New and more efficient multivalent glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes

Article abstract of DOI:10.1016/j.bmc.2011.03.027

New multi-valent, carbohydrate ligands that contain terminal N-acetylgalactosamine (GalNAc) or lactose (Lac) were prepared using a nitrilotriacetic acid (NTA) derivative of l-lysine as scaffold. Tri-valent structures were prepared by attaching an ω-amino

Full text of DOI:10.1016/j.bmc.2011.03.027

Bioorganic & Medicinal Chemistry 19 (2011) 2494–2500
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New and more efficient multivalent glyco-ligands for asialoglycoprotein
receptor of mammalian hepatocytes
Reiko T. Lee a, Mei-Hui Wang b, Wuu-Jyh Lin b, , Yuan C. Lee a,
⇑
⇑
a Department of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA
b Institute of Nuclear Energy Research, Taoyuan 325, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
New multi-valent, carbohydrate ligands that contain terminal N-acetylgalactosamine (GalNAc) or lactose
(Lac) were prepared using a nitrilotriacetic acid (NTA) derivative of -lysine as scaffold. Tri-valent struc-
tures were prepared by attaching an -amino glycoside of GalNAc or Lac to each of the three carboxyl
groups of N -protected N -dicarboxymethyl- -lysine. In addition, a hexa-valent lactoside was synthe-
Received 5 January 2011
Revised 4 March 2011
Accepted 11 March 2011
Available online 15 March 2011
L
x
e
a
L
e
a
sized by attaching N -deprotected trivalent lactoside to each of the carboxyl group of N -(trifluoroace-
tamido)hexanoyl -aspartic acid. Tri-valent GalNAc glycosides and the hexa-valent lactoside had high
L
Keywords:
affinity (dissociation constants approaching nM) for rat hepatocytes. The hexa-valent lactoside, after
Liver-targeting
Tri-valent glyco-ligand
Hexa-valent glyco-ligand
Hepatocytes
e
de-N -protection, was modified with a chelator, diethylenetriaminepentaacetic acid (DTPA), through
which a fluorescent or radioactive tag, such as europium or indium, can be firmly attached. Intravenous
infusion of 111Indium-tagged hexa-valent lactoside to rats and mice resulted in nearly exclusive accumu-
lation of radioactivity in the liver.
Endocytosis
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
and tyronine, respectively. For structures, see Fig. 1), utilizing three
carboxylic acid groups of EE (c-glutamyl-glutamic acid, c-Glu-Glu)
Liver is an important organ for homeostasis that produces many
important biomolecules, including acute-phase proteins. Liver is
also unique in that it is endowed with a powerful carbohydrate-
based receptor, asialoglycoprotein receptor also known as the
Ashwell–Morell receptor (AMR), that is highly specific for galact-
ose- and N-acetylgalactosamine-terminated glycans, and is present
in large numbers on mammalian hepatocyte cell surface.1 Their
efficacy, however, depends on the presence of at least three termi-
nal Gal or GalNAc residues with proper spatial arrangement.2
When terminal Gal residues are suitably arranged in space,
di- and tri-valent Gal-terminated glycans can manifest affinity as
and DD (b-aspartyl-aspartic acid, b-Asp-Asp) as the attachment
sites for amino-terminated GalNAc glycosides. These tri-valent
GalNAc glycosides manifested strong binding affinity to AMR (KD
<10 nM). Merwin et al.5 used 125I-labeled YEE(ahGalNAc)3 linked
to polylysine complexed with the luciferase gene to demonstrate
specific hepatic uptake. Cryoautoradiography showed that the
iodinated glycopeptide complex was preferentially taken up by
hepatocytes rather than by non-parenchymal cells. The same Gal-
NAc ligand, YEE(ahGalNAc)3, was also successfully utilized for
hepatocyte-targeted delivery of antisense material to mask hepatic
mRNA or genomic DNA.6 The examination of biodistribution of
125I-YEE(ahGalNAc)3-labeled material showed that it accumulated
specifically in hepatocytes.7
high as ca. l
M and nM (as KD values), respectively.2 Based on the
chemical and physical information of high-affinity natural ligands
for AMR, we constructed tri-valent GalNAc-containing glycopep-
tides, YEE(ahGalNAc)3 and YDD(GahGalNAc)3 (D, E, G, and Y are
single letter abbreviations for aspartic acid, glutamic acid, glycine,
In this paper, we report synthesis of new multivalent glyco-
ligands based on a single amino acid, lysine, as scaffold. One of
the new ligands has affinity for hepatic AMR higher than
YEE(ahGalNAc)3 and YDD(GahGalNAc)3. In addition, we prepared
lactose (Lac)-containing, hexa-valent ligand, which proved to be
a powerful ligand for AMR that is effective in liver targeting.
3
4
Abbreviations: ah, aminohexyl; AMR, Ashwell–Morell receptor; ASOR, asialo-
orosomucoid; DCM, dicarboxymethyl; DIEA, diisopropylethylamine; DTPA, dieth-
ylenetriaminepentaacetic acid; Gal, D-galactose; GalNAc, N-acetyl-D-galactosamine;
Lac, lactose; NTA, nitrilotriacetic acid; SPECT, single photon emission computed
tomography; TFA, trifluroacetyl or trifluroacetic acid; TLC, thin layer chromatog-
2. Results and discussion
raphy; YDD,
aspartic acid; Z, benzyloxycarbonyl.
