Design and synthesis of novel N-acetylgalactosamine-terminated glycolipids for targeting of lipoproteins to the hepatic asialoglycoprotein receptor

Article abstract of DOI:10.1021/jm049481d

A novel glycolipid has been prepared that contains a cluster glycoside with an unusually high affinity for the asialoglycoprotein receptor (ASGPr) and a bile acid moiety that mediates stable incorporation into lipidic particles. The glycolipid spontaneously associated with low-density lipoproteins (LDL) and high-density lipoproteins (HDL) within human and murine plasma, and loading of lipoproteins with this glycolipid resulted in an efficient dose-dependent recognition and uptake of LDL and HDL by the liver (and not by spleen) upon intravenous injection into wild-type mice. Preinjection with asialoorosomucoid largely inhibited the uptake, establishing that both HDL and LDL were selectively recognized and processed by the ASGPr on liver parenchymal cells. Finally, repeated intravenous administration of the glycolipid to hyperlipidemic LDL receptor-deficient mice evoked an efficient and persistent cholesterol-lowering effect. These results indicate that the glycolipid may be a promising alternative for the treatment of hyperlipidemic patients who do not respond sufficiently to current cholesterol-lowering therapies.

Full text of DOI:10.1021/jm049481d

5798
J. Med. Chem. 2004, 47, 5798-5808
Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids
for Targeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor
Patrick C. N. Rensen,*,† Steven H. van Leeuwen,† Leo A. J. M. Sliedregt, Theo J. C. van Berkel, and
Erik A. L. Biessen
Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, University of Leiden,
Gorlaeus Laboratory, P.O. Box 9502, 2300 RA Leiden, The Netherlands
Received June 30, 2004
A novel glycolipid has been prepared that contains a cluster glycoside with an unusually high
affinity for the asialoglycoprotein receptor (ASGPr) and a bile acid moiety that mediates stable
incorporation into lipidic particles. The glycolipid spontaneously associated with low-density
lipoproteins (LDL) and high-density lipoproteins (HDL) within human and murine plasma,
and loading of lipoproteins with this glycolipid resulted in an efficient dose-dependent
recognition and uptake of LDL and HDL by the liver (and not by spleen) upon intravenous
injection into wild-type mice. Preinjection with asialoorosomucoid largely inhibited the uptake,
establishing that both HDL and LDL were selectively recognized and processed by the ASGPr
on liver parenchymal cells. Finally, repeated intravenous administration of the glycolipid to
hyperlipidemic LDL receptor-deficient mice evoked an efficient and persistent cholesterol-
lowering effect. These results indicate that the glycolipid may be a promising alternative for
the treatment of hyperlipidemic patients who do not respond sufficiently to current cholesterol-
lowering therapies.
Introduction
receptor, galactose/fucose receptor)10,11 and preferen-
tially recognizes a high density of either fucose or
galactose on either proteins11-13 or particles.14,15 Previ-
ous proof-of-concept studies already showed that the
bifunctional glycolipid TG(20Å)C (glycolipid 1, Figure
1), consisting of a cholesteryl moiety for anchorage to
lipoproteins and a triantennary Gal-terminated glyco-
side with moderate affinity for the ASGPr, was able to
associate with LDL and HDL and induce their liver
uptake in rodents. However, due to the relatively high
hydrophilicity of the glycolipid, rapid exchange of the
glycolipid occurred in the blood, leading to only a
moderate 20% liver uptake of these lipoproteins after
preloading with TG(20Å)C.16 To stabilize incorporation
of the glycolipid into lipoproteins, the lipophilicity of the
anchoring moiety was increased by adding a fatty acid
chain to the steroid core structure. In addition, the
intrinsically labile methylene acetals connecting the
glycol spacers to the tris cluster core in TG(20Å)C were
replaced by more stable ether bonds.17 The resulting
glycolipid compound (glycolipid 2, Figure 1) indeed
showed a very stable interaction with lipidic particles
which was retained in the blood and resulted in an
effective targeting of liposomes to the ASGPr on hepa-
tocytes.17,18 However, a major setback still was that high
glycolipid surface densities resulted in elimination of
liposomes by the galactose particle receptor,17,18 and
preliminary data showed that glycolipid 2 redirected
LDL almost exclusively to Kupffer cells.
Clinical data indicate a positive correlation between
LDL cholesterol levels and the occurrence of arterio-
sclerosis,1,2 while a strong inverse correlation has been
demonstrated between HDL cholesterol levels and
cardiovascular disease.3,4 However, recent insights in-
dicate that the antiatherogenic properties of HDL are
not dictated by its plasma levels, but rather by its
functionality (i.e. capability to deliver cholesteryl esters
to the liver). In fact, increasing the flux of HDL to the
liver may also be atheroprotective, as it has been shown
that overexpression of the hepatic HDL receptor SR-BI
in heterozygous LDLr-deficient (ldlr-/-) mice on a high
fat/high cholesterol diet resulted in strong reduction in
plasma HDL cholesterol and concomitantly reduced
atherosclerotic lesion area.5,6 Thus, these studies clearly
illustrate that stimulation of the hepatic uptake of LDL
and HDL will be an effective entry to antiatherosclerotic
therapy and led us to explore the feasibility to induce
effective clearance of these lipoproteins via the asia-
loglycoprotein receptor (ASPGr).7
The ASGPr is a high-capacity receptor uniquely
expressed on the surface of hepatocytes to mediate the
hepatic uptake and subsequent lysosomal processing of
galactose (Gal)- and N-acetylgalactosamine (GalNAc)-
terminated substrates from the serum,8 at which the
affinity for GalNAc is approximately 50-fold higher than
for Gal.9 In addition to the ASGPr on hepatocytes, a
second receptor recognizing galactose and fucose groups
is present in the liver on Kupffer cells (galactose particle
In this paper, we introduce an improved amphiphilic
glycolipid, 3 (see Figure 1), in which, with respect to 2,
two important modifications are present. A tyrosine
moiety was introduced to allow for trace labeling with
125I, providing the possibility to perform kinetic studies
in vivo. In addition, the galactose residues within the
triantennary cluster glycoside were replaced by GalNAc
* Corresponding author’s present address: Department of General
Internal Medicine, LUMC, Leiden, and TNO-Prevention and Health,
Gaubius Laboratory, P.O. Box 2215, 2301 CE Leiden, The Netherlands.
Telephone: + 31 71 51 81406. Fax: + 31 71 51 81904. E-mail:
pcn.rensen@pg.tno.nl.
† Both authors contributed equally to this work.
10.1021/jm049481d CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/06/2004

Novel N-Acetylgalactosamine-Terminated Glycolipids
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5799
Figure 1. Chemical structures of glycolipids TG(20Å)C (1), (3R(oleoyloxy)-5â-cholanoyl)-GABA-Gly-tris(Gal)3 (2), and (3â-
(oleoylamido)-5â-cholanoyl)-Tyr-Gly-tris(GalNAc)3 (3).
residues, which led to a 50-fold increased affinity of the
cluster glycoside toward the ASGPr on hepatocytes (Ki
2 nM).18 The resulting compound was tested for its
ability to associate with plasma lipoproteins and to
induce their liver uptake and for its cholesterol-lowering
capacity in hyperlipidemic ldlr-/- mice. We conclude
from these data that glycolipid 3 is very effective in
promoting cholesterol transport to hepatocytes and thus
may be a promising alternative for hyperlipidemic
individuals who do not respond sufficiently to current
cholesterol-lowering therapies, such as familial hyper-
cholesterolemic patients.19,20
markedly improved association with lipidic particles,
but the galactose-exposing cluster glycoside still induced
particle uptake by Kupffer and splenic cells via the
galactose particle receptor depending on substrate size
and substrate surface density of the glycolipid.17 Previ-
ous studies by us22,23 and others24 have established that
triantennary GalNAc-containing ligands displayed a
higher affinity for the ASGPr than the corresponding
diantennary GalNAcs or triantennary galactosides.
