Varied presentation of the Thomsen-Friedenreich disaccharide tumor-associated carbohydrate antigen on gold nanoparticles

Article abstract of DOI:10.1016/j.carres.2008.05.003

Three-dimensional self-assembled monolayers of gold coated with the Thomsen-Friedenreich antigen (TF_ag) disaccharide (β-Galp-(1→3)-GalpNAc) in a variety of presentations have been prepared and characterized. Anomalies in the size distribution of our originally synthesized TF_ag-bearing nanoparticles as shown in dynamic light scattering experiments prompted us to explore the effect of antigen density on the uniformity of the particles. Gold nanoparticles containing a range of densities 'diluted' with copies of the PEG-thiol spacer unit showed that lower antigen density affords more uniform particles. We also wanted to study the constitution of the actual antigen by synthesizing nanoparticles not only with the linker-extended disaccharide, but also within the context of the surrounding peptide sequence where it may be presented in vivo. The synthesis of TF_ag-containing glycopeptide thiols based on a mucin peptide repeating unit were prepared, assembled into gold nanoparticles and their physical properties evaluated. These novel multivalent tools should prove extremely useful in exploring the binding properties and immune response to this important carbohydrate antigen.

Full text of DOI:10.1016/j.carres.2008.05.003

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1594–1604
Varied presentation of the Thomsen–Friedenreich
disaccharide tumor-associated carbohydrate antigen
on gold nanoparticles
Andreas Sundgren and Joseph J. Barchi, Jr.*
Laboratory of Medicinal Chemistry and, Center for Cancer Research, National Cancer Institute at Frederick,
376 Boyles Street, PO Box B, Building 376, Room 209, Frederick, MD 21702, USA
Received 11 March 2008; received in revised form 23 April 2008; accepted 4 May 2008
Available online 8 May 2008
Abstract—Three-dimensional self-assembled monolayers of gold coated with the Thomsen–Friedenreich antigen (TFag) disaccharide
(b-Galp-(1?3)-GalpNAc) in a variety of presentations have been prepared and characterized. Anomalies in the size distribution of
our originally synthesized TFag-bearing nanoparticles as shown in dynamic light scattering experiments prompted us to explore the
effect of antigen density on the uniformity of the particles. Gold nanoparticles containing a range of densities ‘diluted’ with copies of
the PEG-thiol spacer unit showed that lower antigen density affords more uniform particles. We also wanted to study the consti-
tution of the actual antigen by synthesizing nanoparticles not only with the linker-extended disaccharide, but also within the context
of the surrounding peptide sequence where it may be presented in vivo. The synthesis of TFag-containing glycopeptide thiols based
on a mucin peptide repeating unit were prepared, assembled into gold nanoparticles and their physical properties evaluated. These
novel multivalent tools should prove extremely useful in exploring the binding properties and immune response to this important
carbohydrate antigen.
Published by Elsevier Ltd.
Keywords: Thomsen–Friedenreich antigen; Gold nanoparticles; Glycopeptides; Immunogen
1. Introduction
many different molecules) on their surface, yielding mul-
tivalent constructions that can be used to study and/or
interfere with carbohydrate–protein interactions. Hence,
‘glyconanotechnology’4–8 is a burgeoning field and there
are now a host of published examples of glyconanopar-
ticles (GNP’s) that have shown strong potential to act as
extremely useful tools in glycan science. One of the most
widely used platforms that has often been ‘sugar-coated’
is that which employs spherical self-assembled mono-
layers of gold. These gold NP’s have a strong affinity
for organic thiols and their synthesis has been refined
to where highly stable and relatively monodispersed par-
ticles can be rapidly produced through simple reduction
of gold salts in the presence of a molecule of choice teth-
ered appropriately to a terminal mercapto group.9 Thus,
mono- and oligosaccharides have been displayed on
the surface of gold NP’s and shown to have provoca-
tive characteristics compared with their monovalent
counterparts.10 In addition, carbohydrates have been
Spurred by recent advances in technology development,
there has been a surge in the design, development and
the use of nanoscale platforms for many areas of medi-
cal research. Various materials (such as gold,1 iron
oxide,2 and semiconductor materials3) can be employed
to form self-assembled structures that can be coated
with organic agents or biomacromolecules. These novel
nanoparticles (NP’s) have intriguing physical properties
that may be related to their core materials and nano-
meter size ranges, as well as the chemistry of the coating
itself. The field of carbohydrate chemistry has benefited
from this technology, as many platforms allow the pre-
sentation of multiple copies of a particular molecule (or
*
Corresponding author. Tel.: +1 301 846 5905; fax: +1 301 846
6033; e-mail: barchi@helix.nih.gov
0008-6215/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.carres.2008.05.003

A. Sundgren, J. J. Barchi Jr. / Carbohydrate Research 343 (2008) 1594–1604
1595
attached on quantum dots11–15 for cell imaging applica-
tions and on magnetic NP’s16 for differential separation
of bacterial strains.
prompted us to refine the synthesis of the NP’s to
increase their monodispersity and attempt to determine
the causes of the aforementioned non-uniformity. In
addition, we synthesized the antigen in different forms
including within a tumor-associated glycopeptide con-
struction from a mucin protein (MUC4).
Our interest in this area is targeted to the synthesis
of GNP’s with tumor-associated carbohydrate anti-
gens17–19 (TACA’s) on their surface. TACA’s are aber-
rantly expressed carbohydrate units found on the cell
surface of tumors that derive from the differences in
the way glycans are processed in malignant cells com-
pared to normal phenotypes. Our rationale for the de-
sign of these particles was twofold: (1) They have the
potential to be used as a novel vaccine platform20 to eli-
cit an immune response in vivo, or (2) They could be
useful inhibitors of well-known protein carbohydrate
interactions in which specific TACA’s are involved.