L
-tyrosyl-
c
-
L
-glutamyl-
L
-glutamic acid; YEE,
L
-tyrosyl-b-
L
-aspartyl-
L-
2.1. Syntheses of multi-valent glycosides
Tri-valent GalNAc glycosides for liver-targeting in the past used
⇑
Corresponding authors. Tel.: +886
(W.-J.L.); tel.: +1 410 516 7041; fax: +1 410 516 8716 (Y.C.L.).
3
4711400x7008; fax: +886 3
4711416
dipeptides, c-glutamyl-glutamic acid (EE) and b-aspartyl-aspartic
E-mail addresses: wjlin@iner.gov.tw (W.-J. Lin), yclee@jhu.edu (Y.C. Lee).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.027

R. T. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2494–2500
2495
O
GalNAc
Asp. The
a-amino group of Asp will be a convenient handle for
CO
N
H
attaching a fluorescent or other tag in future. Construction of
HexaLac again uses DCC/1-OH-Bt method, which proceeded
smoothly requiring only 10% excess of tri-valent lactoside for each
carboxyl group.
O
O
GalNAc
YEE(ahGalNAc)3
Glu CO NH
GalNAc
Tyr Glu CO NH
O
GalNAc
GalNAc
GalNAc
CO Gly
Asp Gly
N
H
2.2. Binding affinity of various multi-valent glycosides to rat
hepatocyte cell surface
O
N
YDD(GahGalNAc)3
H
N
H
O
Tyr Asp Gly
The hepatocyte binding assay was carried out at 4° in order to
prevent internalization of the surface-bound ligands. In the past,
we have used inhibition assays with 125I-ASOR as a high affinity li-
gand to compare a number of potential ligands side by side, using
hepatocyte suspension,11 isolated liver membrane preparations,12
and solubilized AMR.13 In the present study, we chose intact, iso-
lated hepatocytes, as this would be closest to the in vivo liver
system.
O
O
Gly
CO
N
N
H
GalNAc
Nε-Z-DCM-Lys(GahGalNAc)3
NHZ
H
N
CO Gly
2, Tri(GalNAc)
GalNAc
O
CO
Gly
N
H
GalNAc
Two modifications were made in the present inhibition assay:
(1) Eu-DTPA-labeled ASOR was used instead of 125I-ASOR; (2) rat
primary hepatocytes on collagen-coated surface were used instead
of hepatocyte suspension. Although the sensitivity of detection of
Eu fluorescence is comparable to that of 125I-radioactivity,14 the
former lacks the versatility of 125I that allows measurement to be
made with wet cell pellet in a plastic tube or radioactive counts
trapped on filter discs. After testing the use of Eu fluorescence in
the cell suspension method, using either centrifugation through
an oil layer11 or filtration using MultiScreen 96-well filters of
O
CO
N
N
H
GalNAc
Nε-Z-DCM-Lys(ahGalNAc)3
3
NHZ
H
CO
N
GalNAc
O
CO
O
N
H
GalNAc
COOH
N
Nε-Z-Nα-DCM-Lys
NHZ
0.45 or 1 lm pore (Millipore, Bedford, MA) to collect hepatocytes,
COOH
we came to a conclusion that the method using the surface-immo-
bilized cells would be simpler for the measurement of Eu fluores-
cence than the centrifugation or filtration method. In order to
compare four inhibitors on a same 24-well hepatocyte plate, single
point determinations were made for inhibitors at concentrations
COOH
OH
OH
O
GalNAc =
spanning 5 decades (0.1 M to 1 lM). Experiments were repeated,
HO
NHAc
however, at least twice to assure the reproducibility of the results.
Typical inhibition curves are shown in Figure 3. For the purpose of
direct comparison with the previously reported trivalent GalNAc
Figure 1. GalNAc-containing trivalent structures.
3
4
glycosides, YEE(ah-GalNAc)3 and YDD(G-ah-GalNAc)3 (Fig. 1)
were also included in the test for direct comparison. The IC50 val-
ues obtained are listed in Table 1. The IC50 values of YEE(ah-Gal-
NAc)3 and YDD (G-ah-GalNAc)3 in the present assay were 10 and
20 nM, respectively, as compared to 8 nM for both compounds
determined in the past by inhibition assay using rat hepatocyte
suspension and 125I-ASOR as reference ligand, indicating that the
IC50 values obtained by the both methods are comparable. Table 1
acid (DD), as scaffold.3,4 In the present communication, we used a
single amino acid, lysine, as scaffold. The new scaffold could, not
only be prepared less expensively, but also was easier to use be-
cause of the better solubility. In order to attach glycosides having
amino-terminated aglycon to the scaffold, Lys was first converted
to an NTA (nitrilotriacetic acid) derivative24 by a facile procedure
e
of carboxymethylation of the
a
-amino group of Lys.10 Conjugation
also suggests that N -Z-DCM-Lys(GahGalNAc)3 (2, Fig. 1) has at
least 10-fold higher affinity than all other GalNAc-containing tri-
valent ligands.
