Novel Tn antigen-containing neoglycopeptides: synthesis and evaluation as anti tumor vaccines.

Article abstract of DOI:10.1016/S0968-0896(01)00433-3

The fully unprotected alpha-C-glycosyl analogue of N-acetylgalactosamine 9 was conjugated by a non-natural oxime bond to the segment peptides (328--340)OVA and (327--339)OVA, affording neoglycopeptides 1--2 and 3, having one or two sugar units, respective

Full text of DOI:10.1016/S0968-0896(01)00433-3

Bioorganic & Medicinal Chemistry 10 (2002) 1639–1646
Novel Tn Antigen-Containing Neoglycopeptides:
Synthesis and Evaluation as Anti Tumor Vaccines
Laura Cipolla,* Maria Rescigno,y Antonella Leone,{ Francesco Peri,
Barbara La Ferla and Francesco Nicotra*
`
Department of Biotechnology and Biosciences, Universita degli Studi di Milano-Bicocca, P.za della Scienza 2, 20126 Milan, Italy
Received 25 September 2001; accepted 17 December 2001
Abstract—The fully unprotected a-C-glycosyl analogue of N-acetylgalactosamine 9 was conjugated by a non-natural oxime bond to
the segment peptides 328À340OVA and 327À339OVA, affording neoglycopeptides 1–2 and 3, having one or two sugar units, respec-
tively. The three neoglycopeptides were tested in vitro in an antigen presentation assay as antitumor vaccines. Neoglycopeptides 1–3
could be presented to and recognized by the T cell receptor; neoglycopeptide 3, bearing two B-epitopes, was presented to the TCR
with higher efficiency, compared to neoglycopeptide 2, having only one B-epitope. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
lymphocytes.5 This argument is also true for vaccines
against tumors since they do not have to provoke only
The Tn antigen (GalNAca-O-Ser/Thr) is a carbohy-
drate epitope overexpressed in various human carcino-
mas1 of epithelial origin, which comprise by far the
largest group of malignant tumors. In most of these
tumors the Tn antigen, one of the blood group pre-
cursor antigens, is found exposed on the external sur-
face membranes. In all other tissues, the Tn antigen is
masked and not accessible to the immune system.
Moreover the extent of Tn expression often correlates
with carcinoma differentiation and aggressiveness.
Thus, carcinomas have antigens recognized as foreign
by the host immune system and that could be exploited
to induce an anticancer immune response.2 The ultimate
goal in the design of an antitumor vaccine is to develop
vaccines specific for a tumor antigen (active specific
immunotherapy, ASI)3 using well characterized chemi-
cally synthesized antigens as immunogens. Unfortu-
nately, synthetic antigens are, usually, poorly or not at
all immunogenic, being low molecular weight com-
pounds or self-antigens.4 In general, a good vaccine has
to induce an immunologic memory of long duration,
that means that it must stimulate memory B and T
an immediate and strong humoral response, such to
eliminate the tumor cells present in the organism, but
also a long-lasting protection against the formation of
micrometastases.
The design of an antitumor vaccine is not that simple: in
order to elicit an IgG humoral response, B cells have to
internalize the antigen through their B cell receptor and
present antigenic peptides on MHC II molecules for T
cell help. Thus, a bivalent antigen constituted by a sac-
charidic B epitope and a T epitope is desirable. To do
so, saccharidic tumor antigens have been coupled to
suitable protein carriers,6 including serum albumin from
different sources, as chicken serum albumin (CSA),7
bovine serum albumin (BSA),8 human serum albumine
(HSA),9 keyhole limpet hemocyanin (KLH),8b,10 or
ovine submaxillary mucin (OSM).11 The latter was used
in a very elegant study by Singhal et al. in the early
1990s in which mice were immunized against a highly
invasive tumor, the adenocarcinoma TA3-Ha, using as
an immunogen the Asialo Ovine Submaxillary Mucin
(A-OSM) displaying a great number of Tn epitopes. The
glycosylated protein elicited protection against the
tumor, whereas the unglycosylated mucine did not, thus
confirming the specificity for the saccharidic Tn antigen
and not for the carrier protein. However, this approach
presents two main drawbacks: the carbohydrate con-
tents are usually low in protein conjugates and the con-
struction has a non-defined chemical structure (i.e., the
*Corresponding author. Tel.: +39-02-6448-3453; fax: +39-02-6448-
3565; e-mail: laura.cipolla@unimib.it
yPresent address: Istituto Oncologico Europeo, Via Ripamonti 435,
20141 Milan, Italy.
{Present address: Biosearch Italia, Via Lepetit 34, 21040 Gerenzano,
Italy.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00433-3