For these reasons we concentrated on the Thomsen–
Friedenreich disaccharide (TFag),21,22 a human TACA
present primarily in carcinomas but rarely expressed in
normal tissues.23 This antigen has been the subject of
many studies attempting to design immunogens or vac-
cine platforms targeted to this TACA.24–26 In addition,
tumors displaying the TFag metastasize through a spe-
cific interaction with endothelial cell-derived galectin-3
(Gal-3).27,28 Both adhesion to Gal-3 and cell growth
are inhibited by TFag function-blocking or anti galec-
tin-3 antibodies. Another report corroborated the
importance of the TFag–Gal-3 interaction by showing
that Gal-3 interacts with the cancer-associated mucin
MUC1 via TFag.29 This interaction caused a polariza-
tion of MUC1 on the cell surface revealing epithelial
adhesion molecules that are otherwise concealed by
MUC1, thus promoting cancer cell adhesion to the
endothelium. The fact that TFag is covalently attached
to the peptide backbone of MUC1 suggests that the sur-
rounding peptide sequence is part of the antigen that is
recognized by the immune system, and hence synthetic
glycopeptides may be better immunogens than the
‘naked’ dissaccharide.
2. Results and discussion
Our synthesis of TFag-coated GNP’s employed the
reduction of gold salts by NaBH4 in the presence of spe-
cific carbohydrate-linked thiols.31,32 This method has
been utilized in many GNP reports cited above and
the results, in general, have been quite successful. In
our previous work, we synthesized TFag-coated GNP’s
with approximately 90 sugar units on the gold surface
1 along with the unnatural b-analogue 2 to compare
the quality and properties of these particles with the a-
linked GNP’s. For our in vivo studies, we had prepared
the control particles 3, which displayed simply the linker
molecule where the TFag disaccharide is essentially
substituted with a simple hydroxyl group (Fig. 1).
The synthesis of the pentenyl-hexaPEG linker and
TFag-linked thiol was as described previously.30 The
GNP’s and linker-coated particles were purified by ultra-
filtration and analyzed by transmission electron micro-
scopy (TEM), NMR spectroscopy, elemental analysis,
and dynamic light scattering (DLS). The core diameter
of the GNP’s 1 and 2 was in the 3–4.5 nm range by
TEM measurements, whereas NP’s 3 with simply the lin-
ker were slightly larger and somewhat more polydis-
persed but in solution looked superior to the
disaccharide-bearing particles (Fig. 2). In comparing
the GNP’s 1–3 with DLS, the a-linked TFag particles 1
were the least uniform, and their hydrodynamic diameter
(HDD) was also twice as large as b-linked TFag GNP’s
when volumes were considered (Fig. 2).
We mentioned in our earlier report30 that TFag-coated
GNP’s could inhibit lung metastasis in the murine 4T1
breast cancer model. The details of this work were never
published since we unfortunately were not able to repro-
duce the original result in several subsequent studies
using different nanoparticle concentrations and control
experiments with particles coated simply with hydro-
xyl-terminated linker groups (vide infra). Although we
showed that TFag-coated GNP’s agglutinated TFag-spe-
cific antibodies, TFag-coated GNP’s were unable to
inhibit TFag-specific antibody binding to immobilized
BSA-conjugated TFag (Barchi, Rittenhouse-Olson,
Heimburg, Sundgren, unpublished results). A closer
examination revealed that the TFag-coated GNP’s were
polydispersed (non-uniform size distribution) in solu-
tion by dynamic light scattering (DLS) experiments
(vide infra). We reasoned that this may be the source
of the problems with our in vivo experiments and
Initially, our hypothesis was that the anomeric stereo-
chemistry of the sugar could cause problems in the self-
assembly process of the nanoparticles. Very few a-linked
sugars have been coated on gold nanoparticles.33 To our
knowledge, only one group has synthesized a-linked
disaccharides on gold and they showed that different
aggregation properties were observed when comparing
a and b-linked disaccharide-coated particles.34,35 If the
directionality of the sugar is angled differently in the
a- and b-linked GNP’s and this is the source of aberra-
tions in the self-assembly process, reducing the carbo-
hydrate density on the surface may facilitate the
production of more uniform particles yielding a tighter
distribution of sizes. Scheme 1 outlines the synthesis of
GNP’s with various ratios of carbohydrate ligand to
underivatized linker thiol used in the reaction mixture.
As the density of the carbohydrate is lowered on the
particle surface, the size distribution also improves

1596
A. Sundgren, J. J. Barchi Jr. / Carbohydrate Research 343 (2008) 1594–1604
HO
OH
O
HO
O
AcHN
OH
O
HO
OH
O
O
5
O
O
OH
O
OH HO
HO
HO
S
S
O
O
AcHN
OH
S
O
5
Au
O
O
OH
HO
O
OH
O
HO
HO
HO
O
O
O
O
5
O
O
AcHN
OH
S
S
1
S
O
5
Au
HO
O
HO
OH
O
OH
HO
O
OH
O
HO
O
HO
O
O
O
NHAc
OH
3
S
OH
HO
HO
HO
O
O
O
O
O
S
Au
O
5
OH
NHAc
S
OH
O
O
HO
O
OH
O
O
HO
HO
O
NHAc
OH
2
Figure 1. TFag (1), b-TFag (2) and linker ‘control’ (3) GNP’s.
Figure 2. DLS data for compounds 1–3. Top trace is intensity versus size while the bottom traces are volumes versus size. The lower row shows TEM
data for each NP with size histograms as an inset.
(seeSupplementary data for DLS data on particles 6–9).