e
a
of glycosides to this scaffold, N -Z-N -DCM-Lys (Fig. 1), by the DCC/
1-OH-Bt method in non-aqueous solvents proceeded smoothly to
produce desired tri-valent glycosides. We used a large excess of
glycosides over the carboxyl group (50% excess for GalNAc glyco-
sides and 80–90% excess for lactoside) in order to force the tri-va-
lent conjugation to completion. Although tri-valent glycosides can
be prepared using smaller excess of glycosides over the Lys scaf-
fold, forcing the conjugation to completion helped in the purifica-
tion of the tri-valent products by gel filtration, in that di- and
mono-conjugated products were virtually absent.
2.3. Whole animal experiments with 111In-labeled HexaLac
The efficiency of loading of 111In to DTPA-HexaLac was >98% as
determined by radio-HPLC and was stable for 72 h. The specific
activity of 111In-HexaLac (Fig. 2) was 2.5 ꢀ 1010 Bq/mg. The
SPECT/CT image showed the radioactivity was accumulated specif-
ically in the liver (Fig. 4). There is virtually no uptake in the other
organs. This specific accumulation of radioactivity in the liver was
It is a well-known fact that AMR of all mammalian species thus
far examined binds GalNAc much better than Gal and lactose. For
this reason, whereas tri-valent GalNAc glycosides have been shown
to be useful ligands for liver targeting via AMR, a tri-valent lacto-
side is expected to be a much poorer ligand. Since AMR on the
hepatocyte surface is known to be densely clustered15 we reasoned
that connecting two molecules of trivalent lactoside by a short lin-
ker should improve the affinity tremendously. For this reason, we
synthesized a hexa-valent lactoside, HexaLac (Fig. 2), by attaching
tri-valent lactoside molecule to each of the two carboxyl groups of
ablated in the presence of
a powerful competitor, bovine
asialofetuin. Distribution of 111In in various organs of mice
15 min after iv injection of 111In-HexaLac is shown in Figure 5,
which again showed the almost exclusive accumulation of radioac-
tivity in the liver. These data indicated that 111In-HexaLac pos-
sesses a highly specific liver targeting property, which makes
HexaLac structure to be useful as liver-specific drug-delivery
vehicle that would enhances the therapeutic effect of drugs and re-
duces the unwanted side effects.

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R. T. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2494–2500
O
NHCOR
OH
HO
CONH(CH2)6OLac
N
NH2
O
CONH(CH2)6OLac
CONH(CH2)6OLac
4
O
NHCOR
LacO(CH2)6HNOC
N
H
N
CONH(CH2)6OLac
N
HN
LacO(CH2)6HNOC
CONH(CH2)6OLac
O
LacO(CH2)6HNOC
CONH(CH2)6OLac
HexaLac
OH
OH
HO
OH
O
O
Lac =
OH
O
OH
HO
OH
Figure 2. Formation of HexaLac from trivalent Lac (4).
Table 1
100
80
60
40
20
0
Characterization of cluster glycosides
Compounds
Molecular mass
IC50 (nM)
Calcd
Founda
e
a
N -Z-N DCM-Lys(ah-GalNAc)3 (3)
1303.47
1474.63
1303.7
1474.7
12
1
e
a
N -Z-N DCM-Lys(G-ah-GalNAc)3 (2),
tri(GalNAc)
e
a
N -Z-N DCM-Lys(ah-Lac)3 (4)
TFA-AH-Asp[Lys(ahLac)3]2,
(5, HexaLac)
1666.23
3371.5
1666.8
3371.0
>1000
9
b
YEE(ahGalNAc)3
YDD(G-ahGalNAc)3
Not analyzed
Not analyzed
10
20
c
a
b
c
By ESI.
See Ref. 3.
See Ref. 4.
0.001
0.01
0.1
1
10
100
1000
10000
Inhibitor concentration, nM
Figure 3. Inhibition curves of five multivalent glycol-ligands generated using
e
a
plated rat hepatocytes as described in Methods: d, N -Z-N -DCM-Lys(ah-GalNAc)3
e
a
(3); N, N -Z-N -DCM-Lys(G-ah-GalNAc)3 (2); s, YEE(ah-GalNAc)3; ., YDD(G-G-ah-
GalNAc)3; and h, ‘HexaLac’.
glyco-ligands of equal to or higher affinity to AMR than the previ-
ously synthesized structures.
Of the three newly synthesized high-affinity ligands, that is,
compound 2, tri(GalNAc), compound 3, and HexaLac, compound
2 had the highest affinity, outperforming our previously reported
YEE- and YDD-derivatives. As expected from the fact of lactose
being much less strongly bound by AMR than GalNAc, tri-valent
lactoside was indeed much weaker ligand than the GalNAc
3. Conclusions
Compounds containing NTA(Nitrilotriacetic acid)-group have
been frequently used for immobilized metal affinity chromatogra-
phy (IMAC), such as for capture of His6-tagged proteins with
Ni-complexed NTA materials. One of the efficient methods for
preparation of NTA structure is by di-carboxymethylating the
counterparts, having IC50 value higher than lM (Table 1). However,
conjoining of two trivalent lactoside molecules produced a hexa-
valent lactoside (HexaLac) which possessed IC50 in the nM range.