Therefore, we now synthesized a third-generation gly-
colipid by combining the lipophilic LCO unit possessing
high affinity for lipidic particles with a triantennary
GalNAc-containing glycoside (glycolipid 3). This novel
triantennary glycoside with 20 Å-spaced terminal Gal-
NAc residues appeared to display a low nanomolar
affinity for the ASGPr (2 nM) and to redirect differently
sized stable unilamellar liposomes to hepatocytes in
vitro and in vivo.18
Results and Discussion
Synthesis. Previously, we synthesized glycolipid 1,
which only modestly induced the hepatic uptake of
lipoproteins, as its cholesterol moiety afforded an only
weak association with lipoproteins.16,21 Replacement of
cholesterol by the highly lipophilic lithocholic oleate
(LCO) residue yielded glycolipid 2, which showed a
In this study we describe the synthesis of this novel
glycolipid 3 and present new biological data indicating

5800 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Scheme 1. Synthesis of the Cluster Galactoside Synthon 13a
Rensen et al.
a Reagents: (i) NaN3, NaI, 62%; (ii) 5, TMSOTf, 58%; (iii) PPh3, THF, 49%; (iv) BocGlyOH, BOP, DiPEA, 93%; (v) aq NaOH, dioxane,
100%; (vi) 8, HOBt, PyBOP, DiPEA, DMF; (vii) TFA/DCM 1/4, 100%.
that this improved glycolipid is able to direct both LDL
and HDL to the ASGPr on hepatocytes in vivo, thereby
avoiding lipoprotein uptake by the galactose particle
receptor on Kupffer cells and spleen. In analogy to
glycolipid 1, we have attached the GalNAc units to the
tris core by a flexible ethylene glycol linker to warrant
an optimal orientation and thus recognition of the
terminal sugar moieties by the ASGPr. Given this
galactosamine synthon terminus at each dendrimer
arm, an eight-atom spacer moiety sufficed to establish
a 20 Å long connection between the separate GalNAcs
and the tris branching point. Thus, 2-(2-(2-chloroet-
hoxy)ethoxy)ethanol 4 was converted to its correspond-
ing azido-derivative 5 and the galactosamine building-
unit 8 was synthesized by glycosylation of the known25
oxazoline 6 with derivative 5 (see Scheme 1). Condensa-
tion in the presence of trimethylsilyl triflate (TMSOTf)
gave compound 7 in good yield, while subsequent
reduction of the azido moiety with triphenylphosphine
afforded the terminal amine derivative 8.
The triantennary core-unit, which has been described
earlier,17 was smoothly functionalized by a N-Boc-
glycine under standard amino acid coupling conditions
to furnish compound 10. Hydrolysis of the terminal
ethyl esters rendered the trivalent carboxylic acid 11.
Purified 11 was condensed with excess of GalNAc 8 in
order to afford compound 12. Removal of the Boc-
protecting group with TFA furnished the trivalent
galactosamine 13 with a free amine, which allowed
functionalization of the cluster with a tyrosine moiety.
The incorporation of this amino acid was performed to
enable the ready introduction of a 125I radiolabel and
thus to enable us track the incorporation efficacy and
pharmacokinetics of the glycolipid per se. Thus, Fmoc-
protected tyrosine(OtBu) was reacted with amine 13
under the agency of TBTU and HOBt to afford com-
pound 21. Subsequent treatment with piperidine (20%
in DMF) provided galactoside 22.
component of the target glycolipid. Lithocholic acid was
converted into its methyl ester 15, followed by the
introduction of a mesyl protecting group at the 3-hy-
droxyl position. The mesylated methyl ester was reacted
with sodium azide to afford, by a nucleophilic substitu-
tion reaction, the required azido derivative 17 under
inversion of the stereochemistry. Reduction of the azide
group readily furnished amine 18. Freshly distilled
oleoyl chloride was used to acylate the free amino group
of compound 18, followed by saponification of the methyl
ester to furnish 3-oleoylamidolithocholic acid, synthon
20 (Scheme 2).
The bile oleoylamido acid 20 was joined in moderate
yield to the cluster GalNAc via TBTU/HOBt-aided
coupling. Deprotection of the fully protected compound
23 was achieved in two steps. The tert-butyl ester was
removed from the tyrosine by acid treament, while
deprotection of the acetyl groups of GalNAc was ac-
complished by incubation with sodium methanolate.
Purification of the target compound was performed by
gel filtration over Sephacryl S100 in methanol. The
homogeneity and identity of 3 was fully confirmed by
NMR spectroscopy and mass spectrometry (Scheme 3).
Glycolipid 3 forms stable micelles with a slightly larger
size than HDL (8-10 nm) as judged from size exclusion
chromatography on a Superose 6 column.18
Association of Glycolipids with Lipoproteins.
Previously, we showed by FPLC that the presently
applied hydrophobic bile acid residue 20 (see Scheme
2) mediates rapid incorporation of glycolipids 217 and
318 into liposomes by intercalation into the liposomal
phospholipid bilayer. We now demonstrate using aga-
rose gel electrophoresis that glycolipid 3 avidly and
quantitatively incorporated into HDL and LDL at all
glycolipid concentrations tested, leading to a reduced
electrophoretic mobility of the lipoproteins by shielding
off their negative surface charge (Figure 2). Since
glycolipid 3 incorporates instantaneously and quanti-
tatively into lipoproteins, even without the need for
incubation at 37 °C (not shown), it indeed has a very
Having the required key synthon 22 in hand, atten-
tion was focused on the construction of the lipophilic

Novel N-Acetylgalactosamine-Terminated Glycolipids
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5801
Scheme 2. Synthesis of the Lipophilic Anchor Synthon 20a
a Reagents: (i) MeOH, HCl, 100%; (ii) MsCl, pyridine; (iii) NaN3, DMF, 70% two steps; (iv) PPh3, THF; (v) oleoyl chloride, Et3N, DCE,
86%; (iv) NaOH, dioxane, H2O, 100%.
Scheme 3. Synthesis of Glycolipid 3a
a Reagents: (i) FmocTyr(tBu)OH, TBTU, HOBt, DiPEA, DMF, 70%; (ii) 20% piperidine/DMF, 75%; (iii) 20, TBTU, HOBt, DiPEA,
DMF, 80%; (iv) DCM/TFA, 100%; (v) NaOMe, MeOH, 90%.
high affinity for lipoproteins. Since glycolipid 2 induced
similar effects on LDL and HDL (not shown), the
orientation of the oleoyl moiety relative to the steroid
skeleton (R for glycolipid 2 and â for glycolipid 3)
apparently does not affect the distribution of glycolipid
over LDL and HDL. Incorporation of the glycolipid did
not significantly alter the lipoprotein size, as judged
from Sepharose 6 elution profiles (not shown). At the
applied amounts of lipoproteins (i.e. equal protein
weight of HDL and LDL), the glycolipid displayed an
increased association with LDL. This enhanced binding
to LDL probably reflects the presence of a larger total
phospholipid surface area (i.e. 1.7-fold) of LDL versus
HDL at the applied particle ratio. Therefore, the gly-
colipid does not show preference for association with
either lipoprotein. Indeed, the glycolipid induced a
similar dose-dependent reduction in the mobility of both
lipoproteins, and the relative distribution of the gly-
colipid over HDL and LDL remained constant over the
whole glycolipid concentration range (Figure 2). At the
highest amount tested, glycolipid 3 did not affect the
composition of LDL (27.2 ( 1.7% protein, 3.3 ( 0.3%
triglycerides, 18.8 ( 1.5% phosphatidylcholine, 42.1 (
1.4% cholesteryl esters, and 8.5 ( 0.2% free cholesterol)

5802 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Rensen et al.
uptake, reaching 70 ( 3% and 78 ( 2% of the dose,
respectively, at 0.5 µg of glycolipid per µg of particle
protein (Figure 3A), similar to the effect of glycolipid 2
on the hepatic clearance of both lipoproteins. At this
glycolipid loading, the ASGPr-specific inhibitor ASOR26
inhibited the glycolipid-induced hepatic clearance of
LDL and HDL for 63% and 94%, respectively. In
contrast, the enhanced liver uptake of glycolipid 2-laden
LDL could not be inhibited by ASOR (not shown). These
data indicate that the triantennary galactose moiety of
glycolipid 2 mediates the uptake of LDL exclusively via
the galactose particle receptor on liver macrophages (i.e.
Kupffer cells), while replacement of the galactose groups
by GalNAcs within the novel glycolipid 3 results in
prominent uptake of both HDL and LDL by the ASGPr
on hepatocytes. This observation is of major importance
for application of glycolipids in cholesterol-lowering
therapy, since only hepatocytes are able to directly
excrete cholesterol from the body via the bile. Also,
whereas glycolipid 2 enhanced the association of LDL
with the spleen by 33.5-fold, glycolipid 3 did not affect
the splenic uptake (and extrahepatic accumulation in
general) of both lipoproteins (Figure 3B). The applica-
tion of glycolipid 3 in cholesterol-lowering therapy will
thus not result in unwanted cholesterol delivery to
tissues other than the liver.
Figure 2. Association of the glycolipid with isolated lipopro-
teins. Glycolipid 3 (0-50 µg) mixed with a trace amount of
[
125I]glycolipid 3 (20 ng) was incubated with HDL and LDL (5
µg of protein each) for 30 min at 37 °C. The mixtures were
subjected to agarose gel electrophoresis, and radioactivity was
visualized. +, anode; -, cathode; Rf 0, position application slots;
Rf 1, position front marker bromophenol blue.