In addition, the optimum uniformity is observed for the
particles prepared where a 5:1 linker-to-sugar ratio is
used (compound 10). This method is being used by us
for the comparison of various particles with different
passivating agents and surface coatings. For example,
we have prepared simple b-galactose-coated GNP’s
from compounds where the linker-to-sugar ratio was
either 0:1, 1:1, or 5:1. Microscopy and sizing data also
showed that the 5:1 linker-to-sugar particles have the

A. Sundgren, J. J. Barchi Jr. / Carbohydrate Research 343 (2008) 1594–1604
1597
3
OH
O
OH
HO
O
HO
HO
i
O
i
AcHN
OH
SH
HO
O
SH
O
O
+
O
O
5
5
5
4
OH
OH
OH
HO
O
TFag:Linker
O
OH
OH
OH
OH
O
6,
7,
1:1
1:2
1:3
1:4
1:5
AcHN
O
HO
O
OH
OH
O
HO
OH
HO
O
OH
O
O
HO
OH
HO
8,
O
O
O
AcHN
O
O
O
AcHN
O
9,
10,
O
O
OH
O
O
O
HO
HO
O
S
S
O
O
S
S
OH
S
HO
S
O
OH
HO
HO
S
S
O
Au
O
O
S
5
O
O
AcHN
OH
S
S
S
O
S
S
O
O
O
O
O
HO
O
O
O
NHAc
O
O
O
OH
O
HO
O
O
HO
O
HO
O
OH
O
HO
OH
NHAc
HO
O
O
HO
OH
NHAc
O
O
HO
HO
HO
OH
HO
HO
O
OH
O
HO
HO
OH
Scheme 1. Reagents and conditions: (i) HAuCl4, NaBH4, H2O, 0 °C; DLS data for compound 10 are shown in the lower right with intensity versus
size shown in the top trace and volume versus size in the bottom trace.
most uniform size by both DLS and TEM analysis, and
the ability for these to inhibit or promote HIV fusion to
mammalian cells changes with carbohydrate density
(unpublished results).
sult. The long protein backbone is comprised of many
repeating motifs of between ꢀ16 and 22 amino acids that
contain several serine and threonine residues of which
the majority are glycosylated.38 This serves to ‘extend’
the protein backbone straight out from the cell surface,
and the inter-digitation of the mucin molecules forms a
protective barrier as part of the glycocalyx. Several mucins
are overexpressed in various cancers; structures related to
both the carbohydrate and the peptide backbone of
mucins have been employed in antitumor vaccines strate-
gies. We chose the 16-amino acid repeating unit of
MUC4 (Fig. 3), the primary mucin found on adenocarci-
nomas of the pancreas,39–42 for our studies to prepare gly-
copeptide-coated nanoparticles (GPNP’s). The Kunz
group had previously synthesized several glycopeptides
of MUC4 by solid phase peptide synthesis for use in immu-
nological studies. We followed similar procedures to make
a repertoire of glycopeptides for assembly on gold
particles.
The scientific literature is replete with reviews about TA-
CA’s along with suggestions on the exact constitution of
the actual antigen ‘structures’. Many (including us) believe
that the recognition element of a specific glycoprotein-
based TACA includes either the amino acid or local pep-
tide sequence to which it is attached in a cellular context.
In addition, this ‘natural’ antigen should elicit a more pow-
erful immune response in synthetic vaccine preparations
since it should more closely resemble the conformation dis-
played in vivo. Recent work has shown that nanoparticle
platforms have the potential to act as single-36,37 or multi-
component20 vaccine scaffolds. We thus extended the
antigen ‘structure’ to the TFag coupled to specific mucin
peptides. Mucins are very large proteins that act as a
lubricant and can protect the cell surface from outside in-

1598
A. Sundgren, J. J. Barchi Jr. / Carbohydrate Research 343 (2008) 1594–1604
N
NH
O
O
OR2
HO
OR1
O
O
O
O
OH
NH2
O
O
O
O
H
N
H
N
H
H
H
N
H
N
R3HN
N
N
N
O
N
H
N
H
OH
N
H
N
H
N
H
N
N
H
N
H
O
O
O
O
O
O
HO
OH
OH
11, R1 = H, R2 = H
OH HO OH
HO
HO
12, R1 = H, R2 =
O
O
O
13, R2 = H, R1 =
AcHN
OH
O
O
R3
=
O
O
O
N
O
O
O
N
H
SH
H
O
Figure 3. Structure of MUC416 peptide and two glycopeptides with TFag at positions Thr6 and Thr10. The linker R3 was used to functionalize the
N-terminus of the glycopeptide for GPNP synthesis.
We have synthesized glycopeptides with the TFag at
two separate positions and developed appropriate linker
chemistry for the synthesis of GPNP’s (Scheme 2).
SPPS of the peptides proceeded smoothly for the
majority of the coupling reactions. We employed Fmoc
chemistry using a mixture of HOBt and HBTU for acti-
vation. Synthesis was performed mostly in the auto-
mated mode on a peptide synthesizer. The glycoamino
acid, however, was coupled manually and depending
on the position of the sugar unit in the peptide sequence,
specific conditions were needed to affect efficient cou-
pling. The two glycopeptides we report on here had
the TFag at the 6th and 10th threonine residues (Fig.
3). We prepared the appropriately functionalized
control peptide (without a covalently attached carbohy-
drate) attached to the linker unit for self assembly onto
gold particles. The linker strategy is one that deserves
mention, since this can be a critical component in the
production of NP’s. The glycopeptide linker described
here incorporated a PEG unit and a fatty portion which
can potentially facilitate packing at the surface. Linkers
consisting solely of PEG units were difficult to couple to
the peptide and did not self assemble efficiently. An ex-
tender glycine amino acid was added to bolster the reac-
tivity of the carboxylate toward coupling with the amino
group of the peptide. The commercially available N-
Fmoc-1-amino-3,6,9,12,15,18-hexaoxahenicosan-21-oic
acid was reacted according to Scheme 2 to yield the
O
H
N
i
O
OH
O
FmocHN
FmocHN
OtBu
6
6
O
O
14
iii, iv
O
O
O
O
H
N
H
N
vi-viii
RS
O
OH
AcS
O
N
N
H
N
H
OR
6
6
6
6
H
O
O
16 R = t-butyl
18 R = Ac
v
17 R = H
R1
R2
ix
H2N TSSASTGHATPLPVTD
R1 = H, R2 = H
O
Ph
O
x, xi
OAc
AcO
AcO
R1 = H, R2
R2 = H, R1
=
=
O
O
O
AcHN
OAc
11, 12 or 13
Scheme 2. Reagents: (i) DIPCDI, HOBt, DMF, dichloromethane (CH2Cl2), 86%; (ii) thiolacetic acid, AIBN, 84%; (iii) piperidine, CH2Cl2; (iv)
AcS(CH2)6COOH (15), DIPCDI, HOBt, DMF, CH2Cl2, 86% in two steps; (v) TFA, 72%; (vi) 2-amino ethanol, DIPCDI, HOBt, DMF, CH2Cl2; (vii)
hydrazine hydrate, EtOH; (viii) dithiothreitol, water, 85% in three steps; (ix) 17, DIPCDI, HOBt, DMF; (x) hydrazine hydrate, EtOH; (xi) 95% TFA,
2.5% 1,2-ethanedithiol.