After modifying HexaLac with DTPA and radiolabelling with 111In,
it was shown to possess excellent liver-targeting activity by both
the whole-body micro-SPET/CT imaging and by the 111In distribu-
tion among major organs. Thus NTA-based multivalent glyco-
a
-amino group of lysine.16,17 Compared to our earlier trivalent
glyco-ligands using dipeptides, c-Glu-Glu or b-Asp-Asp, as scaffold,
our new strategy of using a lysine-based NTA-structure for sugar
conjugation was simpler and more cost effective, and produced

R. T. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2494–2500
2497
prepared as described previously. Orosomucoid (
a1-acid glycopro-
tein) was desialylated to produce asialo-orosomucoid (ASOR) as
described.13 Rat primary hepatocytes on collagen-coated, 24-well
plates and hepatocyte basal medium (HBM) were purchased from
Lonza (Walkersville, MD).
4.2. General methods
Thin-layer chromatography (TLC) on Silica Gel 60 layer with
F254, coated on aluminum sheet (EMD Chemicals), was used
routinely to monitor reaction progress and column elution profile.
UV-absorbing material was detected under a UV-lamp, and carbo-
hydrate-containing material was visualized by spraying TLC plate
with 15% sulfuric acid in 50% ethanol, and heating it on a hot plate
until the carbohydrate material charred. Unmasked amino group
was visualized by spraying with 0.2% ninhydrin solution in 95%
ethanol, and heating on a hotplate briefly. The trifluoroacetyl or
benzyloxycarbonyl masked amino group was detected by first
spraying the TLC plate with 0.2 M NaOH, heating it briefly, then
spraying with glacial acetic acid followed by ninhydrin, and heat-
ing the plate again. Protein was assayed using a micro BCA
(bicinchoninic acid) assay.19
For the determination of Eu-fluorescence, samples in the wells
of a 96-well plate were incubated with 200 lL of Eu-enhancing
Figure 4. Biodistribution of ‘HexaLac’ in Balb/c mice studied with the single-photon
emission computed tomography (SPECT)/computed tomography (CT) after iv
injection of 111In-HexaLac.
solution20 for 20 min, and then counted using the time-resolved
mode with Eu fluorescence setting in a microplate fluorometer
(Wallac Victor 1460 or Perkin-Elmer Envision).21,22 1H NMR spectra
were measured using a Bruker 300 and 400 MHz ultrashield, and
Varian 300 MHz. Molecular mass of various trivalent and hexa-va-
lent glycosides was determined by ESI-MS, using a Finnigan LCQ.
For the DCC-dependent amide linkage formation, the reaction
was carried out in DMF, DMSO, or a mixture of the two, depending
on the solubility of the reactants, using equimolar amounts of ami-
no- and carboxy-containing compounds. 1-OH-Bt was in the equi-
molar amount and DCC was in 15% excess of the carboxyl group.
After stirring the reaction mixture overnight at room temperature,
the precipitate (dicyclohexyl urea) was filtered off, and the filtrate
was evaporated. The residue was purified by gel filtration through
a column of Sephadex G-15 or G-25 (2.5 ꢀ 140 cm) equilibrated in
and eluted with 0.1 M acetic acid. The Sephadex G-15 column was
used for all tri-valent products, and the G-25 column was used for
the hexa-valent product. Removal of N-benzyloxy group was by
hydrogenolysis using a Brown hydrogenator, in either 80–95% eth-
anol or 60% acetic acid, depending on the solubility of the com-
pounds. Catalyst, 10% Pd on carbon, was used in 10% of the
compound to be hydrogenated on the weight basis.
100
80
60
40
20
0
Figure 5. Biodistribution of 111In-HexaLac in various organs of Balb/c mice after
intravenous injection.25
4.3. Synthesis of Asp derivative as a connector
In order to join two molecules of trivalent lactoside to produce a
hexa-valent ligand, we chose an aspartic acid derivative as a con-
nector, since Asp has two carboxyl groups separate by only two
carbons, and it has an amino group that can be used for linkage
of fluorescent or radioactive probe. In order to make the amino
group more accessible, N-protected 6-aminohexanoyl group was
attached to the amino group of dibenzyl ester of Asp. One milli-
ligands promise to be powerful liver-specific carrier for diagnostic
and therapeutic agents.