We have previously observed that a high surface
density of glycolipid 2 leads to a shift in uptake of
liposomes from hepatocytes to Kupffer cells,17 whereas
glycolipid 3 induced the uptake of liposomes by hepa-
tocytes, irrespective of its surface density.18 A similar
phenomenon may underlie the different effect of the
glycolipids on the intrahepatic distribution of LDL over
hepatocytes and Kupffer cells. Whereas glycolipids can
evenly distribute over the phospholipid surface of lip-
osomes, the surface of LDL is largely covered by its
protein constituent apolipoprotein B-100,27,28 leaving
relatively small clefts for the association of glycolipid.
Therefore, the glycolipids will accumulate in these clefts,
resulting in a local high glycolipid density, which, for
glycolipid 2, may lead to the induced LDL uptake by
Kupffer cells. In contrast, the affinity of glycolipid 3 for
the ASGPr is sufficiently high and selective to still
induce the uptake of LDL by hepatocytes.
Figure 3. Liver uptake of control and glycolipid-laden lipo-
proteins in mice. [125I]LDL (5 µg of protein) or [3H]HDL (10
µg of protein) were incubated with the indicated amounts of
glycolipid 3 (A, B) or 2 (B) and injected into anaesthetized
C57Bl/6J mice, without or with previous injection of ASOR
(25 mg/kg) at 1 min before injection of the lipoproteins (A). At
10 min after injection, liver lobules (A) and spleen (B) were
taken, and the amount of radioactivity was determined. Values
are means ( variation of two experiments.
Association of Glycolipid with Lipoproteins In
Vivo. To evaluate whether glycolipid 3 is able to
spontaneously associate with plasma lipoproteins upon
injection prior to elimination by the ASGPr, the tyrosine
moiety of glycolipid 3 was radioiodinated, which allowed
for kinetic studies in mice. The serum decay of glycolipid
3 upon iv injection (3.33 mg/kg) in C57BL/6J mice was
relatively slow (t1/2 ∼30 min) and could be mainly
attributed to a gradually increasing uptake by the liver
(44 ( 3% at 60 min after injection) (Figure 4A). At 60
min after injection, glycolipid 3 was almost completely
(>95%) associated with HDL (1.063 < d < 1.21 g/mL),
as can be appreciated from the overlapping [125I]gly-
colipid and HDL-cholesterol profiles (Figure 4B). Ap-
parently, the affinity of the bile acid anchoring moiety
of the glycolipid for lipoproteins is sufficiently high to
overcome rapid elimination of the glycolipid by the liver.
Instead, the glycolipid selectively accumulated in lipo-
proteins, which thus enables the subsequent delivery
of lipoproteins to hepatocytes.
or HDL (54.9 ( 1.3% protein, 2.2 ( 0.4% triglycerides,
22.9 ( 1.2% phosphatidylcholine, 17.7 ( 1.0% choles-
teryl esters, and 2.4 ( 0.4% free cholesterol), as deter-
mined by incubation of isolated lipoproteins with the
glycolipid and subsequent reisolation of lipoproteins by
density gradient ultracentrifugation.
Liver Uptake of Glycolipid/Lipoprotein Com-
plexes. We have recently shown that the novel trian-
tennary GalNAc-terminated cluster glycoside 13 has a
50-fold increased affinity toward the ASGPr on hepa-
tocytes as compared to triantennary galactose-termi-
nated glycosides (Ki 2 vs 100 nM).18 We now evaluated
the effect of the increased affinity of glycolipids 3 over
2 on the induction of the hepatic uptake of lipoproteins.
Upon intravenous injection into C57BL/6J mice, the
hepatic uptake of [125I]LDL and [3H]HDL was negligible
(<1% of the dose at 10 min after injection) (Figure 3A).
Prior incubation of LDL or HDL with the novel gly-
colipid 3 caused a dose-dependently enhanced liver

Novel N-Acetylgalactosamine-Terminated Glycolipids
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5803
reached only after 48 h after injection. For clinical
application of glycolipids in hyperlipidemia, glycolipids
should be able to show consistent effects upon repeated
administration. Importantly, three consecutive injec-
tions of glycolipid 3 mice at 72 h time intervals resulted
in consistent reductions in total cholesterol levels
(Figure 5B). The glycolipid showed no sign of toxicity
at the applied dose as compared to saline injection. At
3 or 24 h after iv injection into mice, no effects were
observed on the weight of several organs (liver, heart,
kidneys, spleen) and plasma levels of enzymes that
would indicate systemic toxicity (lactate dehydrogenase)
or liver toxicity (alanine aminotransferase, aspartate
aminotransferase, γ-glutamyl transferase). In addition,
the glycolipid did not cause hemolysis after incubation
with heparinized blood at concentrations equivalent to
doses of 100 mg/kg (not shown).
In conclusion, we have prepared glycolipid 3 with an
unusually high affinity and specificity for the hepatic
ASGPr. The glycolipid is capable of associating with
plasma lipoproteins and thereby induces the uptake of
LDL and HDL by hepatocytes, which are able to secrete
excess cholesterol from the body via the bile as neutral
sterols and bile acids, rather than by Kupffer and splen-
ic cells. In this respect, the glycolipid presented here is
superior to the latest glycolipid introduced by our group
for hepatocyte-directed targeting purposes.17 Impor-
tantly, injection of glycolipid 3 resulted in a prolonged
and repeated cholesterol-lowering in a mouse model of
familial hypercholesterolemia. This advantageous prop-
erty of glycolipid 3 opens the way for the employment
of glycolipids for cholesterol-lowering therapy in those
hypercholesterolemic patients who do not respond to
conventional cholesterol-lowering therapies.
Figure 4. Association of the glycolipid with lipoproteins in
plasma in vivo. [125I]Glycolipid 3 (75 µg) was injected into the
vena cava inferior of anaesthetized C57Bl/6J mice, and the
liver uptake and serum decay were determined. Values are
means ( variation of two experiments (A). At 60 min after
injection, the mice were bled, and serum samples (100 µL;
circles) or pure [125I]glycolipid 3 (5 µg; triangles) were subjected
to density gradient ultracentrifugation. The gradients were
subdivided from top (fraction 1) to bottom (fraction 24), and
the fractions were assayed for 125I-activity (b, 2) and total
cholesterol (TC) (O) (B).
Experimental Section
General. Pyridine, N,N-dimethylformamide (DMF), 1,2-
dichloroethane (DCE), dichloromethane (DCM), 1,4-dioxane,
ethanol, tetrahydrofuran (THF), toluene, N,N-diisopropylethy-
lamine (DIPEA), trifluoroacetic acid (TFA), piperidine, tri-
ethylamine (Et3N), and methanol were from Biosolve (Valk-
enswaard, The Netherlands). Methanol was stored over 3 Å
molecular sieves, and DMF, DCE, DCM, and THF were stored
over 4 Å molecular sieves. Trimethylsilyl trifluoromethane-
sulfonate (TMSOTf, Fluka), triphenylphosphine (Acros), N-
Boc-L-glycine (NovaBiochem), (benzotriazol-1-yloxy)tris(dim-
ethylamino)phosphonium hexafluorophosphate (BOP, Nova-
Biochem), 1-hydroxybenzotriazole (HOBt, NovaBiochem),
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluo-
roborate (TBTU, NovaBiochem), N-Fmoc-O-tert-butyl-L-ty-
rosine (NovaBiochem); mesyl chloride (Merck); sodium azide
(Acros); and 1,3-dipropanedithiol (Aldrich) were used as
received. Before use oleoyl chloride (Fluka) was distilled under
reduced pressure to afford a colorless oil. [1R,2R-3H]cholesteryl
oleate and 125I (carrier-free) were from Amersham Biosciences
(Little Chalfont, UK). Asialoorosomucoid (ASOR) was prepared
by enzymatic desialylation (approximately 70%, as judged by
the extent of sialic acid release) of human R1-acid glycoprotein
(orosomucoid) from Cohn Fraction VI (99%; Sigma) as de-
scribed.29 All other chemicals were of analytical grade. Merck
Kieselgel 60 F254 DC Alufolien was used for TLC analysis.
Compounds were visualized with UV light (254 nm). Carbo-
hydrate compounds were visualized by charring with sulfuric
acid/ethanol (1/4, v/v) at ∼150 °C and bile acid esters30 by
charring with MnCl2 at ∼150 °C. Compounds containing NH
groups were visualized after treatment of the plates with
chlorine, spraying with a solution of TDM,31 and subsequent
heating to ∼150 °C or by spraying with a solution of ninhy-
drine in acetic acid/water and subsequent heating. Column
Figure 5. Hypocholesterolemic effect of the glycolipid in mice.
