Design, synthesis and biological evaluation of carbohydrate-functionalized cyclodextrins and liposomes for hepatocyte-specific targeting

Article abstract of DOI:10.1039/c0ob00372g

Targeting glycan-binding receptors is an attractive strategy for cell-specific drug and gene delivery. The C-type lectin asialoglycoprotein receptor (ASGPR) is particularly suitable for liver-specific delivery due to its exclusive expression by parenchymal hepatocytes. In this study, we designed and developed an efficient synthesis of carbohydrate-functionalized β-cyclodextrins (βCDs) and liposomes for hepatocyte-specific delivery. For targeting of ASGPR, rhodamine B-loaded βCDs were functionalized with glycodendrimers. Liposomes were equipped with synthetic glycolipids containing a terminal d-GalNAc residue to mediate binding to ASGPR. Uptake studies in the human hepatocellular carcinoma cell line HepG2 demonstrated that βCDs and liposomes displaying terminal d-Gal/d-GalNAc residues were preferentially endocytosed. In contrast, uptake of βCDs and liposomes with terminal d-Man or D-GlcNAc residues was markedly reduced. The d-Gal/d-GalNAc-functionalized βCDs and liposomes presented here enable hepatocyte-specific targeting. Gal-functionalized βCDs are efficient molecular carriers to deliver doxorubicin in vitro into hepatocytes and induce apoptosis.

Full text of DOI:10.1039/c0ob00372g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Design, synthesis and biological evaluation of carbohydrate-functionalized
cyclodextrins and liposomes for hepatocyte-specific targeting†
Gonc¸alo J. L. Bernardes,‡a,b Raghavendra Kikkeri,‡a,b Maha Maglinao,a,b Paola Laurino,a,b Mayeul Collot,a,b
Sung You Hong,a,b Bernd Lepeniesa,b and Peter H. Seeberger*a,b
Received 2nd July 2010, Accepted 22nd July 2010
DOI: 10.1039/c0ob00372g
Targeting glycan-binding receptors is an attractive strategy for cell-specific drug and gene delivery. The
C-type lectin asialoglycoprotein receptor (ASGPR) is particularly suitable for liver-specific delivery due
to its exclusive expression by parenchymal hepatocytes. In this study, we designed and developed an
efficient synthesis of carbohydrate-functionalized b-cyclodextrins (bCDs) and liposomes for
hepatocyte-specific delivery. For targeting of ASGPR, rhodamine B-loaded bCDs were functionalized
with glycodendrimers. Liposomes were equipped with synthetic glycolipids containing a terminal
D-GalNAc residue to mediate binding to ASGPR. Uptake studies in the human hepatocellular
carcinoma cell line HepG2 demonstrated that bCDs and liposomes displaying terminal
D-Gal/D-GalNAc residues were preferentially endocytosed. In contrast, uptake of bCDs and liposomes
with terminal D-Man or D-GlcNAc residues was markedly reduced. The D-Gal/D-GalNAc-
functionalized bCDs and liposomes presented here enable hepatocyte-specific targeting.
Gal-functionalized bCDs are efficient molecular carriers to deliver doxorubicin in vitro into hepatocytes
and induce apoptosis.
(galactosyl-terminated) glycoproteins.18 Human ASGPR consists
Introduction
of two subunits, H1 and H2. The H1 subunit mediates Ca2+-
Carbohydrate-based therapies hold great promise and glycan-
dependent D-Gal/D-GalNAc recognition whereas the H2 subunit
is responsible for functional configuration.19 Recent studies de-
scribe the use of natural ASGPR ligands or synthetic ligands with
galactosylated cholesterol, glycolipids, or polymers.20–22 Specific
targeting of hepatocytes has enabled gene delivery,23 for example
using galactosylated DNA lipid nanocapsules as the vector.24
Binding of D-Gal and D-GalNAc to ASGPR strongly depends
on the valency of the ligands with a binding hierarchy increasing
from monoantennary to tetraantennary ligands.25 It is also
known that ASGPR exhibits a 10–50 fold higher affinity to
D-GalNAc compared to D-Gal residues.26 Furthermore, the dis-
tance between the terminal D-Gal/D-GalNAc residues is impor-
tant for optimal spatial orientation resulting in maximal binding
to the heterooligomeric ASGPR.27
We previously reported the use of carbohydrate-capped
quantum dots (QDs) for selective targeting of ASGPR on
hepatocytes in vitro and in vivo.28 Intravenous injection of
D-Gal/D-galactosamine-functionalized QDs into mice led to
specific sequestration in the liver indicating that specific targeting
of hepatocytes by multivalent Gal-terminated ligands is possible.
While QDs represent an excellent tool for imaging purposes due
to high quantum yield and non-bleachable fluorescence, they are
not suitable for targeted drug and gene delivery due to their
inherent toxicity. Herein, we report the design and synthesis of
b-cyclodextrins (bCDs) functionalized with Gal dendrimers and
liposomes that displayed D-GalNAc-terminated lipids to target
ASGPR by exploiting multivalent D-Gal/D-GalNAc interactions.
Both bCDs and glycoliposomes were equipped with a fluores-
cent dye to enable detection of hepatocyte binding and uptake
in vitro. Our results indicate that the constructed bCDs and
glycoliposomes displaying multiple D-Gal/D-GalNAc residues for
binding receptors have been targeted for cell-specific drug and gene
delivery.1–3 Specific binding of isolated and synthetic carbohydrates
to endogeneous lectins on cell surfaces resulted in cell-specific
binding and uptake.4 However, due to the relatively low affinity of
proteins to their carbohydrate ligands multivalent interactions are
often required to exert biological effects. Recent reports showed
that lectins can be targeted by either multivalent or chemically-
modified ligands.5–7 Different strategies have been employed
to achieve multivalency such as the use of glyconanoparticles
functionalized with sialyl-Lewisx for selectin targeting,8 polymers
displaying multiple sugar residues9 or cross-linked carbohydrate
antigens10 for siglec CD22 targeting, and glycodendrimers for
high-affinity binding to the C-type lectin DC-SIGN.11 Glycoden-
drimers represent an excellent tool to exploit multivalency for
targeting purposes,12,13 but degradable dendrimers have also been
used in the field of controlled drug delivery systems.14–16
The asialoglycoprotein receptor (ASGPR) is a C-type lectin that
is a particularly attractive target for liver-specific drug delivery
since it is expressed exclusively on parenchymal hepatocytes.17
The main function of ASGPR is to maintain homeostasis of
serum glycoprotein levels by binding and uptake of desialylated
aMax Planck Institute of Colloids and Interfaces, Research Campus-Golm,
14476 Potsdam, Germany
bFree University Berlin, Department of Biology, Chemistry and Pharmacy,
Institute of Chemistry and Biochemistry, Arnimallee 22, 14195 Berlin,
Germany. E-mail: Peter.Seeberger@mpikg.mpg.de; Fax: +49 (0) 30-838-
59302; Tel: +49 (0) 30-838-59301
† Electronic supplementary information (ESI) available: Experimental
details, photophysical properties of dendrimers and NMR data for all
new compounds. See DOI: 10.1039/c0ob00372g
‡ These authors contributed equally to this work.