4. Experimental
4.1. Materials
mole each of dibenzyl ester of L-Asp p-tosylate and 6-N-(trifluro-
e
e
N -Benzyloxycarbonyl-
L
-lysine (N -Z-Lys), N-benzyloxycar-
-aspartic acid
acetyl)aminohexanoic acid (TFA-AHA) were reacted using the
DCC/1-OH-Bt method in DMF with DIEA (1 mmol) to neutralize
the tosylate. The product was purified by silica gel column chroma-
tography using toluene–ethyl acetate as solvent. Fractions that
contained dibenzyl ester of N-(trifluroacetamidohexanoyl)aspartic
acid [TFA-AHA-Asp(OBn)2, 1] (TLC in toluene–ethyl acetate, 1:1;
Rf = 0.46) were combined and evaporated to yield a white solid in
89% yield; mp, 112–113°. 1H NMR in methanol-d4 showed the
presence of two benzyl groups and 5 side chain methylene groups
bonyl glycine (Z-Gly), and dibenzylester of
L
p-tosylate were purchased from Advanced ChemTech (Louisville,
KY). Dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole
(1-OH-Bt), DTPA (diethylenetriaminepentaacetic acid), its dianhy-
dride, and HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid] were from Sigma-Aldrich (St. Louis, MO). 6-Aminohexyl
b-glycosides of GalNAc and lactose (ahGalNAc and ahLac, respec-
tively)8 and 6-trifluroacetamido-hexanoic acid (TFA-AHA)18 were

2498
R. T. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2494–2500
[r
2.9–1.3 ppm]. Methylene group of Asp was at 3.25 ppm, but the
H atoms, respectively. Between r of 3.9 and 3.0 ppm, where 6 su-
methine H appeared to be buried under HOD peak at 4.8 ppm.
Hydrogenolysis of 1 in 95% ethanol produced a single ninhydrin-
positive product, TFA-AHA-Asp, 10, in quantitative yield. 1H NMR
showed that the benzyl signals have disappeared, and the correct
gar Hs and some low-field methylene and a methine signals are lo-
cated, there were 43 (calcd = 41) and 37 (calcd = 35) hydrogens for
2 and 3, respectively. In the high-field methylene region (
1.23) were 31 Hs for both 2 and 3 (calcd = 32).
r = 1.57–
number of side-chain methylene groups were present between
r
13C NMR spectra of 2 and 3 showed that: (1) 2 had four types of
carbonyl carbons at 175.11, 174.97, 171.45 and 158 ppm, whereas
3 had three types at 175.24, 174.06 and 159.2 ppm. Signals
around175 ppm belong to nitriloacetyl group and around
159 ppm to benzyloxycarbonyl group. Signals at 174.06 and
174.97 ppm are from GalNAc carbonyl groups and those around
171 ppm should belong to the glycyl carbonyl groups. (2) Aromatic
C signals were similar for both compounds, with three peaks lo-
cated between 129.7 and 128.24 ppm. (3) Anomeric C signals for
2 and 3 were at 102.24 and 102.64 ppm, respectively. (4) Methyl
signal was at 53.10 and 53.51 ppm for 2 and 3 (note: this signal
is absent in the spectra of lactose-containing multivalent struc-
tures). (5) Six oxygen-linked C signals for both compounds were
between 76 and 61.56 ppm (5 peaks discernible). (6) Eight peaks
belonging to methylene carbons in high field (29.66–22.01 ppm)
were visible (calcd = 9). (7) One type of methylene and a methine
carbons were located between 40.03 and 40.17 ppm, and com-
pound 2 had an extra signal in this region at 42.92 ppm, which
should belong to methylene of glycyl group. The combined 1H
and 13C NMR results suggest both compounds 2 and 3 contain all
the expected elements in correct proportions and no extraneous
material.
2.78 and 1.35 ppm. The methylene group of Asp seemed to be bur-
ied under the methanol peak at 3.24 ppm, whereas the methine sig-
13
nal was visible at 4.7 ppm. Comparison of C NMR of 1 and 10
against TFA-AHA showed that both 1 and 10 had, in addition to
two extra carboxylic acid signals at around 174 ppm, a methylene
signal at 37.00 and 36.92 ppm, and methine signal at 50.42 and
50.23 ppm, respectively. In addition, 1 had two benzyl signals that
were slightly different from each other (e.g., benzyl methylene sig-
nals at 67.69 and 68.26 ppm). All aromatic C signals, other than the
alkyl attachment site, were located around 129.3 ppm.