Glycolipid 3 (20 mg/kg; b) or PBS (O) was injected via the tail
vein into ldlr-/- mice, and plasma samples were taken at the
indicated times (A). Subsequently, mice received two additional
injections of glycolipid 3 (closed bars) or PBS (open bars) at
72 h intervals, and additional plasma samples were taken at
6 h after injection (B). Total cholesterol levels in the plasmas
were determined and expressed as percentage of control
values. The initial cholesterol values (100%) in plasma samples
taken before injection of glycolipids were 37.9 ( 3.6 mmol/L.
Values are means ( SEM (n ) 6). *P < 0.05, **P < 0.01
Cholesterol-Lowering Effect of Glycolipids. Since
glycolipid 3 appeared superior to glycolipid 2 with
respect to its ability to target lipoproteins specifically
to hepatocytes, the ability of glycolipid 3 to reduce
plasma cholesterol (i.e. lipoprotein) levels was evaluated
in hyperlipidemic ldlr-/- mice as a model of familial
hypercholesterolemia. Upon iv injection of glycolipid 3
into ldlr-/- mice, a marked lipoprotein-lowering effect
was detected (Figure 5A). The effect peaked already at
3-6 h (20%; P < 0.01) and was still significant (P <
0.05) up to 12 h after injection. Baseline levels were

5804 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Rensen et al.
chromatography was performed with Kieselgel 60, 230-400
mesh (Merck). Gel filtration was performed with Sephadex LH-
20 (Pharmacia). 13C{1H} NMR spectra (75 MHz) and 1H NMR
spectra (300 MHz) were recorded using a Bruker WM-300
spectrometer. Chemical shifts are given in ppm (δ) relative to
tetramethylsilane as an internal standard. Fast atom bom-
bardment (FAB) mass spectrometry was carried out using a
JEOL JMS SX/SX102A four-sector mass spectrometer, coupled
to a JEOL MS-MP7000 data system.
2-(2-(2-Azidoethoxy)ethoxy)ethanol (5). 2-(2-(2-Chloro-
ethoxy)ethoxy)ethanol 4 (10 g, 59.3 mmol) was dissolved in
dry ethanol (50 mL), and sodium iodide (0.9 g, 6 mmol) and
sodium azide (4.55 g, 70 mmol; preactivated with hydrazine
monohydrate) were added. The resulting mixture was refluxed
for 5 days until TLC analysis (DCM/methanol 9/1, v/v) showed
one product. The reaction mixture was filtered (salts) and
concentrated. The residue was dissolved in DCM (50 mL) and
stored for 16 h at 4 °C. After filtration and concentration,
compound 5 was obtained as clear oil. Yield: 6.42 g (36.6 mmol,
62%). 13C{1H} NMR (CDCl3): δ 50.0 (CH2-N3), 60.6 (CH2OH),
69.2, 69.5, 69.8 (3 × CH2O). 1H NMR (CDCl3): δ 3.44 (m, 2H,
CH2N3), 3.68 (m, 10H, CH2O).
mmol), N-tert-butoxycarbonyl-glycine (N-BocGlyOH) (306 mg,
1.73 mmol), and BOP (920 mg, 2.08 mmol) were dissolved in
DCE (15 mL), and acylation was started by addition of DIPEA
(0.72 mL, 4.15 mmol). The resulting mixture was stirred
overnight at 20 °C. According to TLC analysis (DCM/methanol
9/1, v/v), the reaction was complete. The mixture was taken
up in DCM (50 mL); washed with H2O (50 mL), NaHCO3 (1
M, 50 mL), and brine (50 mL); dried (MgSO4); and concen-
trated. The resulting oil was applied to a silica gel column,
using ethyl acetate/hexane (1/2 f 1/0, v/v) as eluent, affording
compound 10. Yield: 0.90 g (1.61 mmol, 93%). 13C{1H} NMR
(CDCl3): δ 14.0 (CH3 OEt), 28.1 (CH3 Boc), 34.7 (CH2CdO),
44.2 (CH2 Gly), 59.6 (Cq tris), 60.2 (OCH2CH3), 66.6 (OCH2),
68.9 (CH2 tris), 80.5 (Cq Boc), 156.4 (CdO Boc), 169.1 (CdO
1
ester), 171.4 (CdO amide). H NMR (CDCl3): δ 1.26 (t, 9H,
CH3 ethyl), 1.45 (s, 9H, CH3 Boc), 2.03 (d, 2H, CH2 Gly, JH-NH
) 0.6 Hz), 2.53 (t, 6H, CH2CdO, J ) 6.2 Hz), 3.70 (m, 12 H,
OCH2), 4.13 (dq, 6H, OCH2, J ) 7.1 Hz), 5.38 (bs, 1H, NH Boc),
6.39 (d, 1H, NH Gly).
N-(tert-butoxycarbonylglycinyl)tris(carboxyethoxy-
methyl)aminomethane (11). The triester 10 (0.85 g, 1.52
mmol) was dissolved in a mixture of 1,4-dioxane (90 mL) and
water (27 mL), and aqueous NaOH (4 M, 3 mL) was added.
After stirring for 16 h, TLC analysis (DCM/methanol/acetic
acid 18/2/1, v/v/v) showed complete conversion of the starting
material into one product. The mixture was carefully neutral-
ized by the addition of Dowex (50 W x 8, H+ form). The mixture
was filtered, and the filtrate concentrated. The residue was
coevaporated with toluene (2 × 10 mL) and purified by means
of a silica gel column using DCM/methanol/acetic acid (100/
0/1 f 95/5/1, v/v/v) as eluent. Yield: 0.75 g (1.52 mmol, 100%).
13C{1H} NMR (CDCl3): δ 28.7 (CH3 Boc), 36.7 (CH2CdO), 44.9
(CH2 Gly), 61.2 (Cq tris), 68.2 (OCH2), 70.0 (CH2 tris), 80.7 (Cq
Boc), 156.5 (CdO Boc), 172.2 (CdO amide), 176.0, 176.3 (CdO,
2-(2-(2-Azidoethoxy)ethoxy)ethyl 2-Acetamido-3,4,6-
tri-O-acetyl-2-deoxy-â-D-galactopyranoside (7). The known
oxazoline 625 (1.04 g, 3.0 mmol) and 2-(2-(2-azidoethoxy)-
ethoxy)ethanol 5 (0.80 g, 4.6 mmol) were dissolved in 1,2-
dichloroethane (DCE) (10 mL). Molecular sieves (4 Å) were
added and the mixture was stirred for 30 min. Then, TMSOTf23
(0.27 mL, 1.5 mmol) was added, and the reaction was stirred
overnight. According to TLC analysis (DCM/methanol 9/1, v/v),
the oxazoline 6 was completely converted into a lower running
product. The mixture was filtered over Hyflo. The filtrate was
taken up in DCM (50 mL), washed with aq NaHCO3 (1 M, 50
mL) and water (50 mL), dried over MgSO4, and concentrated
to an oil. The crude product was purified by silica gel column
chromatography, with DCM/methanol (1/0 f 94/6, v/v) as
eluent. Yield: 1.29 g (2.56 mmol, 85%). 13C{1H} NMR
(CDCl3): δ 20.3 (CH3 Ac), 22.8 (CH3 NHAc), 50.2 (C-2 Gal),
50.3 (CH2-N3), 61.1 (CH2O), 61.3 (C-6 Gal), 68.2-72.2 (4 ×
CH2O), 66.4, 70.2, 70.5 (C-3,4,5 Gal), 101.7 (C-1 Gal), 169.7,
1
carboxyl). H NMR (CDCl3): δ 1.44 (s, 9H, CH3 Boc), 2.09 (s,
2H, CH2 Gly), 2.57 (t, 6H, CH2CdO), 3.71 (bs, 14 H, OCH2),
6.59 (bs, 1H, NH Gly).