A. Sundgren, J. J. Barchi Jr. / Carbohydrate Research 343 (2008) 1594–1604
1599
linker 17 which was coupled to the peptide on resin.
Deprotection of the acetate groups was also accom-
plished most efficiently while attached to the resin with
hydrazine/ethanol. Coupling of the glycoamino acids
was performed with HOBt–diisopropylcarbodiimide
(DIPCDI), and although efficient, was slow and
required extended reaction times and additional activat-
ing reagents to reach completion. Experimentation with
microwave catalysis to improve the yield and increase
the rate of these coupling steps is in progress. Scheme
3 outlines the method for the preparation of GPNP’s
20–22 from the compounds 12 and 13.
In addition to preparing the peptide-bearing particles
19 and GPNP’s 20 and 21, we prepared the ‘diluted’ par-
ticles with a 1:5 ratio of glycopeptide to linker (22) along
with another ‘control’ particle containing the linker
alone (23). It was necessary to modify the linker 17 used
in the synthesis of 23 by extension with an ethanolamine
tail to produce 18 for the preparation of particles that
expose simple hydroxyl groups on their surface similar
to our approach for linker-coated NP’s 3. The rationale
for producing two ‘control’ particles (19 and 23) is to be
able to dissect the effects of the sugars, the peptide back-
bone, and the linker on the recognition of the GPNP’s.
Physical characterization of particles 19–23 followed
similarly as for the GNP’s above. An estimate as to
the number of copies of glycopeptide exposed on the
GPNP’s was made from elemental analysis and core
diameters that estimate the number of gold atoms in
the particle composition.43 The glycopeptides 20 and
21 contained ca. 180 copies of ligand while for the pep-
tide alone (19) there are an estimated 220 copies. The
peptide and glycopeptide-conjugated gold particles all
looked very uniform by TEM analysis (Fig. 4). As has
been shown in several past reports including from our
laboratory, NMR of the GNP’s or GPNP’s in water
show nearly all signals that are evident in the mono-
meric ligands, but broadened by the high molecular
weight and changes to relaxation times in the NP’s (data
not shown). A surprising discovery during this work was
that, even with identical synthetic procedures used
throughout to prepare NP’s, uniformity was dictated
by the surface chemistry and could not be predicted a
priori. Hence, the disaccharide-coated NP’s that were
prepared typically showed polydispersity unless ‘diluted’
with interstitial spacer PEG linkers to reduce the saccha-
ride density on the surface of the particle. Glycopeptides
were consistently more uniform by microscopic analysis,
but in all particles that we have made to date, DLS anal-
ysis shows intensity bands in the range of 50–150 nm.
Scheme 3. Reagents and condition: (i) HAuCl4, NaBH4, H2O, 0 °C.

1600
A. Sundgren, J. J. Barchi Jr. / Carbohydrate Research 343 (2008) 1594–1604
Figure 4. TEM data for GPNP, peptide- and linker-coated NP’s 19–23.
These usually disappear on conversion of these intensity
maps to volumes or when the particles are first filtered
through membranes that exclude all material above
0.1 lm, indicating that although there may be particles
that are much larger in hydrodynamic diameter
(HDD) and scatter light efficiently, their actual number
is quite low and the bulk of the particles are in the 7–
11 nm HDD range. We are now in the process of testing
these novel particles in several bioassays, and the results
will be reported in due course.
An important conclusion gleaned from this work is that
nanoconstructions with various surface chemistries may
display very different behavior when comparing micros-
copy to sizing measurements in solution. By comparing
measurements performed in various milieus (water,
10 mM NaCl, PBS, data not shown), it is evident that
each particle has its own individual properties when ex-
posed to different solutions. This could have important
consequences when studying these particles in a cellular
or in vivo context. Subsequent reports will outline in de-
tail the ‘idiosyncrasies’ of each GNP(GPNP) and how
these relate to their biological activity and therapeutic
potential.
3. Conclusions and summary
Gold nanoparticles bearing the Thomsen–Friedenreich
antigen at different densities and in different contexts
have been prepared and characterized. Sizing data
showed that certain sugar presentations result in the
assembly of particles that may have reasonable unifor-
mity in their gold core diameters by TEM analysis,
but they can be polydispersed and ‘non-uniform’ in
solution. Glycopeptide-coated nanoparticles result in
more uniform particles as seen by TEM analysis, how-
ever, GNP’s and GPNP’s contain some larger size
elements that could be a consequence of either aberra-
tions in the self-assembly process or aggregation events.
4. Experimental
Flash column chromatography (FCC) was performed
using RediSepÒ silica columns on a CombiFlashÒ
CompanionÒ employing solvent polarity gradient (hex-
ane?ethyl acetate). Reversed phase chromatography
was performed on 900 mg AlltechÒ Maxi-CleanTM C18
Cartridges employing solvent polarity gradient
(water?methanol) unless otherwise noted. Chemicals
were purchased from Aldrich–Sigma (Milwaukee, WI)
and used without further purification. NMR spectra

A. Sundgren, J. J. Barchi Jr. / Carbohydrate Research 343 (2008) 1594–1604
1601
were recorded on a Varian Inova 400 instrument with
residual CHCl3 (7.26 ppm) as the internal standard at
0 °C after H-Gly-OtBu (152 mg, 904 lmol) was added,
stirring was continued for 18 h and a second portion
of 0.5 M HOBt in DMF (4.70 mL, 2.35 mmol) and DIP-
CDI (362 lL, 2.35 mmol) was added. After 48 h the sol-
vents were evaporated and the residue was purified by
column chromatography to give compound 14
(534 mg, 86%). 1H NMR (CDCl3): d 1.46 (s, 9H,
(CH3)3C), 2.51 (t, 2H, J = 6.0 Hz), 3.40 (q, 2H,
J = 5.6 Hz), 3.57 (t, 2H, J = 4.8 Hz), 3.62–3.64 (m, 20
H), 3.74 (t, 2H, J = 5.6 Hz), 3.92 (d, 2H, J = 5.6 Hz),
4.22 (t, 1H, J = 6.8 Hz), 4.40 (d, 2H, J = 6.8 Hz), 7.32
(dt, 2H, J = 1.2 Hz, J = 7.6 Hz, aromatic), 7.40 (dt,
2H, J= 0.8 Hz, J = 7.6 Hz), 7.61 (d, 2H, J = 7.6 Hz),
7.76 (d, 2H, J = 7.6 Hz); 13C NMR (CDCl3): d 28.1,
36.7, 42.0, 47.3, 67.1, 70.30, 70.32, 70.5, 70.6, 81.6,
120.0, 125.1, 127.1, 127.7, 128.2, 129.1, 141.3, 144.0,
169.1, 171.7; HRMS [C36H52N2O11+Na]+: calcd
711.347, found: 711.347.