4.4. Synthesis of multi-valent glycosides
Syntheses of 6-(trifluroacetamido)hexyl b-glycosides of GalNAc
and lactose, ahGalNAc and ahLac, were by the well-established
methods.8,9 To make a longer glycoside of GalNAc, first Z-Gly was
attached to ahGalNAc using the DCC/1-OH-Bt method in DMF
and then the Z-group of the product was removed by hydrogenol-
ysis to yield (6-N-glycyl)-aminohexyl b-GalNAc (GahGalNAc).4 The
product was crystallized from hot 95% ethanol. The NTA derivative
of lysine, to be used as scaffold for attachment of sugars, was
e
prepared by bromoacetylation of N -Z-
L
-Lys10 which produced
1H NMR of tri-valent lactoside in D2O: With the methylene sig-
e
a
e
a
N -Z-N -dicarboxylmethyl derivative of lysine (N -Z-N -DCM-Lys,
see Fig. 1). To this scaffold (0.1 mmol) was conjugated ahGalNAc
(0.45 mmol), GahGalNAc (0.45 mmol), or ahLac (0.57 mmol), using
the DCC/1-OH-Bt method in either DMF for GalNAc derivatives and
DMF-DMSO mixture for lactoside. In order to ensure complete
sugar conjugation, GalNAc glycosides were used in 50% excess for
each carboxyl group of N -Z-N -DCM-Lys, whereas, in the case of
ahLac, it was necessary to use 80–90% excess over each carboxyl
group. Eluted fractions from the Sephadex G-15 column showed,
by TLC using ethyl acetate–acetic acid–water (3:2:1), that the tri-
valent product (Rf = 0.048 for 2, 0.058 for 3, and 0.02 for tri-valent
nal of benzyl group (
signal ( = 7.25) was 4.7 and that of anomeric signal (2 anomeric
hydrogens for each lactose unit) was 5.4. The mid range
= 3.82–2.96), where all the remaining lactose H and some H
r = 4.94) set as 2, the number of H in aromatic
r
(r
atoms of methylene groups of aglycon and lysine side chain are lo-
cated, contained signals for 55 H atoms (calculated number = 53),
and the high-field region for most of methylene groups
e
a
(r = 1.445–1.066) contained signal worth 30 H atoms (calculated
number = 33). 1H NMR of HexaLac in D2O: HexaLac is composed
of 6 aminohexyl lactosyl residues, 2 dicarboxymethylated lysyl res-
idues and 1 TFA-aminohexanoylated aspartyl residue. Setting the
lactoside) eluted well ahead of excess
x
-amino glycoside with
number of H in the methylene signal of aspartyl group (r = 2.554–
little under sugar-derivatized products being produced under such
2.426, m) as 2, anomeric signal of lactose, mid section of spectra
reaction conditions. Yield of 3, 2, and tri-valent lactoside on the
(all the remaining lactose H signals and some low-field methylene
e
a
basis of N -Z-N -DCM-Lys was 70%, 99%, and 30%, respectively.
The de-N-protected trivalent GalNAc derivatives and tri-valent lac-
toside (4) were obtained in quantitative yield by hydrogenolysis.
A hexa-valent lactoside (HexaLac, 5, Fig. 2) was prepared by
and methine signals;
r = 3.83–2.99), and high-field methylene sig-
nals ( = 1.46–1.19) were 11.5, 116.2 and 66.5 H atoms, respec-
r
tively. The calculated H numbers are 12, 115 and 66 for the same
three regions, showing excellent agreement of experimental values
with the projected values.
attaching two molecules of
4 (142 lmol) to TFA-AHA-Asp
(60 mol) by the DCC/1-OH-Bt method in DMSO. The fractionation
l
13C-NMR data of tri-valent lactoside and HexaLac: Similar to the
tri-valent GalNAc glycosides, the tri-valent lactoside had carbonyl
signals from nitriloacetic acid group and benzyloxycarbonyl group,
but lacked the carbonyl group of GalNAc. The same triad of aro-
matic C around 128–129 ppm was present, as well as a much smal-
ler signal at 136.7 ppm that belongs to the C attached to methylene
group. There were two anomeric signals of lactose at 103.1 and
102.2 ppm. As expected, between 78.6 and 60.3 ppm, there were
many more C signals than seen in the GalNAc counterparts; 12
peaks (2 of which were almost totally overlapping) in this region
represent 10 C from lactose and 2 other O-linked carbons. Only 6
peaks were discernible in the high-field methylene region of
of reaction mixture on the Sephadex G-25 column yielded HexaLac
(earliest eluting material, Rf = 0.18 in 80% ethanol) in 69% yield.
All multi-valent products appeared pure by the TLC examina-
tion in the respective solvent system, and ESI-MS examination
indicated that all had the dominant peak of the expected molecular
mass. No incompletely glycoside-conjugated products, namely
mono- and di-valent products, were detected in any of the tri-va-
lent compounds. The MS of the HexaLac showed no peak for the
starting tri-valent lactoside, 4, and only a negligible amount of
Asp derivative with a single tri-valent lactoside attached was
detected.
1H and 13C NMR data for all the multi-valent products are sum-
r = 28.9–23, where nine carbons should be located, as in the
marized below. 1H NMR spectra of tri-valent GalNAc derivatives, 2
GalNAc counterparts.
and 3, showed the following: With aromatic signals (
r
= 7.393 for 2
13C NMR spectrum of HexaLac is quite similar to that of the tri-
valent lactoside, with a few extra signals and a few missing signals.
Carbonyl signals were at 176.5, 174.3, 173.3, 172.2 and 171.4 ppm,
representing two from the aspartyl group and one from amino-
hexanoyl group, in addition to the carbonyls present in the
and 7.351 for 3) set as 5H atoms, benzyl methylene signals at
5.08 ppm and 5.03 ppm were 2 H atoms each for both 2 and 3. Both
had 3 H atoms for anomeric signals at 4.42 and 4.35 ppm. Methyl
signals for 2 (2.013 ppm) and for 3 (1.959 ppm) had 8.87 and 8.8

R. T. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2494–2500
2499
tri-valent lactoside. The carbonyl signal of trifluoroacetyl group
4.7. Radiosynthesis
was not visible, as in the case of TFA-AHA. There were two extra
signals between 39 and 35 ppm. These are likely signals from
aspartic methylene and methine carbons. In the region of
140–127 ppm, the entire aromatic signal is missing, as well as
benzyl C signal at 158–159 ppm. Thus, combined 1H and 13C
NMR data are in good agreement with the structures of tri-valent
lactoside and HexaLac.