N-(tert-butoxycarbonylglycinyl)tris{(1-[2-(2-aminoet-
hoxy)ethoxy]ethoxy-2-acetamido-3,4,6-tetra-O-acetyl-2-
deoxy-â-D-galactosamine)(carboxyethoxymethyl)}meth-
ane (12). To a solution of the tricarboxylate 11 (74 mg, 149
µmol) and the crude amine 8 (<622 µmol) in DMF (5 mL) were
added DIPEA (188 µL, 1.08 mmol), HOBt (73 mg, 0.54 mmol),
and TBTU (173 mg, 0.54 mmol). The mixture was stirred
overnight, after which an extra amount of DIPEA (188 µL, 1.08
mmol) was added and the mixture was stirred again for
another 18 h. According to TLC analysis (DCM/methanol 4/1,
v/v), the reaction was complete. The reaction mixture was
taken up in DCM (40 mL); washed with dilute H3PO4 (1 M,
40 mL), aq NaHCO3 (1 M, 40 mL), and water (40 mL); dried
(MgSO4); and concentrated to an oil. This oil was applied to a
silica gel column, and elution was performed with DCM/
methanol (1/0 f 84/16, v/v). The crude product thus obtained
was further purified by Sephadex LH20 gel filtration, using
DCM/methanol (1/1, v/v) as eluent. Yield: 161 mg (86 µmol,
58%). 13C{1H} NMR (CDCl3): δ 20.3 (CH3 Ac), 22.7 (CH3
NHAc), 27.9 (CH3 Boc), 34.1, 36.1, 38.8 (CH2CdO), 43.1 (CH2
Gly), 50.2 (C-2 Gal), 51.3 (CH2-NH), 59.4 (CH2O), 61.2 (C-6
Gal), 66.4, 70.1 (C-3,4,5 Gal), 67.0 - 70.0 (CH2O), 79.2 (Cq, Boc),
101.0 (C-1 Gal), 155.7 (CdO Boc), 169.5, 169.9, 170.6 (CdO
1
170.1 (CdO). H NMR (CDCl3): δ 1.99, 2.00, 2.05 (3 × s, 9H,
CH3, Ac), 2.16 (s, 3H, CH3, NHAc), 3.48 (t, 2H, CH2N3), 3.61-
3.76 (m, 8H, CH2O), 3.89 (m, 1H, H-5 Gal), 3.90 (t, 2H, CH2O),
4.15 (t, 2H, H-6a, H-6b Gal), 4.22 (m, 1H, H-2 Gal, JN-2 ) 9.3
Hz), 4.78 (d, 1H, H-1 Gal, J1-2 ) 8.6 Hz), 5.06 (dd, 1H, H-3
Gal, J3-4 ) 3.3 Hz, J3-2 ) 11.2 Hz), 5.32 (d, 1H, H-4 Gal), 6.15
(d, 1H, NH).
2-(2-(2-Aminoethoxy)ethoxy)ethyl 2-Acetamido-3,4,6-
tri-O-acetyl-2-deoxy-â-D-galactopyranoside (8). The azide
7 (314 mg, 622 µmol) was dissolved in THF (10 mL), and
triphenylphosphine (196 mg, 746 µmol) was added. The
mixture was stirred for 48 h, until TLC analysis (toluene/
methanol 1/1, v/v) showed the absence of starting material.
Water (32 µL, 1.8 mmol) was added to the reaction, and
stirring was continued for another 24 h. TLC analysis (toluene/
methanol 1/1, v/v and 2-propanol/water 4/1, v/v) showed one
product that was both H2SO4- and ninhydrine-positive. Tri-
fluoroacetic acid (TFA, 96 µL, 1.25 mmol) and toluene (10 mL)
were added, the mixture was concentrated to near dryness and
finally coevaporated with toluene (2 × 10 mL). The crude
amine 8 thus obtained was used immediately for the synthesis
of the protected cluster 12. 13C{1H} NMR (CDCl3): δ 20.2 (CH3
Ac), 22.6 (CH3 NHAc), 42.4 (CH2NH2), 49.9 (C-2 Gal), 61.3 (C-6
Gal), 68.5-70.2 (CH2O), 66.5, 69.9 (C-3,4,5 Gal), 101.1 (C-1
Gal), 169.6, 169.8, 169.9 (CdO), 170.9 (CdO, NHAc). 1H NMR
(CDCl3): δ 1.88, 1.94, 2.03 (3 × s, 9H, CH3, Ac), 2.09 (s, 3H,
CH3, NHAc), 3.20 (m, 2H, CH2NH2), 3.47 (m, 4H, 2 × CH2O),
3.61 (t, 2H, CH2O), 3.66 (t, 2H, CH2O), 3.75 (m, 2H, CH2O),
3.91 (m, 2H, H-5 Gal, NH), 4.11 (m, 3H, H-2, H-6a, H-6b Gal),
4.87 (d, 1H, H-1 Gal, J1-2 ) 8.5 Hz), 5.20 (dd, 1H, H-3 Gal,
J3-4 ) 3.3 Hz, J3-2 ) 11.2 Hz), 5.34 (d, 1H, H-4 Gal).
1
ester, amide), 171.3 (CdO NHAc). H NMR (CDCl3): δ 1.44
(s, 9H, CH3 Boc), 1.96, 1.99, 2.05 (3 × s, 27H, CH3 Ac), 2.09 (s,
2H, CH2 Gly), 2.15 (s, 9H, CH3 NHAc), 2.43, 2.55 (m, 6H,
CH2CdO), 2.97, 3.05, 3.25 (m, 6H, CH2NH), 3.46-3.94 (m, 24H,
OCH2, H-5 Gal), 4.13 (m, 9H, H-6a, H-6b, H-2 Gal), 4.79 (d,
3H, H-1 Gal), 5.21 (dd, 3H, H-3 Gal), 5.34 (d, 3H, H-4 Gal, J4,3
) 3.0 Hz).
Glycinyltris{(1-[2-(2-aminoethoxy)ethoxy]ethoxy-2-ac-
etamido-3,4,6-tetra-O-acetyl-2-deoxy-â-D-galactosamine)-
(carboxyethoxymethyl)}methane (13). The fully protected
derivative 12 (155 mg, 83 µmol) was dissolved in TFA/DCM
(1/4, v/v; 5 mL). After the mixture was stirred for 60 min,
toluene (20 mL) was added. The mixture was concentrated and
the residue coevaporated with toluene (3 × 10 mL). According
N-(tert-butoxycarbonylglycinyl)tris(carboxyethoxy-
methyl)aminomethane triethyl ester (10). Tris(carboxy-
ethoxyethyl)aminoethane triethyl ester 917 (729 mg, 1.73

Novel N-Acetylgalactosamine-Terminated Glycolipids
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5805
to TLC analysis (DCM/methanol 9/1, v/v), the starting material
was completely converted into a lower running product. The
thus obtained crude 13 was used without prior purification
for the synthesis of compound 21. 13C{1H} NMR (CDCl3): δ
20.2 (CH3 Ac), 22.5 (CH3 NHAc), 34.3, 35.9, 38.9 (CH2CdO),
44.7 (CH2 Gly), 50.1 (C-2 Gal), 51.4 (CH2-N3), 60.2, 66.4, 67.0,
68.5, 69.8 (CH2O), 61.3 (C-6 Gal), 66.5, 70.3 (C-3,4,5 Gal), 101.2
(C-1 Gal), 170.2, 170.4 (CdO ester, amide), 171.9 (CdO NHAc).
added. After the mixture had been stirred for 3 h, TLC analysis
(hexane/diethyl ether 2/3, v/v) revealed complete conversion
of starting material. The reaction mixture was taken up in
DCM (40 mL), washed with aq NaHCO3 (1 M, 40 mL) and
water (40 mL), dried (MgSO4), and concentrated to an oil. This
oil was applied to a silica gel column, and elution was
performed with hexane/diethyl ether (4/1 f 3/1, v/v). Yield:
225 mg (344 µmol, 86%). 13C{1H} NMR (CDCl3): δ 11.7 (CH3
C18), 13.7 (CH3 oleoyl), 17.9 (CH3 C21), 20.8 (CH2), 23.5 (CH3
C19), 22.4, 23.9, 24.7, 24.8, 25.9, 26.2, 26.9, 27.8, 28.8, 28.9,
29.1, 29.3, 29.4, 29.5, 30.4, 30.5, 30.7, 31.6, 34.3, 34.5 (CH2),
35.0 (CH C20), 35.4 (CH C8), 37.1 (CH C9), 39.6 (CH C5), 39.9
(Cq C10), 42.4 (Cq C13), 50.8 (OCH3), 55.7 (CH, C17), 56.2 (CH
C14), 69.8 (CH C3 CH-NH), 129.3, 129.5 (CdCH, oleoyl), 172.4,
173.7 (CdO). 1H NMR (CDCl3): δ 0.65 (s, 3H, CH3 C18), 0.83-
0.92 (m, 6H, CH3 C21, oleoyl, 0.96 (s, 3H, CH3 C19), 1.08-
1.16 (m, 8H, CH2), 1.26-1.43 (m, 30H, CH2), 1.52-1.73 (m,
8H, CH2), 1.75-2.05 (m, 7H, CH), 2.17-2.38 (m, 4H, CH2CdO),
3.66 (s, 3H, OCH3), 5.07 (bs, 1H, NH), 5.34 (m, 2H, CdCH
oleoyl).
Lithocholic Acid Methyl Ester (15).32 Lithocholic acid
(3.77 g, 10 mmol) was dissolved in methanol (25 mL). Con-
centrated HCl (0.5 mL, 36-38%) was added, and the solution
was heated at reflux temperature (65 °C). After 30 min, TLC
analysis (toluene/ethanol 85/15, v/v; MnCl2 staining) showed
that the ester formation was complete. The reaction mixture
was cooled overnight at 0 °C. The crystallized solid was
collected by filtration and washed with cold methanol (25 mL).