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Fig. 1 Schematic representation of the delivery systems used in this study. A, bCD glycodendrimer consisting of bCD (cylinder), the encaged fluorescent
dye rhodamine B (red ball), and terminal D-Man 1 or D-Gal 2 residues. B, Glycoliposomes consisting of L-a-phosphatidylcholine (blue), the fluorescent
lipid rhodamine-DHPE (red ball), and synthetic glycolipids containing either a terminal GalNAc or GlcNAc residue (green arrow).
multivalent binding to ASGPR are suitable hepatocyte-specific
targeting systems.
endocytosis into HepG2 cells or D-Man as control (Fig. 1A). For
the liposome-based delivery system, a standard method for prepa-
ration of liposomes was employed.30 Liposomes were composed of
L-a-phosphatidylcholine and the integration of the fluorescent
lipid rhodamine-DHPE into the liposome membrane made the
detection of liposome uptake into HepG2 cells possible. For
hepatocyte-specific delivery glycolipids displaying a terminal
D-GalNAc residue (for targeting ASGPR) or D-GlcNAc (speci-
ficity control) were synthesized (Fig. 1B).
Results and discussion
The delivery systems evaluated in this study are shown in Fig. 1. We
used (1) a b-cyclodextrin (bCD)- and (2) liposome-based systems
where drugs or siRNAs may be encaged, and (2) a liposome-
based system. For detection of bCD uptake into HepG2 cells,
a bCD inclusion complex equipped with the fluorescent dye
rhodamine B (RhB) was assembled. Previous studies indicate that
the formation of a 1 : 1 inclusion complex of RhD with bCD
may be suitable for delivery strategies such as the one proposed
here.29 Additionally, bCDs were equipped with a glycodendrimer
displaying either D-Gal residues to induce ASGPR-mediated
Design and synthesis of targeting systems
Glycodendrimer-functionalized bCDs were prepared using Cu(I)-
catalyzed [2+3] cycloaddition between tripodal sugar azides and
a bCD bearing an alkyne reactive moiety (Scheme 1A).31,32
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Scheme 1 Schematic representation of bCD glycodendrimer and glycolipid synthesis. A, Synthesis of rhodamine B-labeled bCD 3 and its
functionalization with glycodendrimers. Reagents and conditions: (a) (i) acrylonitrile/NaOH (40%); (ii) conc. HCl/EtOH, 51%; (iii) 5-bromovaleric
acid, DIC, HOBT, DCM, 69%; (b) (i) NaN3, DMF, 72%; (ii) NaOH/MeOH; (iii) pentafluorophenol, DIC, HOBT, DCM, 86% (2 steps); (c) tripod-Man
or tripod-Gal, DCM, TEA, 52–59%; (d) compound 8 or 9, CuSO4, ascorbic acid, THF–H2O (1 : 1), 82–84%; (e) NaOMe/MeOH 77–78%; (f) compound
11 or 12 or bCD, rhodamine B, H2O. B, Synthesis of glycolipids 20 and 25. Reagents and conditions: (I) (a) POCl3, hexane, Et3N, 2 h, RT; then
acetone : H2O : Et3N (85 : 10 : 5), overnight, 80% (2 steps). (II) (a) Dibenzylphosphate, 1,2-dichloroethane, 41–69%; (b) H2, Pd/C, MeOH, TEA, 88–100%;
(c) 1,1¢-carbonyldiimidazole (CDI), 15, DMF–MeOH, 72 h, 40–66%; (d) NaOMe, DCM–MeOH, 30 min, 100%.
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Tripodal sugar-azides were prepared by coupling tris(hydroxyl-
methyl)aminomethane with acrylonitrile, followed by treatment
with HCl in ethanol and amide bond formation with 5-
bromovaleric acid yielded 69% of 5. Bromide substitution with
azide, saponification and further esterification with pentafluo-
rophenol afforded 7 in 86% yield. The pentafluorophenol ester 7
was further reacted with tripod-Man or tripod-Gal to obtain azide
dendrons 8 or 9 (Scheme 1, ESI†). Dendrons 8, 9 were coupled
with stoichiometric amounts of alkynyl substituted bCD 10 by
Cu(I)-catalyzed [2+3] cycloaddition and deacetylated to yield 77–
78% of dendrimer 13 or 14 respectively (Scheme 2, ESI†). Finally,
functionalized bCD-inclusion complexes 1–3 of dendrimers 13,
14 and bCD with rhodamine B (RhB) were prepared (Scheme 3,
ESI†) and used for uptake studies in HepG2 cells. Inclusion
complexation of RhB and dendrimer (13, 14 and bCD) was verified
by 1H NOESY experiments (see Fig. 22–24 in the ESI†). An
equimolar mixture of dendrimer 13 and RhB displayed a clear
nuclear Overhauser effect (NOE) correction between the –CH
proton of bCD and methyl protons of the diethylamino groups of
RhB and the aromatic protein of the diethylaminophenyl group of
RhB. Similar results were also observed with 2 and 3. Inclusion of
RhB and 13 showed a slight increase in quantum yield, indicating
the encapsulation of the fluorescent probe by the dendrimer.
Glycolipids 20 and 25 were chosen for incorporation into
liposomes and the synthetic approach selected for their syn-
thesis is shown in Fig. 2B. To increase targeting specificity,
glycolipids were equipped with terminal D-GalNAc residues since
ASGPR displays a preference for D-GalNAc over D-Gal.26 Sugar
oxazolines 16 and 21 were synthesized according to literature
procedures33,34 and were converted to the corresponding a-
glycosyl dibenzylphosphate derivatives 17 and 22 in moderate
yield (17: 41%, 22: 69%). Hydrogenolytic deprotection produced
the corresponding a-glycosyl phosphates 18 and 23 in excellent
yields. Lipid monophosphate 15, that was generated by treating
citronellol with phosphorus oxychloride was activated using 1,1¢-
carbonyldiimidazole (CDI) and subsequently coupled to the
a-glycosyl phosphates 18 and 23 to produce 19 and 24 in moderate
yields (19: 40%, 24: 66%).35 Finally, removal of the acetate
groups by treatment with sodium methoxide in a mixture of
DCM–methanol36 yielded the desired deprotected a-diphosphate
glycolipids 20 and 25. We chose citronellol, a short, saturated
isoprenol lipid, due to difficulties in handling long lipid chains
and instability of allylic pyrophosphates. Glycoliposomes were
prepared by dissolving L-a-phosphatidylcholine, the fluorescent
lipid rhodamine-DHPE and glycolipid 20 (terminal D-GlcNAc,
control) or glycolipid 25 (terminal D-GalNAc) in ethanol, evap-
orating the ethanol and dissolving the lipid film in PBS buffer.
To ensure that the liposomes were comparable in shape and
size, liposomes were extruded through a 100 nm polycarbonate
membrane. TEM measurements of the liposome preparations were
performed after staining them with an aqueous solution of uranyl
acetate and indicated that the liposomes had a size of about
100 nm or less, spherical shape and exhibited a high degree of
monodispersity (Fig. 2B).