1640
L. Cipolla et al. / Bioorg. Med. Chem. 10 (2002) 1639–1646
number of saccharide units linked to the carrier protein).
To date, several studies with conjugate cancer vaccines
containing natural or synthetic antigens have been
reported and many approaches have been explored to
increase the immunogenicity of these molecules.6À10,12 In
particular, a novel concept for the antigen construction
of antitumour vaccines has been recently developed;
according to the reported strategy,12j a tumor-asso-
ciated glycopeptide antigen (MUC1) was combined with
a T-cell epitope of tetanus toxin. Moreover, multiple
antigen glycopeptides (MAG)13 have been developed:
since the antigens occur on the cell surface as multiple,
not single, molecules, it is not surprising that the multi-
ple antigen presentation on synthetic glycoconjugates
results in their enhanced immunogenicity.14
aqueous solution; to date, different reactions have pro-
ven useful for chemoselective ligation.18 In our case,
chemoselectivity was achieved coupling an aminoxy
group properly introduced in the peptide backbone with
a keto function present on the C-glycosyl analogue of
the Tn antigen, affording the corresponding oxime.19
Results and Discussion
The synthesis of three possible vaccines, 1–3, was per-
formed by a convergent approach based on the chemo-
selective coupling of a-C-glycosyl ketone 920 analogue
of N-acetyl-galactosamine with peptides 6–8 (Scheme 1).
The aminooxy acetic acid linker allowed the conjuga-
tion of the T-epitope to the B-epitope (Fig. 1). The T-
cell epitope of choice is the short peptide fragment 327–
339 of ovalbumin (327À339OVA) from chicken.21
In this context, we focused our attention on the synth-
esis of neoglycopeptides 1–3 (Fig. 1) as potential small
molecular weight antitumor vaccines, constructed by
covalently linking a short peptide, as the T-epitope, to
the B-epitope, the Tn antigen; in order to achieve
increased stability toward in vivo degradation, the two
moieties are connected by a C-glycosidic bond, in place
of the natural O-glycosidic linkage.
Due to contradictory numbering of the protein, two
different sequences were initially synthesized for the
attachment of one sugar unit. Fragment peptide 6
derived from previous work by Grey et al. in vitro
immunological studies,22 while peptide 7 corresponded
to NCBI database.21,23
C-Glycosyl conjugates are, in fact, stable against both
chemical and enzymatic degradation and have gained
considerable attention in recent years,15 but their use in
biological investigation has been quite rare.16 In addi-
tion, a technique based on the concept of chemoselec-
tive ligation17 has been applied to the site-specific
attachment of the sugar moiety to peptides. Chemo-
selective ligation involves the coupling of two uniquely
reactive functional groups with such selectivity that no
protecting groups are needed, and can be performed in
The peptides were assembled using standard Fmoc pro-
tocol for solid-phase peptide synthesis.24 All amino
acids were attached to the resin carrying an Na-Fmoc
protective group and acid labile side-chain protecting
groups. The attachment of the growing chain to the
resin was made by an acid labile linker, in order to
cleave the peptides from the solid support and simulta-
neously hydrolyze the side-chain protecting groups. The
Figure 1. Structure of neoglycopeptides as potential anticancer vac-
cines.
Scheme 1. Synthetic strategy for the preparation of neoglycopeptides
1–3.