The white solid 15 was dried at room temperature. Yield: 2.97
g (7.6 mmol, 76%). 13C{1H} NMR (CDCl3): δ 11.9 (CH3 C18),
18.1 (CH3 C21), 20.7 (CH2), 23.3 (CH3 C19), 24.1, 26.3, 27.1,
28.0, 30.4, 30.8, 30.9, 34.4, 35.3 (CH2), 35.2 (CH C20), 35.7
(CH C8), 40.0 (Cq C10), 40.3 (CH C9), 42.0 (CH C5), 42.6 (Cq
C13), 51.4 (OCH3), 55.8 (CH C17), 56.3 (CH C14), 71.6 (CHOH
3-Oleoylamidolithocholic Acid (20). The methyl ester 19
(200 mg, 306 µmol) was dissolved in a mixture of 1,4-dioxane
(30 mL) and water (9 mL), and deprotection was started by
adding aq NaOH (4 M, 1 mL). The mixture was stirred
overnight. TLC analysis (hexane/diethyl ether 2/3, v/v) re-
vealed that the starting material was completely converted
into a product with zero mobility. The mixture was carefully
neutralized by the addition of Dowex (50 W x 8, H+ form) and
then filtered. The filtrate was concentrated to a small volume
and applied to a silica gel column. Elution with hexane/diethyl
ether (1/0 f 1/1, v/v) furnished pure 20 as a white solid.
Yield: 196 mg (306 µmol, >99%). 13C{1H} NMR (CDCl3): δ
12.0 (CH3 C18), 14.1 (CH3 oleoyl), 18.2 (CH3 C21), 20.8 (CH2),
23.3 (CH3 C19), 22.7, 24.2, 24.7, 25.1, 26.3, 26.7, 27.0, 27.2,
28.1, 29.1, 29.3, 29.5, 29.7, 30.8, 31.0, 31.9, 32.3, 34.0, 34.6,
34.8, 35.0 (CH2), 35.3 (CH C20), 35.8 (CH C8), 40.1 (Cq C10),
40.4 (CH, C9), 41.9 (CH C5), 42.7 (Cq C13), 56.0 (CH, C17),
56.5 (CH C14), 74.1 (CH C3 CH-NH), 129.7, 130.0 (CdCH
1
C3), 174.7 (CdO C24). H NMR (CDCl3): δ 0.64 (s, 3H, CH3
C18), 0.90 (s, 3H, CH3 C21), 0.92 (s, 3H, CH3 C19), 0.97-1.98
(m, 27H, CH, CH2), 2.28 (m, 2H, CH2CdO C23), 3.33 (bs, 1H,
OH), 3.66 (s, 3H, OCH3).
3-Azidolithocholic Acid Methyl Ester (17). Lithocholic
acid methyl ester 15 (0.78 g, 2.0 mmol) was dissolved in DCE
(10 mL) and concentrated. Next, the methyl ester was dis-
solved in pyridine (20 mL) and cooled to 0 °C, and following
addition of mesyl chloride (186 µL, 2.4 mmol), the resulting
mixture was allowed to warm to room temperature. After
incubation for 3 h, TLC analysis (toluene/ethanol 85/15, v/v;
MnCl2 staining) showed complete disappearance of the starting
compound 15. The reaction mixture was concentrated and
coevaporated with toluene (3 × 10 mL). The intermediate
compound 16 was dissolved in DMF (20 mL), sodium azide
(156 mg, 2.4 mmol) was added, and the mixture was subse-
quently heated overnight at 100 °C. The reaction mixture was
concentrated in vacuo and taken up in DCM (30 mL). The
organic layer was washed with water (20 mL), NaHCO3 (1 M,
20 mL), and brine (20 mL); dried (MgSO4); and concentrated.
The crude brown solid was recrystallized from DCM/hexane,
affording a white solid. Yield: 0.67 g (1.62 mmol, 81%). 13C{1H}
NMR (CDCl3): δ 11.8 (CH3 C18), 18.1 (CH3 C21), 20.8 (CH2),
23.6 (CH3, C19), 23.9, 24.5, 26.1, 26.3, 28.0, 30.0, 30.5, 30.8,
34.7 (CH2), 35.1 (CH C20), 35.4 (CH C8), 37.1 (CH, C9), 39.9
(CH C5), 40.0 (Cq C10), 42.5 (Cq C13), 51.2 (CH, C17), 55.8
(CH C14), 56.4 (OCH3), 58.5 (CH C3 CH-N3), 174.4 (CdO C24).
1H NMR (CDCl3): δ 0.65 (s, 3H, CH3 C18), 0.90 (d, 3H, CH3
C21 J ) 6.4 Hz), 0.95 (s, 3H, CH3 C19), 1.03-2.00 (m, 27H,
CH, CH2), 2.28 (m, 2H, CH2CdO C23), 3.66 (s, 3H, OCH3).
3-Aminolithocholic Acid Methyl Ester (18). The azido
derivative 17 (0.15 g, 0.36 mmol) was dissolved in methanol
(1.8 mL) to a concentration of 0.2 M. Et3N (0.25 mL, 1.8 mmol)
and 1.3-propanedithiol (0.18 mL, 1.8 mmol) were added, and
the resulting mixture was heated at 60 °C for 2 days. TLC
analysis (hexane/diethyl ether 3/1, v/v) established that the
conversion was complete. After addition of toluene (2 mL) the
reaction mixture was concentrated and coevaporated with
toluene (3 × 10 mL), resulting in a yellowish solid. The crude
product was purified by means of silica gel chromatography
using hexane/diethyl ether (5/1 f 1/1, v/v) as eluent, affording
18 as a white solid. Yield: 101 mg (0.26 mmol, 72%). 13C{1H}
NMR (CDCl3): δ 11.7 (CH3 C18), 18.0 (CH3 C21), 20.8 (CH2),
23.6 (CH3 C19), 23.9, 26.0, 26.4, 27.5, 28.9, 29.6, 30.7, 33.2,
34.7, 36.6 (CH2), 35.0 (CH C20), 35.3 (CH C8), 36.2 (CH C9),
39.5 (CH C5), 39.9 (Cq C10), 42.4 (Cq C13), 51.1 (OCH3), 55.7
(CH C17), 56.3 (CH C14), 66.5 (CH C3 CH-NH2), 174.4 (CdO
C24).
1
oleoyl), 179.9, 180.4 (CdO). H NMR (CDCl3): δ 0.65 (s, 3H,
CH3 C18), 0.86-0.91 (m, 9H, CH3), 1.07-1.11 (m, 8H, CH2),
1.26-1.30 (m, 30H, CH2), 1.61-1.63 (m, 8H, CH2), 1.80-1.83
(m, 2H, CH), 2.00-2.05 (m, 5H, CH), 2.23-2.37 (m, 4H,
CH2CdO), 5.35 (m, 2H, CdCH oleoyl).
O-tert-Butyl-N-Fmoc-tyrosinyl-glycinyl-tris{(1-[2-(2-
aminoethoxy)ethoxy]ethox y-2-acetamido-3,4,6-tetra-O-
acetyl-2-deoxy-â-D-galactosamine)(carboxyethoxymeth-
yl)}methane (21). The crude amine 13 (155 mg, <83 µmol)
and N-FmocTyr(tBu)OH (57 mg, 125 µmol) were dissolved in
DMF (10 mL). To the solution were added DIPEA (30 µL, 170
µmol), HOBT (17 mg, 125 µmol), and TBTU (40 mg, 125 µmol),
and the mixture was stirred for 3 h until TLC analysis (DCM/
methanol 85/15, v/v) indicated the reaction to be complete. The
reaction mixture was taken up in DCM (40 mL); washed with
dilute H3PO4 (1 M, 40 mL), aq NaHCO3 (1 M, 40 mL), and
water (40 mL); dried (MgSO4); and concentrated to an oil. This
oil was applied to a silica gel column, and elution was
performed with DCM/methanol (1/0 f 88/12, v/v), followed by
Sephadex LH-20 gel filtration, using DCM/methanol (1/1, v/v)
as eluent. Yield: 129 mg (58 µmol, 70%). 13C{1H} NMR
(CDCl3): δ 20.4 (CH3 Ac), 22.8 (CH3 NHAc), 28.5 (CH3 tBu),
34.4, 36.4, 39.0 (CH2CdO), 37.7 (CH2 Tyr), 43.1 (CH2 Gly), 50.3
(C-2 Gal), 51.6 (CH2-NH), 56.2 (CH Tyr), 59.9 (CH2O), 61.4
(C-6 Gal), 66.7, 70.4 (C-3,4,5 Gal), 66.9-70.3 (CH2O), 79.2 (Cq
tBu), 101.4 (C-1 Gal), 119.8, 124.9, 126.9, 127.6 (CH Fmoc),
124.0, 129.6 (CH Tyr), 131.3, 154.1 (Cq Tyr Fmoc), 141.1, 143.6
(Cq Fmoc), 155.9 (CdO Fmoc), 170.3, 170.5, 170.6 (CdO ester,
1
amide), 171.6 (CdO NHAc). H NMR (CDCl3): δ 1.28 (s, 9H,
CH3 tBu), 1.94, 1.98, 2.02 (3 × s, 27H, CH3 Ac), 2.07 (s, 2H,
CH2 Gly), 2.13 (s, 9H, CH3 NHAc), 2.41-2.53 (m, 6H, CH2CdO),
2.92-3.05 (m, 6H, CH2NH), 3.14 (m, 2H, âCH2 Tyr), 3.59-
3.92 (m, 45H, OCH2, H-5 Gal), 4.14 (m, 9H, H-6a, H-6b, H-2
Gal), 4.36 (m, 2H, RCH Tyr, CH Fmoc), 4.49 (m, 2H, OCH2
Fmoc), 4.74 (d, 3H, H-1 Gal J ) 8.3 Hz), 5.18 (dd, 3H, H-3 Gal
3-Oleoylamidolithocholic Acid Methyl Ester (19). The
amine 18 (156 mg, 400 µmol) was dissolved in dry DCE (10
mL). The solution was cooled to 0 °C, and triethylamine (88
µL, 0.60 mmol) and oleoyl chloride (0.20 mL, 0.60 mmol) were

5806 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Rensen et al.