The trivalent GalNAc derivatives were de-N-protected by
hydrogenolysis, and HexaLac was de-N-protected in 10% TEA in
10% ethanol, and leaving the mixture at room temperature
overnight, followed by thorough evaporation. The amino-exposed
tri-valent GalNAc derivative and HexaLac were reacted with 5-fold
excess of DTPA dianhydride in 0.2 M sodium borate buffer (pH 8.5),
with NaOH added as needed to maintain the pH slightly alkaline.
DTPA derivatives were purified with the Sephadex G-15 column
(for the GalNAc derivative) or with the Sephadex G-25 column
(for HexaLac). The DTPA-conjugated products no longer moved
on TLC plate using ethyl acetate–acetic acid–water (2:1:1) or 80%
ethanol, and eluted well ahead of free DTPA (see Section 4.5).
Labeling of DTPA-HexaLac with 111In for biodistribution study
was according to the published method.24 Briefly, DTPA-modified
HexaLac or DTPA-modified tri(GalNAc) (0.012 nmol, each) was
mixed with 1.85 ꢀ 107 Bq of 111In in 0.1 M citric acid (pH 2.1) for
30 min. The labeling efficiency was analyzed by silica gel impreg-
nated glass fiber (ITLC-SG, Gelman) developed with 10 mM sodium
acetate and confirmed with radio-HPLC.
4.8. Biodistribution of 111In-DTPA HexaLac in normal Balb/c
mices
Four male mice, averaging 20 g, were sacrificed by cervical
translocation 15 min after iv injection of 0.35 MBq/100 lL
111In-
DTPA HexaLac. The chosen organs were excised out as whole organ
when possible. The organs were rinsed with saline, blotted dry,
weighed and then counted using Cobra II Auto-Gamma counter
(Packard). The radioactivity in the organ was expressed as percent-
age of the injected dose (% ID).
4.9. Small animal microSPECT imaging
4.5. Determination of DTPA and mono-conjugated DTPA
derivatives
For the micro-SPECT/CT image of 111In-DTPA-HexaLac, each
mouse received iv injection of 44 l
Ci of 111In-DTPA HexaLac. The
Reaction of DTPA dianhydride with amino-containing com-
pounds in aqueous solution inevitably produces free DTPA. In order
to locate DTPA-derivative and free DTPA in the column effluent, a
facile assay method using TPTZ (2,4,6-tri-2-pyridyl-s-triazine)
was devised. In this assay, DTPA is allowed to compete against
TPTZ for the binding of ferrous ion. The TPTZ–ferrous sulfate solu-
tion described by Avigad23 was diluted 2-fold to give A593nm of
mice were anesthetized with 1.5% isoflurane (positioned prone in
the scanner) and 60-min image was obtained by micro-SPECT/CT
2 h after iv injection with urine squeezed out. The SPECT images
and CT images were acquired using a micro-SPECT/CT scanner
(X-SPECT/CT, Gamma Medica, Northridge, CA). The SPECT images
were acquired using a parallel hole collimator, with the center of
the field of view (FOV) focused on the abdomen of the mouse.
The radius of rotation (ROR) was 1 cm with an FOV of 1.37 cm.
The imaging was accomplished using 64 projections at 60 s per
projection. The SPECT imaging were reconstructed to produce im-
age sizes of 56 ꢀ 56 ꢀ 56 (pixels) with an image resolution of
1.2 mm. The CT images were also reconstructed in image sizes of
512 ꢀ 512 ꢀ 512 (pixels) with 0.15 mm image resolution.
0.95–1.0. A sample containing up to 0.2 lmol of DTPA or DTPA
derivative was mixed with the diluted TPTZ solution (3 ml), incu-
bated at 37° for 30 min, and A593nm was measured. The presence
of DTPA or its derivative lowers the absorbance, which is approxi-
mately proportional to the DTPA concentration.
4.6. Competition assay for determination of binding affinity of
various multi-valent glycosides to rat hepatocytes
Acknowledgments
The binding affinities of multivalent glyco-ligands 2, 3, 4, and
HexaLac to rat hepatocytes were determined by competitive assay,
using Eu-labeled asialo-orosomucoid (Eu-ASOR) as the reference
compound. The assay was carried out with a 24-well plate of rat
hepatocyte monolayer half immersed in ice–water mixture. HBM
in the well was removed by gentle suction, and replaced with
0.5 mL of five different concentrations of multivalent ligands
We thank Dr. H.-M. Yu and Mr. C.-Y. Chein for technical assis-
tance in biodistribution study. We thank Mr. K. Mathos, E. Lloyd,
and J.-T. Wang for recording of NMR spectra. The animal experi-
ments approved by the Animal Care and Use Committee of the
Institute of Nuclear Energy Research in Taiwan were also gratefully
acknowledged.