Hepatocyte-specificity studies
To analyze ASGPR-mediated uptake of bCD functionalized
with glycodendrimers, the hepatocellular carcinoma cell line
HepG2 was incubated with 1, 2 and 3. The HepG2 cell line
has been previously employed to investigate ASGPR-mediated
uptake of galactosylated polymers and liposomes.37–39 Uptake
by HepG2 cells was analyzed by flow cytometry to enable a
quantitative analysis of targeting specificity. As expected, the
incubation of HepG2 cells with non-functionalized rhodamine
B-labeled bCD did not induce uptake (Fig. 2A). bCD displaying
a Man dendron was used as a specificity control since ASGPR
exhibits specificity for galactosyl-terminated structures. Indeed,
only marginal unspecific uptake of Man-functionalized bCD
was observed (Fig. 2A). In contrast, functionalization of bCD
with Gal dendrons dramatically increased the uptake by HepG2
cells indicating receptor-mediated endocytosis. Recently, we have
targeted hepatocytes in vitro and in vivo through ASGPR-mediated
uptake of D-Gal-capped quantum dots.28 Knock-down of ASGPR
by siRNA led to a reduction in D-Gal QDs uptake indicating
specificity of the hepatocyte targeting. While the use of QDs as
drug/gene delivery system is limited, bCD have been synthesized
and successfully used as carriers.40–44
Glycoliposomes were assembled from L-a-phosphatidylcholine,
the respective glycolipid 20 or 25 and rhodamine-DHPE.
D-GlcNAc-functionalized liposomes were used as specificity con-
trol. In contrast to the bCD glycodendrimers where almost no
unspecific uptake was observed, background fusion of control
liposomes with HepG2 cells was detected to a limited extent
(Fig. 2C). However, preferential uptake of D-GalNAc-capped
liposomes was observed again indicating specificity of D-GalNAc-
mediated hepatocyte targeting. Liposome-based delivery systems
Fig. 2 Targeting of the hepatocellular carcinoma cell line HepG2 with
bCD glycodendrimers and glycoliposomes. A, HepG2 cells were left
untreated (grey area), incubated with 250 mmol rhodamine B-labeled bCD
(dotted line, negative control) or labeled bCD glycodendrimers containing
terminal D-Man (green line, specificity control) or D-Gal residues (red
line). Uptake was measured by flow cytometry. Specific uptake of bCD
displaying a D-Gal dendrimer by HepG2 cells was observed. B, Typical
TEM images of glycoliposomes used in this study. C, HepG2 cells
were left untreated (gray area), incubated with rhodamine-DHPE-labeled
liposomes (dotted line, negative control) or labeled liposomes containing
glycolipids with terminal D-GlcNAc (green line, specificity control) or
D-GalNAc residues (red line). Uptake was measured by flow cytometry.
Preferential uptake of liposomes functionalized with the D-GalNAc
glycolipid was observed.
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are widely used since liposomes can be loaded with drugs or
siRNAs and thus may be used as efficient carrier systems.
Consistently, recent studies have focused on liposomal delivery
systems for targeting ASGPR.20,21
In vitro evaluation of doxorubicin-loaded bCD-Gal 26
bCD functionalized systems have been widely explored as molec-
ular vectors for gene45–47 and drug delivery.48 To evaluate the
ability of our Gal functionalized bCD systems to deliver phar-
macologically active species into hepatocytes, bCD-Gal 14 was
loaded with doxorubicin, a potent chemotherapy drug used in
cancer treatment.49 This system was chosen as a model because
the physico-chemical properties of inclusion complexes of anthra-
cycline antibiotics and bCDs are well described.50 An equimolar
inclusion complex of bCD-Gal 14 and doxorubicin hydrochloride
was prepared over 20 min with sonication and using water as
the solvent yielding doxorubicin-loaded bCD-Gal 26 (Scheme 2).
Cross peaks between H-3 and H-5 of bCD (~3.75 ppm) and
protons in the aromatic region of doxorubicin (~7.60 ppm) were
detected by nuclear overhauser effect spectroscopy (NOESY),
indicating inclusion of doxorubicin into bCD-Gal 14 (Fig. S31,
ESI†).51 The potential of this system to induce apoptosis in HepG2
cells was evaluated. Cells were left untreated or were treated
with doxorubicin only, bCD-Gal 14 or doxorubicin-loaded bCD-
Gal 26 for 24 h. The highest degree of apoptosis was observed
in HepG2 cells incubated with doxorubicin-loaded bCD-Gal 26
whereas bCD-Gal 14 alone induced no apoptosis (Fig. 3). This
result encourages the use of Gal functionalized bCDs as a drug-
delivery system.
Scheme 2 Synthesis of doxorubicin-loaded bCD-Gal 26.
gene delivery and Gal functionalized bCD-based systems for drug-
delivery is currently under investigation in our laboratory.
Conclusions
In summary, we have developed a practical synthesis of delivery
systems based on synthetic carbohydrates for multivalent receptor
interactions that enable targeting of ASGPR expressed by hepa-
tocytes. The Gal functionalized bCD-based system is a molecular
carrier for doxorubicin inducing a high degree of apoptosis
in vitro. The use of liposomal-based systems in hepatocyte-specific
Experimental
Synthesis of dendrons 8 and 9 as well as bCD 10
Compound 4, tripod-Man, tripod-Gal, bCD-amine were synthe-
sized according to published procedures.52,53
Fig. 3 Induction of apoptosis in HepG2 cells treated with doxorubicin-loaded bCD-Gal 26. Cells were left untreated or were treated with doxorubicin
only, bCD-Gal 14 or doxorubicin-loaded bCD-Gal 26 for 24 h. Apoptosis was detected by Annexin-V-APC staining and subsequent measurement by
flow cytometry. Bars indicate the frequency of apoptotic cells present in the whole HepG2 cell population. The highest degree of apoptosis was observed
in HepG2 cells incubated with doxorubicin-loaded bCD-Gal 26 whereas bCD-Gal 14 alone induced no apoptosis. Data are representative of three
independent experiments.
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General procedure A: synthesis of sugar-nonapods
(10 mL) and 2,3,4,5,6-pentafluorophenol (4.2 g, 17.8 mmol)
was added. After cooling to 0 ◦C, N,N¢-diisopropylcarbodiimide
(DIC) (3.95 mL, 19.9 mmol) was added and the reaction mixture
was stirred for 12 h at room temperature. The reaction mixture
was concentrated in vacuo and purified by silica column flash
chromatography to afford 7 (3.7 g, 86%). Rf 0.6 (CH2Cl2–EtOH,
The Boc-protected tripod-sugar (4 eq) was dissolved in 10 mL
dichloromethane/trifluoroacetic acid (3 : 1) and stirred at room
temperature for 1 h. The solvent was evaporated under re-
duced pressure and the resulting oil was dissolved in anhydrous
dichloromethane (20 mL). To this mixture was added tert-butoxy-
carbonyl-3-{N -{tris[3-[pentafluorophenylcarboxyl-ethoxy)me-
thyl]}methylamine}-3-b-alanine (1 eq), adjusted to pH 8 with tri-
ethylamine (TEA) and the mixture was stirred at room temperature
for 12 h. The solvent was evaporated in vacuo and purified by flash
silica column chromatography.
1
88 : 12); H NMR (300 MHz, CDCl3): d 3.83 (br. s, 12H), 3.24
(t, J = 6.6 Hz, 2H), 2.92 (t, J = 6.0 Hz, 7H), 2.17 (t, J = 6.6 Hz,
2H), 1.71–1.62 (m, 6H), 13C NMR (75 MHz, CDCl3): d 167.2,
69.9, 65.9, 59.6, 36.6, 34.1, 28.2, 24.2. FTIR(CHCl3): 3688, 3385,
1749, 1658, 1522, 1359 cm-1. HRMS (MALDI-ToF) (m/z) calcd.
for C36H27F15N4O10Na 983.1385, found: 983.1388.
