 8a on deacetylation
with sodium methoxide in methanol and acetate ion exchange
over Amberlite1 IR-120H+ resin afforded the title glycolipid (2a).
Furthermore, it is possible to synthesize analogs of 2a by adding
fatty acids of different chain length with or without unsaturation.
The synthetic glycolipid 2b, was synthesized from intermediate,
acetyl-p-aminophenyl-
2,3,4,5-tetra-O-acetyl-p-aminophenyl-
(6b) (Fig. 3) were prepared conventionally in two steps. Briefly,
peracetylation of p-nitrophenyl- -mannopyranoside (4a)
Fig. 2) or p-nitrophenyl- -galactopyranoside (4b) (Fig. 3), with
a
-D
-mannopyranoside (6a) (Fig. 2) or
a-D-galactopyranoside
a
-D
(
a-D
acetic anhydride in the presence of catalytic amount of 4-
dimethylaminopyridine (DMAP) and anhydrous pyridine, facilitate
O-acetylation. This furnished mainly per acetylated nitro product
5a (Fig. 2) in quantitative yield. A simpler and one-step conversion
2,3,4,5-tetraacetyl-p-aminophenyl-
a
-D-galactopyranoside
(6b)
(Fig. 4), similar to the procedure described above. For comparative
study, we have synthesized both type of glycolipid 2a and b
carrying homogenous fatty acids (C14:0), and established their
chemical structures by IR, NMR and HRMS. Lipid based delivery
system have been widely used to improve the delivery of many
anticancer, antibiotic, and antifungal drugs (Sparano et al., 2001;
Bellott et al., 2001; Mayer et al., 1993; Vaage et al., 1992; Barjtburg
to 2,3,4,5-tetra-O-acetyl-p-aminophenyl-a-D-mannopyranoside
(6a) from the corresponding p-nitro derivative 5a was accom-
plished by transfer hydrogenation using ammonium formate and
catalytic amount of Pd/C at room temperature. The structure of the
amino derivative 6a (Fig. 2) and 6b (Fig. 3) were supported by the
upfield shift of aromatic protons in the NMR spectra. The amino
Fig. 3. Reagents: (a) Ac2O, pyridine, rt (b) amm. formate, Pd-C, MeOH, EtOAc (c) (CH3)2N(CH2)3CO2H, DCC, DMAP, CH2Cl2 (d) NaOMe/MeOH.
Please cite this article in press as: Ahmad, M.U., et al., Carbohydrate mediated drug delivery: Synthesis and characterization of new lipid-
conjugates. Chem. Phys. Lipids (2014), http://dx.doi.org/10.1016/j.chemphyslip.2014.10.003

G Model
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M.U. Ahmad et al. / Chemistry and Physics of Lipids xxx (2014) xxx–xxx
7
Fig. 4. Reagents: (a) myristic acid, EDC, DMAP, CH2Cl2 (b) NaOMe/MeOH.
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and Bolard, 1996; Kawakami et al., 2000a,b; Sheikh et al., 2010; Ali
et al., 2010). However, the aqueous dispersion of liposome and its
treatment with cholesteryl chloroformate in the presence of
triethylamine was converted into the respective glycolipid–
cholesteryl intermediate (9a), which on deacetylation with sodium
methoxide in methanol and neutralization over ion exchange
Amberlite IR 120H+ resin afforded p-(30cholesterylimido) phenyl-
stability is
a major issue for various types of therapeutic
applications (Lasic and Papahadjopolos, 1995). Addition of steroi-
dal compounds in liposome preparations such as cholesterol and
cholesteryl sulfate is known to enhance the stability of liposomes
but phospholipids–vesicle becomes more rigid and sustains severe
stress after adding cholesterol (Liu et al., 2000). Therefore there is a
need to synthesize new molecules of pharmaceutical interest to
develop stable nanoparticles and effective drug delivery system
(Ahmad et al., 2010). In order to overcome the stability issue, we
a
-
D
-mannopyranoside (3a). The other novel glycolipid–cholesteryl
conjugate (3b), was synthesized from intermediate, 2,3,4,5-
tetraacetyl-p-aminophenyl- -galactopyranoside (6b) similar to
a-D
the procedure described above. The structures of both 3a and b
were supported by their IR, NMR and HRMS spectra. These novel
conjugates are appealing for further development to explore
potential uses in the liposomal drug delivery.
synthesized cholesterol-conjugates of p-aminophenyl-
a
-D-man-
nopyranoside (3a) and p-aminophenyl-
a-D
-galactopyranoside
A binding inhibition study of p-(dimethylamino butylamido)
(3b) (Fig. 5).
phenyl-
noside (
Binding with terminal mannose residues of invertase to Con A
a
-D-mannopyranoside (1a) and methyl-D-a-mannopyra-
The common intermediate 2,3,4,5-tetra-O-acetyl-p-amino-
phenyl- -mannopyranoside (6a) in methylene chloride upon
a-MM) with Con A was performed using invertase enzyme.
a-D
Fig. 5. Reagents: (a) cholesterol chloroformate, Et3N, CH2Cl2 (b) NaOMe/MeOH.