J3-2 ) 10.5 Hz), 5.33 (d, 3H, H-4 Gal), 6.86-7.11 (4H, CH Tyr),
7.30-7.76 (8H, CH Fmoc).
coevaporated with toluene (2 × 10 mL). The crude product 24
was applied to a silica gel column, using DCM/methanol (1/0
f 82/18, v/v) as eluent. Yield: 27 mg (11 µmol, 91%). 13C{1H}
NMR (CDCl3): δ 11.8 (C18 LCO), 13.9 (CH3 oleoyl), 18.0 (C21
LCO), 20.4 (CH3 Ac), 22.4 (CH3 NHAc), 23.7 (C19 CH LCO),
23.9, 24.9, 25.4, 25.9, 26.3, 27.0, 27.9, 28.9, 29.1, 29.3, 29.5,
30.5, 31.7, 32.9 (CH2 LCO), 34.6, 36.1, 39.3 (CH2CdO), 35.4
(C20 CH LCO), 37.2 (C8 CH LCO), 37.9 (CH Tyr), 39.6 (C9
CH LCO), 39.9 (C10 Cq LCO), 42.5 (CH2 Gly), 43.1 (C13 Cq
LCO), 50.7 (C-2 Gal), 51.6 (CH2-NH), 54.7, 55.6, 56.3 (C14,
C17 CH LCO, CH Tyr), 57.9, 60.1, 60.5 (CH2O), 61.4 (C-6 Gal),
66.5, 70.4 (C-3,4,5 Gal), 63.4-70.2 (CH2O), 101.2 (C-1 Gal),
115.4 (CH Tyr), 129.5, 129.8, 130.1 (CH Tyr, CdCH oleoyl),
155.7, 159.0 (Cq Tyr), 170.3, 170.5, 172.6, 172.9, 173.5 (CdO).
Compound 24 was dissolved in methanolic sodium metha-
nolate (0.05 M, 2 mL). After overnight stirring, TLC analysis
(DCM/methanol 85/15, v/v and 2-propanol/25% NH4OH 1/1,
v/v) showed complete conversion of the starting material into
a single product (compound 3). The crude deprotected product
thus obtained was purified using a Sephacryl S100 gel filtra-
tion column, with water as eluent. The product fractions were
pooled, concentrated, and lyophilized. Yield: 8.2 mg (3.8 µmol,
32% based on 23). 13C{1H} NMR (CDCl3): δ 12.2 (C18 LCO),
14.2 (CH3 oleoyl), 18.4 (C21 LCO), 23.0 (CH3 NHAc), 24.1 (C19
CH LCO), 21.9, 22.9, 25.4, 26.4, 26.8, 27.4, 28.4, 29.3, 29.5,
29.9, 30.0, 31.0, 32.1, 33.4 (CH2 LCO), 34.0, 35.6, 36.9, 40.4
(CH2CdO), 35.8 (C20 CH LCO), 35.9 (C8 CH LCO), 38.0 (CH
Tyr), 40.2 (C9 CH LCO), 41.5 (CH2, Gly), 41.9 (C10 Cq LCO),
43.5 (C13 Cq LCO), 52.2 (C-2 Gal), 54.3 (CH2-NH), 56.2 (C17
CH LCO), 57.4 (CH Tyr), 57.9 (C14 CH LCO), 60.5 (CH2O),
61.7 (C-6 Gal), 68.6, 72.7, 75.3 (C-3,4,5 Gal), 67.0-70.8 (CH2O),
101.8 (C-1 Gal), 115.6 (CH Tyr), 127.8 (Cq Tyr), 129.9, 130.2
(CdCH oleoyl), 130.5 (CH Tyr), 156.1 (Cq Tyr), 169.8, 172.9,
173.4, 174.4, 175.2 (CdO).1H NMR (CDCl3): δ 0.65 (s, 3H,
C18), 0.87-1.61 (m, 63H, CH3, CH2 LCO), 2.01 (s, 9H, CH3,
NHAc), 2.33 (t, 3H, CH3 LCO), 2.43-2.55 (m, 8H, CH2CdO),
2.93-3.27 (m, 6H, CH2NH), 3.34-3.83 (48H, OCH2, Gal), 4.43
(d, 3H, H-1 Gal, J ) 8.4 Hz), 4.54 (m, 3H, H-5 Gal), 5.05 (m,
3H, H-3 Gal), 5.33 (m, 5H, H-4 Gal, CdCH LCO), 6.69 (d, 2H
CH Tyr), 7.04 (d, 2H, CH Tyr). Mass (FAB+): m/e 2206.6 [M
+ Na]+.
O-tert-Butyl-N-tyrosinylglycinyltris{(1-[2-(2-aminoet-
hoxy)ethoxy]ethoxy-2-acetamido-3,4,6-tetra-O-acetyl-2-
deoxy-â-D-galactosamine)(carboxyethoxymethyl)}meth-
ane (22). The fully protected derivative 21 (120 mg, 54 µmol)
was dissolved in piperidine/THF (5/95, v/v; 10 mL) and the
mixture was stirred for 1 h. TLC analysis (DCM/methanol 85/
15, v/v) revealed complete conversion of starting material into
a single ninhydrine-positive product with lower mobility. Tolu-
ene (10 mL) was added, the mixture was concentrated to a
small volume and coevaporated with toluene (2 × 10 mL). The
residue was purified by means of silica gel column using DCM/
methanol (100/1 f 8/2) as eluent. Yield: 81 mg (41 µmol, 75%).
13C{1H} NMR (CDCl3): δ 20.4 (CH3 Ac), 22.9 (CH3 NHAc), 28.5
(CH3 tBu), 34.3, 36.2, 38.9 (CH2CdO), 37.3 (CH2 Tyr), 44.3
(CH2 Gly), 50.2 (C-2 Gal), 51.4 (CH2-NH), 56.1 (CH Tyr), 59.8
(CH2O), 61.3 (C-6 Gal), 67.1, 70.2 (C-3,4,5 Gal), 66.5-69.9
(CH2O), 78.1 (Cq tBu), 101.2 (C-1 Gal), 124.0, 124.9, (CH Tyr),
131.3, 153.9 (Cq Tyr), 170.1, 170.2, 170.8 (CdO ester, amide),
171.5 (CdO, NHAc). 1H NMR (CDCl3): δ 1.34 (s, 9H, CH3 tBu),
1.96, 1.99, 2.04 (3 × s, 27H, CH3 Ac), 2.09 (s, 2H, CH2 Gly),
2.14 (s, 9H, CH3 NHAc), 2.35-2.58 (m, 6H, CH2CdO), 2.92-
3.05 (m, 6H, CH2NH), 3.25 (m, 2H, âCH2 Tyr), 3.57-4.16 (m,
54H, OCH2, H-5, H-6a, H-6b, H-2 Gal), 4.23 (m, 1H, RCH Tyr),
4.80 (m, 3H, H-1 Gal), 5.20 (m, 3H, H-3 Gal), 5.35 (m, 3H,
H-4 Gal), 6.93 (d, 2H CH Tyr), 7.12 (d, 2H, CH Tyr).