1
frequencies of 399.74 MHz for H and 100.51 MHz for
13C. Assignments were based on gCOSY, TOCSY,
1
ROESY, and 13C/DEPT experiments. H NMR data
are tabulated in the order of multiplicity (s, singlet; d,
doublet; dd, doublet of doublets; dt, double of triplets;
t, triplet; q, quartet; m, multiplet; br s, broad singlet),
number of protons, and coupling constant(s) in Hertz.
Specific optical rotations were determined using JAS-
CO-P1010 polarimeter in 0.5 dm cuvette at 589 nm in
chloroform. Five consecutive measurements were taken
and the average value is reported. High resolution mass
spectra were performed by Mass Spectrometry Facility
at University of California, Riverside. Elemental analy-
ses were performed by Atlantic Microlab, Inc., Nor-
cross, GA and Galbraith Laboratories Inc., Knoxville,
TN. Transmission electron micrographs were performed
on a Hitachi H-7000 microscope equipped with a Gatan
digital camera operating at 75 kV. A Malvern Zetasizer
Nano ZS instrument (Southborough, MA) with back
scattering detector was used for measuring the hydro-
dynamic size (diameter) in batch mode at 25 °C in a dis-
posable low volume polystyrene microcuvette. Samples
were measured at a concentration of 0.4 mg/mL or
0.2 mg/mL in both H2O and PBS. Samples were filtered
through a 0.1 lm filter before a minimum of ten mea-
surements were made. Hydrodynamic size is reported
as the intensity-weighted average over all size popula-
tions (Z-avg), and the volume-weighted average over a
particular range of size populations corresponding to
the most prominent peak in the % volume distribution
(Vol-Peak).
4.3. 7-(Thioacetyl)heptanoic acid (15)
Heptenoic acid (100 mg, 781 lmol) and AIBN (20 mg)
were dissolved in MeOH (5 mL) containing thioacetic
acid (200 lL). The flask was then irradiated with a
350 W UV lamp for 6 h, concentrated, and azeotroped
with toluene. The residue was purified by column chro-
1
matography to yield compound 15 (133 mg, 84%). H
NMR (CDCl3): d 1.36–1.39 (m, 4H), 1.55–1.58 (m,
2H), 1.61–1.67 (m, 2H), 2.32 (s, 3H, CH3COS), 2.35 (t,
2H, J = 7.6 Hz), 2.86 (t, 2H, J = 7.2 Hz); 13C NMR
(CDCl3): d 24.6, 28.5, 28.6, 29.1, 29.4, 30.8, 34.1,
180.2, 196.2. HRMS [C9H16O3S+Na]+: calcd 227.072,
found: 227.072.
4.1. General procedure for synthesis of gold nanoparticles
4.4. tert-Butyl 3,25-dioxo-6,9,12,15,18,21-hexaoxa-31-
acetylthio-24-azahentriacontan-1-oate (16)
Thiol (1 equiv) and 58 mM HAuCl4 (2.75 equiv) were
added to water (10 mL/lmol thiol) and the obtained yel-
low solution was cooled to 0 °C. 0.1% NaBH4 in water
(1 mL/lmol thiol) was then added over 10 min where-
upon the color changed to red or purple over the first
minute of addition. This solution was then stirred at
0 °C for 2 h and at rt for another 16 h. The solution
was concentrated to about 5 mL, purified utilizing a Cen-
triplus 30K filter, and the obtained pure solution of the
particles was lyophilized to obtain a dark-purple solid.
Compound 14 (518 mg, 753 lmol) was dissolved in
CH2Cl2 (8 mL) and piperidine (3 mL) and the solution
was stirred for 3 h, concentrated, and azeotroped with
toluene. The residue was filtered through a short silica
column to give the crude amine (386 mg), which was dis-
solved in CH2Cl2 (5 mL) together with 14 (154 mg,
753 lmol) followed by addition of 0.5 M HOBt in
DMF (1.96 mL, 979 lmol) and DIPCDI (150 (150 lL,
979 lmol). The reaction mixture was stirred for 18 h,
concentrated and the residue purified by column chro-
matography to give 16 (421 mg, 86%). 1H NMR
(CDCl3): d 1.33–1.37 (m, 4H), 1.46 (s, 9H, (CH3)3C)),
1.55–1.61 (m, 2H), 1.62–1.67 (m, 2H), 2.18 (t, 2H,
J = 7.6 Hz). 2.33 (s, 3H, CH3COS), 2.53 (t, 2H,
J = 6.0 Hz), 2.86 (t, 2H, J = 7.2 Hz), 3.42–3.47 (m,
2H), 3.55 (t, 2H, J = 4.8 Hz), 3.62–3.64 (m, 22 H),
3.76 (t, 2H, J = 5.6 Hz), 3.93 (d, 2H, J = 5.6 Hz); Anal.
Calcd for C30H56N2O11S: C, 55.19; H, 8.65; N, 4.29.
Found: C, 54.91; H, 8.42; N, 4.17.