N-{Tris[3-[ethylcarboxyl-ethoxy)methyl]}methylamide}-5-bromo
3-{Tris[3-carboxylethoxy]methyl]3¢-{tris-[2-ethoxy-2,3,4,6-tetra-
O-acetyl-a-D-manno pyranoside-ethoxy]methyl]methylamide}-
5-azido pentamide 8
pentamide 5
To
a
solution of N-{tris[(3-[ethylcarboxyl-ethoxy)methyl]}-
methylamine (5 g, 11.8 mmol) and 5-bromovaleric acid (2.1 g,
11.8 mmol) in dichloromethane (20 mL) at 0 ◦C, were added
N,N¢-diisopropylcarbodiimide (DIC) (2.25 ml, 12.2 mmol) and
1-hydroxybenzotriazole (0.15 g, 1.18 mmol) The reaction mixture
was stirred at room temperature for 12 h and concentrated
in vacuo. The crude residue was purified by flash silica column
chromatography to yield 5 (4.8 g, 69%). Rf 0.5 (CH2Cl2–MeOH,
98 : 2); 1H NMR (300 MHz, CDCl3): d 4.14 (q, J = 7.2 Hz, 6H),
3.67 (br. s, 11H), 3.41 (t, J = 6.6 Hz, 2H), 2.52 (t, J = 6.0 Hz, 6H),
2.17 (t, J = 6.6 Hz, 2H), 1.9–1.7 (m, 4H), 1.6–1.4 (m, 2H), 1.24 (t,
J = 6.9 Hz, 9H), 13C NMR (75 MHz, CDCl3): d 172.4, 171.4, 69.5,
66.5, 60.7, 60.3, 35.2, 33.4, 33.3, 24.5, 14.1, FTIR(CHCl3): 3390,
2982, 2873, 1734, 1726, 1643, 1521 cm-1. HRMS (MALDI-ToF)
(m/z) calcd. for C24H42BrNO10Na 606.1884, found: 606.1878.
Using general procedure A, tert-butoxycarbonyl-3-{tris[3-
[2-ethoxy-2,3,4,6-tetra-O-acetyl-a-D-mannopyranoside-ethoxy]-
methylamide}-3-b-alanine (0.3 g, 0.18 mmol) and 7 (44 mg,
0.045 mmol), followed by purification using flash chromatography
yielded 8 (0.21 g, 59%). [a]D = +19.4 (c = 1.0, CHCl3); 1H NMR
r.t
(300 MHz,CDCl3): d 7.27 (br, 1H), 5.27 5.2 (m, 27H), 4.75 (s, 9H),
4.15 (dd, J = 9.0 Hz, 9 H), 4.05 (br, 9H), 3.81–3.24 (m, 127H),
3.24 (br, 8H), 2.47 (br, 32H), 2.13 (s, 27H), 2.06 (s, 27H), 2.04 (s,
27H), 1.9 (s, 27H), 1.7–1.5 (m, 6H), 13C-NMR (75 MHz, CDCl3):
d 173.92, 172.2, 171.4, 171.3, 98.7, 70.7, 70.6, 70.4, 69.8, 68.5,
67.6, 67.0, 63.4, 54.6, 39.0, 37.2, 30.5, 20.6, FTIR(CHCl3): 3332,
2734, 1745,1365, cm-1; HRMS-MALDI (m/z): [M+ Na]+ Calcd
for C210H315N19O118Na 5013.913; Found : 5013.945.
3-{Tris[3-carboxyl ethoxy]methyl] 3¢-{tris[2¢-ethoxy-2,3,4,6-tetra-
O-acetyl-b-D-galactopyranoside-ethoxy]methyl]
methylamide}-5-azido pentamide 9
N-{Tris[3-[ethylcarboxyl-ethoxy)methyl]}methylamide}-5-azido
pentamide 6
5 (3 g, 5.14 mmol) and sodium azide (1.34 g, 20.58 mmol) in
anhydrous DMF (20 mL) were mixed and stirred at 70 ◦C for
24 h. The crude residue was dissolved in 20 mL water and extracted
with 40 mL ethyl acetate. The organic layer was washed five times
with 20 mL water and concentrated in vacuo. The crude product
was purified by flash silica column chromatography to yield 6
(2.35 g, 72%). Rf 0.5 (CH2Cl2–MeOH, 98 : 3); 1H NMR (300 MHz,
CDCl3): d 4.14 (q, J = 7.2 Hz, 6H), 3.69 (br. s, 11H), 3.31 (t, J =
6.6 Hz, 2H), 2.52 (t, J = 6.0 Hz, 6H), 2.18 (t, J = 6.6 Hz, 2H), 1.71–
1.62 (m, 6H), 1.27 (t, J = 6.9 Hz, 9H), 13C NMR (75 MHz, CDCl3):
d 172.4, 171.5, 69.1, 66.6, 60.3, 51.1, 36.2, 35.3, 28.2, 32.6, 20.8,
14.1, FTIR(CHCl3): 3390, 2982, 2873, 1734, 1726, 1643, 1521 cm-1.
HRMS (MALDI-ToF) (m/z) calcd. for C24H42N4O10Na 569.2793,
found: 569.2802.
Using general procedure A, tert-butoxycarbonyl-3-{tris[3-
[2-ethoxy-2,3,4,6-tetra-O-acetyl-b-D-galactopyranoside-ethoxy]-
methyl] methylamide}-3-b-alanine (0.45 g, 0.27 mmol) and 7,
followed by purification using flash silica column chromatography
and CH2Cl2–CH3OH (12–13%) as the eluent yielded 0.21 g (52%)
of 9. Rf 0.55 (CH2Cl2–MeOH = 90 : 10); [a]rDt = +0.8 (c = 1.0,
1
CHCl3); H NMR (300 MHz, MeOD): d 5.37 (d, J = 3.3 Hz,
9H), 5.22–5.12 (m, 27H), 4.99 (d, J = 4.2 Hz, 9H), 4.51–4.45 (m,
2H), 4.14 (s, 16H), 3.95–3.92 (m, 9H), 3.84–3.78 (m, 9H), 3.66–
3.56 (m, 74H), 3.41–3.36 (m, 39H), 2.41 (t, J = 5.4 Hz, 36H),
2.14 (s, 27H), 2.04 (s, 27H), 2.02 (s, 27H), 1.96 (s, 27H), 1.9–1.7
(m, 6H); 13C NMR (75 MHz, CDCl3): d171.3, 170.2, 170.0, 169.8,
169.6, 101.2, 70.7, 68.9, 67.4, 67.3, 67.0, 61.2, 39.3, 36.4, 28.9, 20.9.
FTIR(CHCl3): 3385, 3019, 1749, 1658, 1522, 1232, 1205 cm-1;
HRMS-MALDI (m/z): [M+ Na]+ Calcd for C210H315N19O118Na
5013.913; Found : 5013.911.