3-Oleoylamidolithocholic Acid [O-tert-Butyl-N-tyrosi-
nylglycinyltris{(1-[2-(2-aminoethoxy)ethoxy]ethoxy-2-
acetamido-3,4,6-tetra-O-acetyl-2-deoxy-â-D-galactosa-
mine)(carboxyethoxymethyl)}methane] (23). Amine 22
(75 mg, 38 µmol) and acid 20 (36 mg, 57 µmol) were dissolved
in DMF (5 mL), and acylation was commenced by addition of
DIPEA (14 µL, 80 µmol), HOBT (8 mg, 56 µmol), and TBTU
(18 mg, 56 µmol). The mixture was stirred overnight after
which TLC analysis (DCM/methanol 85/15, v/v) showed that
the reaction has completed. The reaction mixture was taken
up in DCM (40 mL); washed with dilute H3PO4 (1 M, 40 mL),
aq NaHCO3 (1 M, 40 mL), and water (40 mL); dried (MgSO4);
and concentrated to an oil. This oil was applied to a silica gel
column, and elution was performed with DCM/methanol (1/0
f 88/12, v/v). The crude product thus obtained was further
purified by Sephadex LH20 gel filtration, using DCM/methanol
(1/1, v/v) as eluent. Yield: 80 mg (30 µmol, 80%). 13C{1H} NMR
(CDCl3): δ 11.7 (C18 LCO), 13.8 (CH3 oleoyl), 18.0 (C21 LCO),
20.3 (CH3, Ac), 20.7 (CH2 LCO), 22.8 (CH3, NHAc), 23.5 (C19
CH LCO), 23.8, 24.8, 25.3, 25.8, 26.1, 26.8, 27.8 (CH2 LCO),
28.5 (CH3 tBu), 28.7, 28.8, 28.9, 29.1, 29.3, 29.4, 30.4, 31.3,
31.5, 32.8 (CH2 LCO), 34.5, 36.2, 38.8 (CH2CdO), 35.1 (C20
CH LCO), 35.3 (C8 CH LCO), 37.2 (CH2 Tyr), 39.5 (C9 CH
LCO), 39.8 (C10 Cq LCO), 42.4 (C13 Cq LCO), 43.1 (CH2 Gly),
50.2 (C-2 Gal), 51.3 (CH2-NH), 55.5 (C17 CH LCO), 55.8 (CH,
Tyr), 56.2 (C14 CH LCO), 59.7, 60.2 (CH2O), 61.3 (C-6 Gal),
66.5, 70.0, 70.2 (C-3,4,5 Gal), 67.1-70.1 (CH2O), 77.9 (Cq tBu),
101.2 (C-1 Gal), 123.8, 129.4, 129.6 (CH Tyr, CdCH oleoyl),
131.3, 153.8 (Cq Tyr), 170.0, 170.1, 170.6, 171.4, 173.0 (CdO).
1H NMR (CDCl3): δ 0.63 (s, 3H, C18), 0.88 (s, 3H, CH3 oleoyl),
0.89 (d, 3H, C21 J ) 6.6 Hz), 0.96 (s, 3H, C19), 1.13-1.63 (m,
53H, CH, CH2 LCO), 1.28 (s, 9H, CH3 tBu), 1.95, 1.99, 2.04 (3
× s, 27H, CH3 Ac), 2.08 (s, 2H, CH2 Gly), 2.14 (s, 9H, CH3
NHAc), 2.27-2.54 (m, 10H, CH2CdO), 2.93-3.44 (m, 8H,
CH2NH, âCH2 Tyr), 3.62-3.95 (m, 45H, OCH2, H-5 Gal), 4.13-
Animals. Male C57Bl/6J mice (12-14 week old, 24-28 g)
were obtained from Broekman Instituut BV (Someren, The
Netherlands) and fed ad libitum with standard rat/mouse chow
(SRM-A, Hope Farms). Homozygous LDL-receptor deficient
(ldlr-/-) mice have originally been obtained from the Jackson
Laboratory (Bar Harbor, ME) and are maintained as a colony
at our local housing facility at the Gorlaeus Laboratory. Three
weeks before use, female ldlr-/- mice (20 week old, 25-30 g)
were fed a Western-type diet (Hope Farms) ad libitum for three
weeks. All experiments were approved by the Ethics Commit-
tee on Animal Experiments of the University of Leiden.
Isolation of Lipoproteins. Human LDL and HDL were
isolated from freshly isolated serum of healthy volunteers as
described33 and dialyzed at 4 °C against PBS, 1 mmol/L EDTA,
pH 7.4. Protein concentrations were determined according to
Lowry et al.34 using BSA as a standard.
Glycolipid. The freeze-dried glycolipids 2 and 3 were
dissolved in phosphate-buffered saline (PBS) at a final con-
centration of 50 µg/µL and stored at -80 °C under Argon. Their
stability was routinely checked by TLC (n-butanol/n-propanol/
25% NH4OH/H2O ) 15/40/30/15, v/v/v/v) and subsequent
staining for cholesterol (MnCl2) and sugar (H2SO4) moieties.
4.19 (m, 9H, H-6a, H-6b, H-2), 4.77 (m, 3H, H-1 Gal J1-2
)
8.3 Hz), 5.16 (dd, 3H, H-3 Gal J3-4 ) 2.2 Hz, J3-2 ) 11.3 Hz),
5.35 (m, 5H, CdCH LCO, H-4 Gal), 6.90 (d, 2H CH Tyr), 7.09
(d, 2H, CH Tyr).
Radiolabeling of Lipoproteins and Glycolipids. HDL
was labeled with [3H]cholesteryl oleate (∼25 dpm/ng protein)
and LDL was radioiodinated and purified as described,35 while
glycolipid 3 was radiolabeled by the Iodogen method using a
Iodogen (10 µg)-coated reaction tube. More than 97% of the
radiolabel in [125I]TC-LDL was 10% TCA precipitable, and the
specific 125I-activity was ∼150 dpm/ng of protein. [125I]glycolipid
3 (1300 dpm/ng) migrated as a single band (Rf 0.64) on TLC
(n-butanol/n-propanol/25% NH4OH/H2O ) 15/40/30/15, v/v/v/v).
3-Oleoylamidolithocholic Acid [Tyrosinylglycinyltris-
{(1-[2-(2-aminoethoxy)ethoxy]ethoxy-2-acetamido-2-de-
oxy-â-D-galactosamine)(carboxyethoxymethyl)}meth-
ane] (3). The fully protected glycolipid 23 (32 mg, 12 µmol)
was dissolved in DCM/TFA (4/1, v/v; 5 mL). The mixture was
stirred for 1 h, when TLC analysis (DCM/methanol 85/15, v/v)
revealed the reaction to be complete. The mixture was diluted
with toluene (10 mL), concentrated to a small volume, and
Association of Glycolipids with Lipoproteins. Human
HDL and LDL (5 µg of protein each) were incubated (30 min

Novel N-Acetylgalactosamine-Terminated Glycolipids
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5807
at 37 °C) with 125I-labeled or unlabeled glycolipid 3 in PBS,
pH 7.4. The mixtures were electrophoresed on a 0.75% (w/w)
agarose gel at pH 8.8, and the gels were fixed and dried. Lipids
were visualized by Sudan Black and 125I-activity was moni-
tored using a Packard Instant Imager (Hewlett-Packard Co.,
Palo Alto, CA).
Kinetic Studies in Mice. Mice were anesthetized,17 and
the abdomens were opened. [125I]glycolipid 3 or radiolabeled
lipoproteins, previously incubated with PBS or glycolipid 3 (30
min at 37 °C), were injected via the inferior vena cava. Blood
and liver samples were taken and processed as previously
described.36 When indicated, mice received a preinjection of
ASOR (25 mg/kg) at 1 min before injection of the lipoproteins.
At 60 min after injection of [125I]glycolipid 3, mice were bled,
and 100 µL serum samples were subjected to density gradient
ultracentrifugation.33 Tubes were fractionated (24 × 0.5 mL)
using a Multiprobe 104DT Robotic System (Packard Instru-
ment Co.), and fractions were assayed for 125I-activity and for
cholesterol using the Roche Molecular Biochemicals enzymatic
kit for cholesterol.
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Acknowledgment. This study was supported by a
grant from The Netherlands Heart Foundation (NHS
grant 95.128) and The Netherlands Organization for
Scientific Research (NWO STIGON grant 014-81-102).
We thank Roel H. Fokkens (Institute of Mass Specto-
metry, University of Amsterdam, The Netherlands) for
performing MALDI-TOF mass spectometry, and Mar-
ijke Fro¨lich (Central Clinical Chemical Laboratory,
Leiden University Medical Center, The Netherlands) for
measuring plasma enzymes.
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