4.2. t-Butyl N-Fmoc-1-amino-21-oxo-3,6,9,12,15,18-
hexaoxa-22-azatetracosan-24-oate (14)
N-Fmoc-1-amino-3,6,9,12,15,18-hexaoxahenicosan-21-
oic acid (Available from NeoMPS Inc., San Diego, CA)
(520 mg, 904 lmol) was dissolved in CH2Cl2 (3 mL),
cooled to 0 °C and treated with a solution of 0.5 M
HOBt in DMF (2.36 mL, 1.18 mmol) followed by DIP-
CDI (180 lL, 1.18 mmol). After stirring for 30 min at

1602
A. Sundgren, J. J. Barchi Jr. / Carbohydrate Research 343 (2008) 1594–1604
4.5. 3,25-Dioxo-6,9,12,15,18,21-hexaoxa-31-mercapto-
heptanamido-24-azahentriacontan-1-oic acid (17)
dine for Fmoc-deprotection. Coupling of the glycosyl-
ated amino acid (1R,2S)-N-Fmoc-1-amino-1-carboxy-
propan-2-yl 2,3,4,6-tetra-O-acetyl-b-D-galactoporano-
syl-(1?3)-2-acetamido- 4,6-O-benzylidene-2-deoxy-a-
D-galactopyranoside and the linker 17 to the N-terminus
was performed by dissolving the acid in DMF followed
by addition of HOBt (2 equiv) and DIPCDI (2 equiv).
This was stirred for 30 min, added to the resin, and
the resin was then shaken for 5–6 h. Another portion
of HOBt (5 equiv) and DIPCDI (5 equiv) was added
and the resin was shaken for an additional 18 h. Re-
moval of the sugar and thioacetate groups was per-
formed on the resin by treatment with 10% hydrazine
hydrate in EtOH for 18 h. The peptides were cleaved
by treating the resin with a solution of 2.5% 1,2-ethane-
dithiol and 2.5% water in TFA for 2 h. The peptide was
then precipitated by pouring the TFA solution into
ice-cooled Et2O (10 mL) whereupon the peptides
precipitated. The obtained mixture was centrifuged
and the organic phase removed. The residue was puri-
fied by HPLC (water–acetonitrile gradient, each con-
taining 0.1% TFA) yielding the pure peptide/
glycopeptides as a white solid.
Compound 16 (200 mg, 307 lmol) was dissolved in 90%
TFA (5 mL) and the obtained solution was stirred for
40 min. The reaction mixture was concentrated and
the residue was purified by column chromatography to
1
give 17 (132 mg, 72%). H NMR (CDCl3): d 1.29–1.39
(m, 4H), 1.53–1.59 (m, 2H), 163 (p, 2H, J = 6.8 Hz),
2.21 (t, 2H, J = 7.6 Hz), 2.32 (s, 3H, CH3COS), 2.54
(t, 2H, J = 5.6 Hz), 2.85 (t, 2H, J = 7.6 Hz), 3.42–3.46
(m, 2H), 3.57 (t, 2H, J = 5.2 Hz), 3.64–3.67 (m, 20H),
3.74 (t, 2H, J = 5.2 Hz), 4.07 (d, 2H, J = 5.2 Hz); 13C
NMR (CDCl3): d 25.6, 28.5, 28.8, 29.1, 29.4, 30.7,
36.5, 39.4, 41.6, 67.1, 70.0, 70.1, 70.27, 70.33, 70.4,
70.47, 70.50, 70.52, 70.6, 174.0, 196.2; HRMS
[C26H48N2O11S+Na]+: calcd 619.288, found: 619.288;
Anal. Calcd for C26H48N2O11S: C, 52.33; H, 8.11; N,
4.69. Found: C, 52.02; H, 7.89; N, 4.71.
4.6. N-(2-(2-Hydroxyethylamino)-2-oxoethyl)-1-(7-mer-
captoheptanamido)-3,6,9,12,15,18-hexaoxahenicosan-21-
amide (19)
Compound 17 (97 mg, 163 lmol) was dissolved in
CH2Cl2 (1 mL), a solution of 0.5 M HOBt in DMF
(976 lL, 488 lmol) and DIPCDI (61 mg, 488 lmol)
was added and the obtained mixture was stirred for
10 min. 2-Amino-ethanol (30 lL, 488 lmol) was added
and the reaction mixture was stirred for 18 h. After con-
centration, the residue was filtered through a silica col-
umn (gradient from hexane to 10% MeOH in EtOAc)
and the crude product was dissolved in 10% hydrazine
hydrate in EtOH (3 mL). The solution was stirred for
16 h, concentrated, and azeotroped with toluene. The
residue was dissolved in water (1 mL) containing dithio-
threitol (10 mg), stirred under argon for 14 h, and then
filtered through a reversed phase column to give 19
(91 mg, 85%). 1H NMR (CDCl3): d 1.14 (p, 2H,
J = 7.2 Hz), 1.23 (p, 2H, J = 7.2 Hz), 1.41–1.47 (m,
4H), 2.10 (t, 2H, J = 7.6 Hz), 2.38 (t, 2H, J = 7.2 Hz),
2.46 (t, 2H, J = 6.0 Hz), 2.78 (s, 2H), 2.89 (s, 2H), 3.23
(t, 2H, J = 5.6 Hz), 3.46 (t, 2H, J = 5.6 Hz), 3.53–3.54
(m, 24H), 3.65 (t, 2H, J = 6.0 Hz), 3.76 (s, 1H), 3.94
(s, 1H); 13C NMR (CDCl3): d 23.6, 25.2, 27.1, 27.5,
32.8, 35.6, 38.8, 40.8, 41.4, 66.5, 68.8, 69.3, 69.4, 69.46,
69.53, 169.8, 174.1, 176.9; HRMS [C26H51N3O10S+
Na]+: calcd 620.319, found: 620.311.
4.7.1. HS-linker-TSSASTGHATPLPVTD (11). Fol-
lowing the general procedure yielded 11 (43 mg, 63%).