N-{Tris[3-[pentafluorophenylcarboxyl-
ethoxy)methyl]}methylamide}-5-azido pentamide 7
Mono-(2,3-di-O-acetyl-6-deoxy-6-(5-hexynyl)-amido)-hexakis-
(2,3,6-tri-O-acetyl)-bCD 10
6 (2.5 g, 3.57 mmol) was dissolved in ethanol (20 mL) and sodium
hydroxide solution (aqueous, 1 N, 2 mL) was added and the
mixture was stirred at room temperature for 2 h, concentrated
in vacuo, the adjusted to pH 5 with hydrochloric acid (aqueous
1 N, 3 mL) and extracted with ethyl acetate. The organic layer
was dried with sodium sulfate and concentrated to dryness under
reduced pressure. The residue was dissolved in dichloromethane
To
a solution of bCD-amine (0.36 g, 0.18 mmol) in
dichloromethane (2 mL) under argon were added 5-hexynoic
acid (40 mg, 0.36 mmol, 2 eq), HOBt monohydrate (61 mg,
0.40 mmol, 2.2 eq) and DIPC (61 mg, 0.40 mmol, 2.2 eq). The
reaction was allowed to stir for 12 h at room temperature. The
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solvent was evaporated and the residue was purified by column
chromatography on silica gel (EtOAc) to give a mixture of 2 and
diisopropyl urea. This mixture was purified by LH-20 column (1 : 1
DCM–MeOH) to give 309 mg of 2 (82%) as an amorphous solid.
Rf : 0.32 (EtOAc). [a]20 +96 (C = 2.3, DCM). 1H NMR (400 MHz,
CDCl3) d: 6.04 (t, J =D6.2 Hz, 1H), 5.34–5.24 (m, 6H), 5.17 (dd, J =
6.8, 8.8 Hz, 1H), 5.14–4.97 (m, 7H), 4.85–4.73 (m, 7H), 4.60–4.05
(m, 20H), 3.99 (ddd, J = 9.2, 6.1, 3.1 Hz, 1H), 3.76–3.58 (m, 6H),
3.48 (dd, J = 9.2, 7.0 Hz, 1H), 2.33 (m, 2H), 2.25–2.21 (m, 2H),
28H), 2.47 (br, 32H), 2.13–1.99 (m, 168H), 1.74–1.57 (m, 6H).
13C NMR (175 MHz, MeOD): d 173.5; 171.1, 170.3, 170.2, 102.3,
98.5, 77.5, 70.9–65.2, 63.4, 61.4, 54.6, 38,6, 36.5, 28.9, 24.5, 14.2
FTIR(CHCl3): 3362, 2711, 1745, 1365, cm-1.
bCD-Man dendrimer 13
Using general procedure C, 11 (60 mg, 8.5 mmol) and sodium
methoxide (4.6 mg, 85 mmol) gave 31 mg (78%) of 13. [a]Drt
=
1
-12.3 (c = 1.0, H2O); H NMR (700 MHz, D2O): d 4.95 (s, 7H),
4.65 (s, 9H), 3.81–3.55 (m, 106H), 3.46–3.30 (m, 7H), 2.47–2.39
(m, 14H); 1.9–1.43 (m, 12H);13C NMR (125 MHz, CD3OD): d
181.1, 174.4, 104.3, 100.2, 82.3, 73.8, 73.5, 71.8, 70.8, 68.5, 67.6,
66.6, 62.4, 61.4, 39.1, 36.8, 17.3. ESI-MS (m/z): [M]+ Calcd for
C186H319N20O117 4704.595; Found 2375.731 [M+Na/2]+.
2.13–1.99 (m, 60H), 1.98 (t, J = 2.6 Hz, 1H), 1.90–1.78 (m, 2H). 13
C
NMR (100 MHz, CDCl3) d: 172–169, 97.2–96.3, 78.1–76.2, 71.0–
70.6, 70.4–70.0, 69.5–69.3, 69.1 5, 62.7–62.4, 34.7, 24.0, 20.8–20.6,
17.7. FT-IR = 2901, 2848, 2264, 1709 cm-1 MS ESI+ HRMS m/z
[M+Na]+ calcd for C88H117NO55Na 2091.6315, found 2091.6302.
Synthesis of complexes 1–3
bCD-Gal dendrimer 14
General procedure B. [2+3] Cycloaddition. The azide-nonapod
(1 eq) and acetylene-bCD (1 eq) was dissolved in 10 mL
tetrahydrofuran–water (2 : 1). To this mixture, copper sulfate (2
eq) and ascorbic acid (2 eq) were added and stirred at room
temperature for 12 h. The solvent was evaporated in vacuo and
purified by flash silica column chromatography.
Using general procedure C, 12 (65 mg, 9.2 mmol) and sodium
methoxide (5 mg, 92m mol) gave 34 mg (77%) of 14. [a]rDt = -34.8
(c = 1.0, H2O); 1H NMR (700 MHz, MeOD): d 4.95 (s, 7H), 4.43
(s, 9H), 4.11–3.35 (m, 102H), 2.47–2.35 (m, 14H), 1.9–1.43 (m,
12H);13C NMR (175 MHz, CD3OD): d 181.1, 174.4, 171.7, 104.3,
97.2, 78.3, 76.2, 75.8, 74.2, 73.8, 73.5, 71.8, 68.5, 67.6, 66.6, 61.2,
39.1, 36.8, 17.3. ESI-MS (m/z): [M]+ Calcd for C186H320N20O117
4704.595; Found 2375.731 [M+Na/2]+.
General procedure C. Synthesis of fluorescent-sugar complex.
CD-glycodendrimer (1 eq) and sodium methoxide (10 eq) were
dissolved in methanol (10 mL) and stirred at room temperature
for 12 h. The solvent was then evaporated in vacuo, the residue
was washed with Amberline-H+ resin and re-dissolved in water
and dialyzed against water using 500 molecular weight cut-off
resin. After two days of dialysis, the sample was lyophilized.
Synthesis of 1–3
A solution of 1eq of bCD-glycodendrimer 13, 14 and 1 eq of
rhodamine B in 1 mL distilled water was prepared and sonicated
for 10 min. Complexation was established by fluorescent and 1H-
NOESY experiment.