Please cite this article in press as: Ahmad, M.U., et al., Carbohydrate mediated drug delivery: Synthesis and characterization of new lipid-
conjugates. Chem. Phys. Lipids (2014), http://dx.doi.org/10.1016/j.chemphyslip.2014.10.003

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M.U. Ahmad et al. / Chemistry and Physics of Lipids xxx (2014) xxx–xxx
Fig. 8. Transfection of pSV-b-gal control vector gene with compound 1a in
A549 cells. Cells were seeded in 96-well plate 24 h before transfection. Cells were
incubated with DNA-compound 1a mixture of different charge ratios for 6 h. The
total amount of reporter gene was 0.2 mg/well.
Fig. 6. Inhibition of invertase and Con A aggregation by 1a and
a-MM after
incubation at 24.5 ꢀC for 7 min. The inhibition was monitored at OD 320 nm.
544
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transfection agents for decades and achieved great success for
in-vitro transfection of gene, antisense and siRNA. However, off-
target effect and immune stimulation limit the systemic applica-
tion of these agents that are otherwise highly efficient transfection
agents. Compound 1a is a small, cationic mannose-derivative. The
coexistence of both mannose group and a net positive charge may
render the molecule with improved transfection efficiency in cells
expressing mannose binding proteins/receptors. To this end, the
525
526
527
528
529
530
531
532
533
534
535
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540
541
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543
causes aggregation of the complexes in solution, resulting in
increase in turbidity at 320 nm (OD320 nm). Fig. 6 shows the percent
inhibition of aggregation at 320 nm when 2
added to a solution of Con A containing different amount of
or compound 1a. Addition of compound 1a and -MM resulted in
mM of invertase was
a
-MM
a
the inhibition of precipitation reaction between Con A and
invertase. The maximum inhibition was obtained at 0.833 mM
concentration (Fig. 6).
transfection of a reporter gene, pSV-
binding protein/receptor expressing A549 cells. The pSV-
b
-gal, was tested in mannose
-gal
b
Fig. 7 shows the changes in absorbance (i.e., turbidity) at
reporter gene when mixed with compound 1a was transfected in
A549 cells (Fig. 8). The greatest transfection efficiency was found at
a (ꢃ) charge ratio of 8:1.
320 nm when 2
(20 M) containing different amounts of
was interesting to observe that the rate of the precipitation
reaction decrease with increasing concentration of -MM. The
m
M invertase was added to a solution of Con A
m
a-MM or compound 1a. It
a
557
4. Conclusion
relative magnitude of inhibition of precipitation reaction was
higher with the synthesized compound 1a. Evidently this resulted
from a decrease in the available concentration of con A for the
reaction with polymannan of invertase.
The transfection efficiency of 1a being a cationic compound was
investigated. Cationic lipids have been used as non-viral
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565
566
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568
We report here synthesis of a series of glycolipid conjugates for
applications in liposomal drug delivery system and for targeting
drugs to receptors. We have designed and synthesized two novel
cationic glycolipids with p-aminophenyl-a-D-mannopyranoside
(PAPM) and p-aminophenyl- -galactopyranoside (PAPG) with
a-D
three methylene units’ spacer in between the quaternary nitrogen
atom and the sugar unit. This study reveals a different chemistry to
conjugate lipids such as fatty acids or cholesterol, in view of
targeting to specific sites. The binding inhibition study of
compound 1a suggests that conjugates of PAPM can be potentially
used to target mannose receptors.
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Conflict of interest
The authors declare that there are no conflicts of interest.
Transparency document
572
573
The Transparency document associated with this article can be
found in the online version.
574
Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.chemphy-
slip.2014.10.003.
Fig. 7. The kinetics of precipitation reaction between invertase and Con A in the
presence of varying amount of
a
-MM or compound 1a. (A)
a
-MM (0.833 mM), Con
578
References
A and invertase (B) -MM (0.416 mM), Con A and invertase (C)
a
a
-MM (0.208 mM),
Con A and invertase (D) compound 1a (0.833 mM), con A and invertase (E)
compound 1a (0.416 mM), Con A and invertase (F) compound 1a (0.208 mM), Con A
and invertase. The final concentration of Con A and Invertase in each test sample
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