1H NMR (selected data) (CDCl3): d 2.38 (t, 2H,
J = 6.8 Hz), 2.47 (t, 2H, J = 6.0 Hz), 2.71 (dt, 1H,
J = 7.6 Hz, J = 16.8 Hz), 2.76 (dt, 1H, J = 5.6 Hz,
J = 17.2 Hz), 2.98 (dd, 1H, J = 8.4 Hz, J = 15.6 Hz),
3.14 (dd, 1H, J = 5.6 Hz, J = 15.6 Hz), 3.23 (t, 2H,
J = 5.6 Hz), 3.46 (t, 2H, J = 5.2 Hz), 3.65 (t, 2H,
J = 6.0 Hz), 3.87 (s, 2H), 4.57 (dd, 1H, J = 5.2 Hz,
J = 7.6 Hz), 7.14 (d, 1H, J = 1.2 Hz), 8.47 (d, 1H,
J = 1.2 Hz); HRMS [C88H150N21O34S]: calcd 2077.04,
found: 2077.04.
4.7.2. HS-linker-TSSASTGHAT(Galb1?3GalNAca)-
PLPVTD (12). Following the general procedure
yielded 13 (38 mg, 47%). 1H NMR (selected data)
(CDCl3): d 2.38 (t, 2H, J = 6.8 Hz), 2.47 (t, 2H,
J = 6.0 Hz), 2.71 (dt, 1H, J = 7.6 Hz, J = 16.8 Hz),
2.76 (dt, 1H, J = 5.6 Hz, J = 17.2 Hz), 2.98 (dd, 1H,
J = 8.4 Hz, J = 15.6 Hz), 3.14 (dd, 1H, J = 5.6 Hz,
J = 15.6 Hz), 3.23 (t, 2H, J = 5.6 Hz), 3.35 (t, 1H,
J = 2.0 Hz, J = 7.6 Hz), 3.46 (t, 2H, J = 5.2 Hz), 3.65
(t, 2H, J = 6.0 Hz), 3.87 (s, 2H), 4.74 (d, 1H,
J = 3.6 Hz, GalNAc H-1I), 7.15 (d, 1H, J = 1.2 Hz),
8.47 (d, 1H, J = 1.2 Hz); HRMS [C102H173N22O44S]:
calcd 2442.170, found: 2442.170.
4.7. General procedure for synthesis of peptides/
glycopeptides
4.7.3.
HS-linker-TSSAST(Galb1?3GalNAca)GHA-
Rink Amide AM Resin (33 lmol loading) was used for
all peptides. All common amino acids were coupled on
an Applied Biosystems 433A Peptide Synthesizer using
HBTU–HOBt–DIEA with NMP as solvent and piperi-
TPLPVTD (13). Following the general procedure
yielded 12 (41 mg, 51%). 1H NMR (selected data)
(CDCl3): d 2.38 (t, 2H, J = 6.8 Hz), 2.47 (t, 2H,
J = 6.0 Hz), 2.71 (dt, 1H, J = 7.6 Hz, J = 16.8 Hz),

A. Sundgren, J. J. Barchi Jr. / Carbohydrate Research 343 (2008) 1594–1604
1603
13. Chen, Y. F.; Ji, T. H.; Rosenzweig, Z. Nano Lett. 2003, 3,
581–584.
14. de la Fuente, J. D. M.; Penades, S. Tetrahedron: Asym-
metry 2005, 16, 387–391.
15. Svarovsky, S. A.; Barchi, J. J., Jr. De Novo Synthesis of
Biofunctional Carbohydrate-Encapsulated Quantum
Dots. In Frontiers in Modern Carbohydrate Chemistry;
Demchenko, A. V., Ed., 960 ed.; Oxford University Press:
New York, NY, 2007; pp 375–394.
2.76 (dt, 1H, J = 5.6 Hz, J = 17.2 Hz), 2.98 (dd, 1H,
J = 8.4 Hz, J = 15.6 Hz), 3.14 (dd, 1H, J = 5.6 Hz,
J = 15.6 Hz), 3.23 (t, 2H, J = 5.6 Hz), 3.35 (t, 1H,
J = 2.0 Hz, J = 7.6 Hz), 3.46 (t, 2H, J = 5.2 Hz), 3.65
(t, 2H, J = 6.0 Hz), 3.87 (s, 2H), 4.78 (d, 1H,
J = 3.6 Hz, GalNAc H-1I), 7.15 (d, 1H, J = 1.2 Hz),
8.47 (d, 1H, J = 1.2 Hz); HRMS [C102H173N22O44S]:
calcd 2442.170, found: 2442.170.
16. El-Boubbou, K.; Gruden, C.; Huang, X. J. Am. Chem.
Soc. 2007, 129, 13392–13393.
17. Ito, M.; Suzuki, E.; Naiki, M.; Sendo, F.; Arai, S. Int. J.
Cancer 1984, 34, 689–697.
18. Hakomori, S. I. Adv. Cancer Res. 1989, 52, 257–331.
19. Hakomori, S.; Zhang, Y. M. Chem. Biol. 1997, 4, 97–
104.
20. Ojeda, R.; de Paz, J. L.; Barrientos, A. G.; Martin-
Lomas, M.; Penades, S. Carbohydr. Res. 2007, 342, 448–
459.
21. Springer, G. F. Crit. Rev. Oncogesis 1995, 6, 57–85.
22. Zanetti, M.; Lenert, G.; Springer, G. F. Int. Immunol.
1993, 5, 113–119.
23. Cao, Y.; Stosiek, P.; Springer, G. F.; Karsten, U.
Histochem. Cell Biol. 1996, 106, 197–207.
24. Dziadek, S.; Kunz, H. Chem. Rec. 2004, 3, 308–321.
25. Dziadek, S.; Hobel, A.; Schmitt, E.; Kunz, H. Angew.
Chem., Int. Ed. 2005, 44, 7630–7635.
Acknowledgments
The authors thank Anil Patri, Jeff Clogston, and Jiwen
Zheng of the Nanotechnology Characterization Labora-
tory (DLS data and analysis) and Kunio Nagashima
and Jason de la Cruz of the Image Analysis Laboratory
(TEM data and analysis) for their work and helpful
discussions. Part of this work was supported by the
Intramural Research Program of the National Cancer
Institute.