Peracetylated bCD-Man dendrimer 11
Synthesis of glycolipids 20 and 25
Using general procedure B, 8 (70 mg, 0.014 mmol), 10 (40 mg,
0.019 mmol), ascorbic acid (5 mg, 0.028 mmol) and copper sulfate
(5 mg, 0.031 mmol), followed by purification using flash silica
column chromatography and CH2Cl2–CH3OH (13–17%) as the
eluent yielded 81 mg (82%) of 11. Rf 0.5 (CH2Cl2 : MeOH = 90 : 10);
Citronellylphosphate 15. Citronellol (0.29 mL, 1.60 mmol) was
dissolved in hexane (5 mL) and triethylamine (0.22 mL, 1.60 mmol)
was added. The mixture was stirred under argon and cooled
to 0 ◦C. Phosphorus oxychloride (0.15 mL, 1.60 mmol) was
added dropwise to the above solution and the resulting mixture
allowed to warm to room temperature. The reaction was stirred
for a 2 h period. The reaction was quenched by the addition
of a acetone : water : triethylamine (85 : 10 : 5) mixture and stirred
overnight. The solution was concentrated in vacuo, diluted with
toluene (10 mL) and washed with water (2 ¥ 6 mL). The organic
layer was concentrated in vacuo to afford 15 (300 mg, 80%) as a
1
[a]rDt = +35.7 (c = 1.0, CHCl3); H NMR (700 MHz, MeOD): d
7.64 (s, 1H), 5.44–5.24 (m, 14H), 5.26–5.05 (m, 41H), 4.85 (d, J =
9.1 Hz, 7H), 4.73 (d, J = 6.8 hz, 7H), 4.51 (d, J = 9.0 Hz, 4H),
4.32 (t, J = 3.1 Hz, 6H), 4.25(t, J = 3.5 Hz, 6H), 4.18 (brs, 38H),
3.81 (s, 12H), 3.74–3.51 (m, 62 H), 3.23 (t, J = 5.0 Hz, 28H),
2.47 (br, 32H), 2.13–1.99 (m, 168H), 1.8–1.57 (m, 6H); 13C NMR
(175 MHz, MeOD): d 173.2; 171.3, 170.5, 170.2, 98.5, 97.6, 77.9,
72.3–70.1, 69.4, 68.5, 67.6, 67.0, 63.4, 61.4, 54.6, 39,6, 36.5, 28.9,
20.5, FTIR(CHCl3): 3332, 2734, 1745, 1365, cm-1.
white foam; FTIR (KBr disc) 1259, 1066, 1017 cm-1; H NMR
1
(400 MHz, MeOD) d 5.12 (d, J = 7.1 Hz, 1H), 4.05–3.92 (m,
2H), 2.08–1.96 (m, 2H), 1.68 (s, 3H), 1.62 (s, 3H), 1.52–1.15 (m,
5H), 0.94 (d, J = 6.6 Hz, 3H); 13C NMR (100.6 MHz, CDCl3) d
131.1, 124.8, 64.6, 37.7, 37.3, 29.3, 25.5, 25.0, 18.8, 16.8; 31P-NMR
(162 MHz, MeOD) d 0.29; m/z (ESI) 235 [M - H]-; HRMS (ESI)
Calcd. For C10H20O4P [M - H]- 235.1094. Found: 235.1091.
Peracetylated bCD-Gal dendrimer 12
Using general procedure B, 9 (40 mg, 0.008 mmol), 10 (17 mg,
0.008 mmol), ascorbic acid (3.5 mg, 0.02 mmol) and copper sulfate
(3 mg, 0.02 mmol), followed by purification using flash silica
column chromatography and CH2Cl2–CH3OH (13–17%) as the
eluent yielded 48 mg (84%) of 12. Rf 0.5 (CH2Cl2 : MeOH = 88 : 12);
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranose 1-
phosphate, monotriethyl-ammonium salt 18. A catalytic amount
of palladium on activated charcoal was added to a stirred solution
1
[a]rDt = +22.7 (c = 1.0, CHCl3); H NMR (700 MHz, MeOD): d
5.44–5.24 (m, 14H), 5.36–5.25 (m, 34H), 5.23 (s, 7H), 4.75–4.56
(m, 7H), 4.45–4.41 (m, 14H), 4.51 (d, J = 9.0 Hz, 4H), 4.32–
4.03 (m, 21H), 3.87–3.77 (m, 24H), 3.74–3.51 (m, 71H), 3.23 (m,
of
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranose
1-dibenzyl-phosphate54 17 (122 mg, 0.20 mmol) in anhydrous
methanol (4 mL). The resulting solution was degassed and
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purged with hydrogen gas, then left to stir under an atmosphere
of hydrogen. After a 3 h period, TLC (diethyl ether–methanol,
98 : 2) indicated the formation of a product (Rf 0) with complete
consumption of starting material (Rf 0.3). The solution was
7.4 Hz, 1H); 5.11 (d, J = 7.1 Hz, 1H), 4.09–4.04 (m, 2H), 4.00
(d, J = 2.8, 10.4 Hz, 1H), 3.93 (ddd, J = 9.9, 2.1, 5.1 Hz, 1H),
3.84 (dd, J = 2.3, 11.9 Hz, 1H), 3.74 (d, J = 9.7 Hz, 1H), 3.70
(dd, J = 5.3, 11.9 Hz, 1H), 3.41 (d, J = 9.5 Hz, 1H), 2.17 (s,
3H), 2.00–1.94 (m, 2H), 1.75–1.69 (m, 2H), 1.68 (s, 3H), 1.61 (s,
3H), 1.48–1.41 (m, 1H), 1.39–1.32 (m, 1H), 1.30–1.23 (m, 1H),
0.92 (d, J = 6.6 Hz, 3H), 13C NMR (100.6 MHz, CDCl3 : MeOD;
3 : 97) d 174.0, 131.9, 125.9, 96.4, 75.0, 73.3, 72.0, 65.6, 62.8, 55.5,
38.7, 38.4, 30.5, 26.5, 25.9, 23.0, 19.8, 17.6; 31P NMR (162 MHz,
CDCl3 : MeOD; 3 : 97) d -12.6 (d), -10.3 (d); m/z (ESI) 518 [M -
H]-; HRMS (ESI) Calcd. For C18H34NO12P2 [M - H]- 518.1551.
Found: 518.1548.
R
filtered through Celiteꢀ. Triethylamine (1 eq) was added and the
resulting solution stirred for further 30 min. The reaction mixture
was concentrated in vacuo to afford 18 (110 mg, 100%) as a white
amorphous solid; [a]2D5 +28.2 (c, 1 in MeOH); FTIR (KBr disc)
3447, 1749, 1240, 1033 cm-1; 1H NMR (400 MHz, CDCl3) d 6.48
(br d, J = 9.3 Hz, 1H), 5.49 (dd, J = 2.9, 7.4 Hz, 1H), 5.27 (dd,
J = 9.9, 10.1 Hz, 1H), 5.15 (d, J = 9.9 Hz, 1H), 4.33 (ddd, J = 2.2,
7.6, 9.8 Hz, 1H), 4.25 (br d, J = 10.0 Hz, 1H), 4.16 (dd, J = 3.1,
12.0 Hz, 1H), 4.09 (dd, J = 1.7, 12.2 Hz, 1H), 2.95 (q, J = 6.9 Hz,
6H), 2.04 (s, 3H), 1.97 (s, 3H), 1.96 (s, 3H), 1.93 (s, 3H), 1.25 (d,
J = 6.8 Hz, 9H), 13C NMR (100.6 MHz, CDCl3) d 171.0, 170.9,
170.8, 169.4, 91.5, 71.7, 68.3, 68.2, 61.8, 52.4, 45.5, 23.1, 20.8,
20.7, 20.6, 9.0; 31P-NMR (162 MHz, CDCl3) d -10.0; m/z (ESI)