(0.8 nM to 1 lM), followed by 0.5 mL of 10 nM of Eu-ASOR. All
References and notes
solutions were made in HBM with 5 mM calcium chloride. After
gentle shaking for 1 h in the cold, the unbound ligand was removed
and the wells were washed with HBM containing calcium chloride.
Europium was stripped and solubilized from Eu-ASOR bound to
hepatocytes by adding 0.3 mL of enhancement solution20 in each
1. Pricer, W. E., Jr.; Hudgin, R. L.; Ashwell, G.; Stockert, R. J.; Morell, A. G. Methods
Enzymol. 1974, 34, 688.
2. Rice, K. G.; Lee, Y. C. Adv. Enzymol. Relat. Areas 1993, 66, 41.
3. Lee, R. T.; Lee, Y. C. Glycoconjugate J. 1987, 4, 317.
4. Lee, R. T.; Lee, Y. C. Bioconjugate Chem. 1997, 8, 762.
5. Merwin, J. R.; Noell, G. S.; Thomas, W. L.; Chiou, H. C.; DeRome, M. E.; McKee, T.
D.; Spitalny, G. L.; Findeis, M. A. Bioconjugate Chem. 1994, 5, 612.
6. Hangeland, J.; Levis, J.; Lee, Y.; Ts’o, P. Bioconjugate Chem. 1995, 6, 695.
7. Hangeland, J. J.; Flesher, J. E.; Deamond, S. F.; Lee, Y. C.; Ts, O. P.; Frost, J. J.
Antisense Nucleic Acid Drug Dev. 1997, 7, 141.
8. Weigel, P. H.; Naoi, M.; Roseman, S.; Lee, Y. C. Carbohydr. Res. 1979, 70, 83.
9. Lee, R. T.; Wong, T. C.; Lee, Y. C. J. Carbohydr. Chem. 1986, 5, 343.
10. Altin, J. G.; Banwell, M. G.; Coghlan, P. A.; Easton, C. J.; Nairn, M. R.; Offermann,
D. A. Aust. J. Chem. 2006, 59, 302.
11. Kawaguchi, K.; Kuhlenschmidt, M.; Roseman, S.; Lee, Y. C. J. Biol. Chem. 1981,
256, 2230.
12. Stowell, C. P.; Lee, R. T.; Lee, Y. C. Biochemistry 1980, 19, 4904.
13. Connolly, D. T.; Hoppe, C. A.; Hobish, M. K.; Lee, Y. C. J. Biol. Chem. 1981, 256,
12940.
well and swirling the plate gently for 20 min. An aliquot (200 lL)
from each well of this solubilized Eu in a highly fluorogenic milieu
was transferred into a 96-well plate and Eu-fluorescence was
determined by time-resolved fluorometry. After subtracting the
blank fluorescence (fluorescence obtained in the presence of
2 lM ASOR), the residual fluorescence values were converted to
the % of fluorescence obtained in the absence of inhibitor. This fluo-
rescence value at each inhibitor concentration was plotted on y
axis with the inhibitor concentration in logarithmic scale on x axis.
IC50 (concentration at which 50% reduction in the bound fluores-
cence occurred) for each compound was determined from the
respective curve.
14. Lee, Y. C.; Kawasaki, N.; Lee, R. T.; Suzuki, N. Glycobiology 1998, 8, 849.

2500
R. T. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2494–2500
15. (a) Connolly, D. T.; Townsend, R. R.; Kawaguchi, K.; Hobish, M. K.; Bell, W. R.;
Lee, Y. C. Biochem. J. 1983, 214, 421; (b) Hardy, M. R.; Townsend, R. R.;
Parkhurst, S. M.; Lee, Y. C. Biochemistry 1985, 24, 22.
16. Hochuli, E.; Dobeli, H.; Schacher, A. J. Chromatogr. 1987, 411, 177.
17. Hainfeld, J. F.; Liu, W.; Halsey, C. M.; Freimuth, P.; Powell, R. D. J. Struct. Biol.
1999, 127, 185.
20. Hemmila, I.; Dakubu, S.; Mukkala, V. M.; Siitari, H.; Lovgren, T. Anal. Biochem.
1984, 137, 335.
21. Kawasaki, N.; Lee, Y. C. Anal. Biochem. 1997, 250, 260.
22. Deras, I.; Kawasaki, N.; Lee, Y. C. Carbohydr. Res. 1998, 306, 469.
23. Avigad, G. Rapid, Sensitive Determination Periodate 1969, 11, 119.
24. Yu, H. M.; Wang, M.-H.; Chen, J.-T.; Lin, W.-J. J. Labelled Compd. Radiopharm.
2010, 53, 417.
18. Lee, R. T.; Lee, Y. C. Carbohydr. Res. 1974, 34, 151.
19. Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.;
Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal.
Biochem. 1985, 150, 76.
25. Wang, M. H.; Lee, R. T.; Yu, H. M.; Chien, C. Y.; Lee, Y. C.; Lin, W. J. Eur. J. Nucl.
Med. Mol. Imaging 2009, 36, S470.