26. Xu, Y. F.; Sette, A.; Sidney, J.; Gendler, S. J.; Franco, A.
Immunol. Cell Biol. 2005, 83, 440–448.
Supplementary data
27. Glinsky, V. V.; Glinsky, G. V.; Rittenhouse-Olson, K.;
Huflejt, M. E.; Glinskii, O. V.; Deutscher, S. L.; Quinn, T.
P. Cancer Res. 2001, 61, 4851–4857.
28. Khaldoyanidi, S. K.; Glinsky, V. V.; Sikora, L.; Glinskii,
A. B.; Mossine, V. V.; Quinn, T. P.; Glinsky, G. V.;
Sriramarao, P. J. Biol. Chem. 2003, 278, 4127–4134.
29. Yu, L. G.; Andrews, N.; Zhao, Q.; McKean, D.; Williams,
J. F.; Connor, L. J.; Gerasimenko, O. V.; Hilkens, J.;
Hirabayashi, J.; Kasai, K.; Rhodes, J. M. J. Biol. Chem.
2007, 282, 773–781.
30. Svarovsky, S. A.; Szekely, Z.; Barchi, J. J. Tetrahedron:
Asymmetry 2005, 16, 587–598.
31. Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.;
Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801–
802.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2008.05.003.
References
1. Sonvico, F.; Dubernet, C.; Colombo, P.; Couvreur, P.
Curr. Pharm. Des. 2005, 11, 2091–2105.
2. Thorek, D. L. J.; Chen, A.; Czupryna, J.; Tsourkas, A.
Ann. Biomed. Eng. 2006, 34, 23–38.
3. Fu, A. H.; Gu, W. W.; Larabell, C.; Alivisatos, A. P. Curr.
Opin. Neurobiol. 2005, 15, 568–575.
32. Brust, M.; Fink, J.; Bethell, D.; Schiffrin, D. J.; Kiely, C. J.
Chem. Soc., Chem. Commun. 1995, 1655–1656.
33. Lin, C. C.; Yeh, Y. C.; Yang, C. Y.; Chen, G. F.; Chen, Y.
C.; Wu, Y. C.; Chen, C. C. Chem. Commun. 2003, 2920–
2921.
34. de Souza, A. C.; Halkes, K. M.; Meeldijk, J. D.; Verkleij,
A. J.; Vliegenthart, J. F. G.; Kamerling, J. P. E. J. Org.
Chem. 2004, 4323–4339.
35. de Souza, A. C. ; Halkes, K. M.; Meeldijk, J. D.; Verkleij,
A. J.; Vliegenthart, J. F. G.; Kamerling, J. P. ChemBio-
Chem 2005, 6, 828–831.
36. Fifis, T.; Mottram, P.; Bogdanoska, V.; Hanley, J.;
Plebanski, M. Vaccine 2004, 23, 258–266.
37. Fifis, T.; Gamvrellis, A.; Crimeen-Irwin, B.; Pietersz, G.
A.; Li, J.; Mottram, P. L.; McKenzie, I. F. C.; Plebanski,
M. J. Immunol. 2004, 173, 3148–3154.
38. Hollingsworth, M. A.; Swanson, B. J. Nat. Rev. Cancer
2004, 4, 45–60.
39. Choudhury, A.; Moniaux, N.; Winpenny, J. P.; Hollings-
worth, M. A.; Aubert, J. P.; Batra, S. K. J. Biochem.
(Tokyo) 2000, 128, 233–243.
40. Andrianifahanana, M.; Moniaux, N.; Schmied, B. M.;
Ringel, J.; Friess, H.; Hollingsworth, M. A.; Buchler, M.
4. Barrientos, A. G.; de la Fuente, J. M.; Rojas, T. C.;
Fernandez, A.; Penades, S. Chem.-A Eur. J. 2003, 9, 1909–
1921.
5. de la Fuente, J. M.; Barrientos, A. G.; Rojas, T. C.; Rojo,
J.; Canada, J.; Fernandez, A.; Penades, S. Angew. Chem.,
Int. Ed. 2001, 40, 2257–2261.
6. de Paz, J. L.; Ojeda, R.; Barrientos, A. G.; Penades, S.;
Martin-Lomas, M. Tetrahedron: Asymmetry 2005, 16,
149–158.
7. Rojas, T. C.; de la Fuente, J. M.; Barrientos, A. G.;
Penades, S.; Ponsonnet, L.; Fernandez, A. Adv. Mater.
2002, 14, 585–588.
8. Rojo, J.; Diaz, V.; de la Fuente, J. M.; Segura, I.;
Barrientos, A. G.; Riese, H. H.; Bernade, A.; Penades, S.
ChemBioChem 2004, 5, 291–297.
9. Daniel, M. C.; Astruc, D. Chem. Rev. 2004, 104, 293–
346.
10. Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan,
M. J. Anal. Chem. 1995, 67, 735–743.
11. Robinson, A.; Fang, J. M.; Chou, P. T.; Liao, K. W.; Chu,
R. M.; Lee, S. J. ChemBioChem 2005, 6, 1899–1905.
12. Sun, X. L.; Cui, W. X.; Haller, C.; Chaikof, E. L. Chem-
BioChem 2004, 5, 1593–1596.

1604
A. Sundgren, J. J. Barchi Jr. / Carbohydrate Research 343 (2008) 1594–1604
W.; Aubert, J. P.; Batra, S. K. Clin. Cancer Res. 2001, 7,
4033–4040.
41. Khorrami, A. M.; Choudhury, A.; Andrianifahanana, M.;
Varshney, G. C.; Bhattacharyya, S. N.; Hollingsworth, M.
A.; Kaufman, B.; Batra, S. K. J. Biochem. (Tokyo). 2002,
131, 21–29.
42. Choudhury, A.; Moniaux, N.; Ulrich, A. B.; Schmied, B.
M.; Standop, J.; Pour, P. M.; Gendler, S. J.; Hollings-
worth, M. A.; Aubert, J. P.; Batra, S. K. Br. J. Cancer
2004, 90, 657–664.
43. Liu, X. O.; Atwater, M.; Wang, J. H.; Huo, Q. Colloids
Surf., B 2007, 58, 3–7.