426 [M - H]-; HRMS (ESI) Calcd. For C14H21NO12P [M - H]-
426.0796. Found: 426.0795.
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-galactopyranose 1-
phosphate, monotriethyl-ammonium salt 23. A catalytic amount
of palladium on activated charcoal was added to a stirred solution
of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-galactopyranose
1-dibenzylphosphate55 22 (0.47 g, 7.74 mmol) in anhydrous
methanol (10 mL). The resulting solution was degassed and
purged with hydrogen gas, then left to stir under an atmosphere of
hydrogen. After 2 h, TLC (diethyl ether–methanol, 98 : 2) indicated
the formation of a product (Rf 0) with complete consumption
of starting material (Rf 0.2). The solution was filtered through
P1-Citronellyl P2-[2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-
glucopyranosyl] diphosphate 19. To a suspension of 15 (83 mg,
0.35 mmol, dried over P2O5 overnight) in anhydrous DMF
(2.5 mL) was added 1,1¢-carbonyldiimidazole (CDI, 0.28 g,
1.77 mmol) in one portion. After stirring at room temperature
for 2 h, anhydrous methanol (60 mL) was added and the reaction
stirred for another 1 h to destroy excess CDI. Methanol was
removed under reduced pressure at room temperature. In a
separate flask, 18 (150 mg, 0.35 mmol, dried over P2O5 overnight)
was dissolved in anhydrous DMF (2.5 mL), and transferred to the
above flask. The resulting mixture was stirred at room temperature
under an argon atmosphere for 72 h. The reaction mixture was
concentrated in vacuo, and the residue purified over a C-18 column
(5–100% gradient, water–acetonitrile) to afford 19 (87 mg, 40%)
as a foam; [a]2D2 +8.4 (c, 1 in CHCl3); FTIR (thin film) 3447, 1748,
R
Celiteꢀ. Triethylamine (1 eq) was added and the resulting solution
stirred for a further 30 min period. The reaction mixture was
concentrated in vacuo to afford 23 (0.41 g, 88%) as a white
amorphous solid; FTIR (KBr disc) 3446, 1747, 1242, 1031 cm-1;
1H NMR (400 MHz, CDCl3) d 7.13 (br d, J = 9.2 Hz, 1H), 5.56
(dd, J = 3.1, 7.4 Hz, 1H), 5.40 (br d, J = 2.1 Hz,1H), 5.24 (dd,
J = 3.2, 11.2 Hz, 1H), 4.60 (ddd, J = 1.4, 9.4, 10.8 Hz, 1H), 4.49
(br, J = 6.5 Hz, 1H), 4.16 (dd, J = 7.5, 11.2 Hz, 1H), 4.02 (dd, J =
5.9, 10.9 Hz, 1H), 2.83 (q, J = 7.3 Hz, 6H), 2.14 (s, 3H), 2.00 (s,
3H), 1.96 (s, 3H), 1.95 (s, 3H), 1.19 (br, J = 7.3 Hz, 9H). 13C NMR
(100.6 MHz, CDCl3) d 171.2, 170.6, 170.5, 170.4, 94.2, 68.6, 67.5,
31
67.1, 61.5, 48.0, 45.7, 23.2, 20.8, 20.7, 20.6, 9.6; dP (162 MHz,
1
1232, 1063 cm-1; H NMR (400 MHz, CDCl3 : MeOD; 3 : 97) d
CDCl3) -0.29; m/z (ESI) 426 [M - H]-; HRMS (ESI) Calcd. For
C14H21NO12P [M - H]- 426.0796. Found: 426.0807.
6.11 (br d, J = 9.4 Hz, 1H), 5.66 (dd, J = 2.7, 7.4 Hz, 1H), 5.23 (d,
J = 10.1 Hz, 1H), 5.17 (d, J = 9.7 Hz, 1H), 5.04 (d, J = 7.1 Hz, 1H),
4.40 (d, J = 9.9 Hz, 1H), 4.24 (br d, J = 9.9 Hz, 1H), 4.19–4.09 (m,
2H), 4.02–3.97 (m, 2H), 2.07 (s, 3H), 2.02 (s, 3H), 1.99 (s, 3H), 1.98
(s, 3H), 1.94–1.86 (m, 2H), 1.71–1.66 (m, 2H), 1.64 (s, 3H), 1.56
(s, 3H), 1.45–1.38 (m, 1H), 1.31–1.25 (m, 1H), 1.16–1.09 (m,1H)
0.85 (d, J = 6.6 Hz, 3H), 13C NMR (100.6 MHz, CDCl3 : MeOD;
3 : 97) d 171.4, 170.9, 170.8, 169.3, 131.2, 124.5, 94.7, 71.0, 68.8,
67.8, 64.9, 61.4, 52.3, 37.9, 37.1, 29.2, 25.7, 25.4, 22.8, 20.8, 20.7,
20.6, 19.1, 17.6; 31P NMR (162 MHz, CDCl3 : MeOD; 3 : 97) d
-12.6 (d), -9.9 (d); m/z (ESI) 644 [M - H]-; HRMS (ESI) Calcd.
For C24H41NNaO15P2 [M+Na]+ 668.1844. Found: 668.1836.
P1-Citronellyl P2-[2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-
galactopyranosyl] diphosphate 24. To a suspension of 15 (0.28 g,
1.16 mmol, dried over P2O5 overnight) in anhydrous DMF (7 mL)
was added 1,1¢-carbonyldiimidazole (CDI, 0.78 g, 4.85 mmol) in
one portion. After stirring at room temperature for 3 hs, anhydrous
methanol (150 mL) was added and the reaction was stirred for
another 1 h to destroy the excess CDI. Methanol was removed
under reduced pressure at room temperature. In a separate flask,
23 (0.41 g, 9.69 mmol, dried over P2O5 overnight) was dissolved
in anhydrous DMF (7 mL), and transferred to the above flask.
The resulting mixture was stirred at room temperature under an
argon atmosphere for 72 h. The reaction mixture was concentrated
in vacuo, and the residue purified over a C-18 column (5–100%
gradient, water–acetonitrile) to afford 24 (0.42 g, 66%) as a foam;
[a]2D2 +5.1 (c, 1 in CHCl3); FTIR (thin film) 3445, 1746, 1229,
P1-Citronellyl
P2-[2-acetamido-2-deoxy-a-D-glucopyranosyl]
diphosphate 20. To a stirred solution of 19 (25 mg, 39 mmol)
in anhydrous DCM (3 mL) at room temperature was added a
solution of sodium methoxide (1 mL of a 110 mg mL-1 solution
in methanol). After 30 min, cation exchange resin (DOWEX
50WX8-200, pyridinium form, 300 mg) was added and the
mixture was stirred for 10 min. The resin was then filtered and
washed with chloroform–methanol (2/1, v/v). The filtrate was
concentrated in vacuo to afford 20 (20 mg, 100%) as a foam;
[a]2D2 +10.3 (c, 1 in MeOH); FTIR (thin film) 3356, 1135 cm-1;
1H NMR (400 MHz, CDCl3 : MeOD; 3 : 97) d 5.57 (dd, J = 3.2,
1
1067 cm-1; H -NMR (500 MHz, CDCl3 : MeOD; 3 : 97) d 5.57
(dd, J = 3.2, 7.4 Hz, 1H), 5.32 (br d, J = 2.5 Hz, 1H), 5.10 (dd,
J = 3.1, 11.3 Hz, 1H), 5.00 (br.d, J = 7.1 Hz, 1H), 4.43–4.50 (m,
2H), 4.19–4.09 (m, 1H), 4.09 (dd, J = 8.0, 10.8 Hz, 1H), 4.01–
3.91 (m, 2H), 2.04 (s, 3H), 1.91 (s, 3H), 1.83 (s, 3H), 1.90 (s,
3H), 1.62–1.57 (m, 2H), 1.50 (m, 2H), 1.45 (s, 3H), 1.46 (s, 3H),
1.32–1.38 (m, 1H), 1.29–1.21 (m, 1H), 1.10–1.02 (m, 1H), 0.81
4994 | Org. Biomol. Chem., 2010, 8, 4987–4996
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(d, J = 6.6 Hz, 3H); 13C NMR (100.6 MHz, CDCl3) d 174.2,
172.3, 172.1, 171.8, 131.9, 125.8, 96.4, 70.2, 68.9, 68.5, 65.7, 62.4,
49.3, 39.0, 38.3, 30.4, 26.5, 25.9, 22.8, 20.8, 20.7, 20.6, 19.8, 17.8;
31P NMR (202 MHz, CDCl3 : MeOD; 3 : 97) d -13.0 (d), -10.3 (d);
m/z (ESI) 644 [M - H]-; HRMS (ESI) Calcd. For C24H41NNaO15P2
[M+Na]+ 668.1844. Found: 668.1844.
were then collected with PBS containing 1% FCS by shearing
force. Uptake of bCD was measured by flow cytometry using
a FACSCantoTM II flow cytometer (Becton Dickinson, Franklin
Lakes, NJ). Excitation wavelength in the FACS experiments was
540 nm, which is the excitation wavelength of Rhodamine B. Cells
were gated on living cells and fluorescence channel FL-2 (PE-A)
was used to detect HepG2 cells that had endocytosed RhB-labeled
bCD molecules. All data were analyzed with the FlowJo software
(Tree Star, Inc., Ashland, OR).
For analysis of liposome uptake by the HepG2 cell
line, cells were incubated with liposomes consisting of L-a-
phosphatidylcholine and rhodamine-DHPE (negative control)
or liposomes displaying additionally glycolipid 20 (GlcNAc,
specificity control) or 25 (GalNAc) on their surface. After
incubation times between one to six hours, cells were washed with
PBS and collected from the plate. Detection of liposome uptake
was performed as described above.
P1-Citronellyl P2-[2-acetamido-2-deoxy-a-D-galactopyranosyl]
diphosphate 25. To a stirred solution of 24 (0.27 g, 0.43 mmol)
in anhydrous DCM (25 mL) at room temperature was added a
solution of sodium methoxide (3 mL of a 150 mg mL-1 solution in
methanol). After 30 min, cation exchange resin (DOWEX 50WX8-
200, pyridinium form) was added and the mixture stirred for
10 min. The resin was then filtered and washed with chloroform–
methanol (2/1, v/v). The filtrate was concentrated in vacuo to
afford 25 (0.27 g, 100%) as a foam; [a]2D2 +8.3 (c, 1 in MeOH); FTIR
(thin film) 3352, 1131 cm-1; 1H NMR (500 MHz, CDCl3 : MeOD;
3 : 97) d 5.62 (br s,1H), 5.07 (d, J = 7.2 Hz, 1H), 4.38 (br d, J =
6.3 Hz, 1H), 4.18–3.46 (m, 7H), 2.04 (s, 3H), 1.93–1.99 (m, 2H),
1.66 (m, 2H), 1.59, 1.55 (s, 6H), 1.38–1.32 (m, 1H), 1.39–1.29 (m,
1H), 1.13 (m, 1H), 0.91 (d, J = 6.3 Hz, 3H); 13C NMR (100.6 MHz,
CDCl3) d 174.6, 130.6, 124.4, 96.4, 72.3, 70.9, 69.0, 68.4, 61.6, 45.6,
36.9, 31.6, 29.0, 25.1, 24.6, 22.3, 22.1, 21.6;31P NMR (162 MHz,
CDCl3 : MeOD; 3 : 97) dP -12.8 (d), -10.1 (d); m/z (ESI) 518 [M
- H]-; HRMS (ESI) Calcd. For C18H34NO12P2 [M - H]- 518.1551.
Found: 518.1556.
Synthesis of doxorubicin-loaded bCD-Gal 26
A solution of bCD-glycodendrimer 14 (1 eq) and doxorubicin-
HCl (1 eq) in 1 mL distilled water was prepared and sonicated for
20 min. The complexation was established by 1H-NOESY (see the
ESI for full details†).
Apoptosis studies in HepG2 cells
For analysis of apoptosis, HepG2 cells were incubated with
doxorubicin-loaded bCD-Gal 26 for 24 h at concentrations of
1 mg ml-1, 5 mg ml-1, and 10 mg ml-1 of the incorporated drug. The
following controls were used: untreated cells (negative control),
cells incubated with doxorubicin alone (positive control), and
cells treated with bCD-Gal 14 only. The concentrations for
doxorubicin and empty bCD-Gal 14 were the same as those of
doxorubicin-loaded bCD-Gal 26. After incubation, the adherent
cells were detached from the growth surface using Trypsin/EDTA
(Pan Biotech, Germany). The staining procedure was performed
according to the protocol in the Annexin V-APC Apoptosis
Detection Kit purchased from eBioscience (Frankfurt, Germany).
Detection of apoptopic cells was done by flow cytometry as
described above. Cells were gated on living cells. Additionally,
fluorescence channel FL-3 (APC) was used to detect apoptotic
cells.
Preparation of liposomes
L-a-Phosphatidylcholine (18 mM) and glycolipid 20 (specificity
control) or glycolipid 25 (4 mM or 10 mM) were added as solids
to a 10 mL round bottomed flask. A solution of rhodamine
DHPE (0.5 mM) in ethanol (0.25 mL) was added. The lipids were
dissolved in ethanol (2.75 mL) and stirred at room temperature
for 2 h. The reaction mixture was concentrated in vacuo and
placed under high vacuum for additional 2 h. The lipid film was
dissolved in sodium phosphate buffer (pH 7, 50 mM, 0.5 mL) and
R
submitted to extrusion through the Avantiꢀ Extruder (100 nm
polycarbonate membrane) 11 times. The glycoliposomes were
stored after preparation at 4 ◦C until further use.
Transmission electron microscopy (TEM) measurements
After extrusion, a drop of a 50 mM solution of glycolyposomes
was diluted with ultrapure water (1 : 10 v/v) and deposited on
the carbon membrane of a copper grid. The grids were stained
subsequently with a 1% aqueous solution of uranyl acetate for
30 s at room temperature. After drying, the grid was transferred to
a transmission electron microscope (EM 912 Omega/Carl-Zeiss
Oberkochen, Germany), and TEM measurements were performed.
Acknowledgements
We thank the Max Planck Society for very generous support.
Funding by the European Union (Marie Curie Intra-European
Fellowship to G.J.L.B.), the German Federal Ministry of Ed-
ucation and Research (BMBF, Fkz. 0315446 to B.L.) and the
European Research Council (ERC Advanced Grant to P.H.S.) is
also gratefully acknowledged.
Uptake studies in HepG2 cells
Human hepatocellular carcinoma cell line, HepG2, was plated in
6-well plates and cultivated in D-MEM medium supplemented
with 10% fetal calf serum (FCS) and 1% penicillin–streptomycin.
When cells were 70% confluent, different concentrations (10 mmol,
50 mmol, and 250 mmol) of CD 1, 2 or 3 were added to the
cells. After incubation times between one and six hours, cells
were washed with phosphate buffered saline (PBS). The cells
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