L. Cipolla et al. / Bioorg. Med. Chem. 10 (2002) 1639–1646
1641
last step of the solid-phase synthesis was the introduc-
tion of one or two linkers, namely the N-Fmoc ami-
nooxy acetic acid 4, by in situ preactivation or as the
corresponding succinate derivative 5, which introduce in
the peptide the required function for the chemoselective
ligation with the C-glycosidic B-epitope.20 In order to
have a multiple antigen presentation, Na,Ne-Fmoc-
lysine is added to the N-terminus of the peptide as the
last amino acid; deprotection of the two amino function
of lysine by standard conditions (piperidine in DMF)
and coupling with N-Fmoc aminooxy acetic acid
allowed the introduction of two sugar moieties into the
327À339OVA peptide. Conjugation of the a-C-glycosyl
ketone 9 to the peptides 6–8 was performed in acetate
buffer at pH 4.5 and monitored by reversed-phase
HPLC (RP-HPLC). All the neoglycopeptides presented
two isomeric forms in equilibrium, due to the cis/trans
isomerism of the oxime bond, detectable by HPLC
analysis and characteristic of oxime-linked glycoconju-
gates.19
manner with T-cell hybridomas in 96-well tissue culture
plates; as a control the same proliferation assay was
performed with synthetic peptide 327À339OVA. In order
to test whether the neoglycopeptides needed to be
internalized and processed to be exposed on the cell
surface in association with MHC molecules, DC were
fixed with glutaraldehyde to block internalization and
processing. In other words, if the antigen needed to be
processed in order to be presented and recognised by the
TCR, it would not be presented correctly with fixed
APC. This evidence is clearly represented in Figure 2:
the whole ovalbumin needs to be processed and reduced
to fragment peptide 327À339OVA in order to form the
peptide–MHC II complex and stimulate T-cells. When
DC are fixed, the peptide fragment cannot be formed
and there is no production of IL-2.
Hence, in order to check if the aminooxy acetic linker
and the additional lysine residue could interfere with the
binding to MHC II and with presentation to T cells,
peptides 6–8 were incubated both with fixed and non-
fixed APC. It resulted that for peptides 7 and 8 the
Neoglycopeptides 1–3 were tested in vitro in an antigen
presentation assay either to the T-cell hybridoma BO
97.10,25 or T cell receptor transgenic T cells from DO
11.10mice, 25 both specific for the peptide 327À339OVA in
association with the molecule MHC of class II I-Ab on
antigen presenting cells (APC). Dendritic cells (DC) are
the only APC able to activate naive T cells, that is, T
cells that have never seen the antigen.26 Recognition of
peptide–MHC II complexes on the dendritic cell surface
results in secretion of interleukin-2 (IL-2) by T-cell
hybridomas or in transgenic T-cell proliferation.
Interleukin production correlates with the ability of
the T-cell receptor (TCR) to recognize the complex
MHC–peptide on APC, that is the interleukin produced
by the hybridomas reflects the magnitude of the immune
response. Secreted IL-2 was then determined in a
radioactive proliferation assay by measuring the incor-
poration of [3H]-thymidine by the IL-2 dependent T-cell
clone CTLL.27 When transgenic T cells were used for
the assay, their proliferation was measured directly as
[3H]-thymidine incorporation.
Different assays were then performed (Fig. 3). First, we
assessed if the additional aminooxy linker would some-
how interfere in the recognition between the MHC II
molecule and the 327À339OVA-specific TCR. Thus, the
three peptides 6–8 were incubated in a dose dependent
Figure 3. Proliferation of CTLL cells dependent on IL-2 production
by hybridoma BO 97.10, incubated with both fixed and non-fixed DC,
and the antigen at increasing concentrations: (a) petide 6 and neogly-
copeptide 1 showed weaker response compared to the reference pep-
tide 327À339OVA; (b) neoglycopeptide 2 showed comparable activity
than the reference ovalbumin fragment; (c) neoglycopeptide 3 showed
a higher response than the 327À339OVA when using non-fixed cells.
Figure 2. Proliferation of CTLL cells dependent on IL-2 production
by hybridoma BO 97.10incubated with APC and whole ovalbumin.

1642
L. Cipolla et al. / Bioorg. Med. Chem. 10 (2002) 1639–1646
addition of the linker and of the additional lysine at the
N-terminus of the sequence did not interfere with the
correct presentation of the peptide–MHC II complex to
the TCR (data not shown). A different conclusion could
be drawn for peptide 6 (Fig. 3a): in this case a lower
efficiency in the presentation was observed both with
fixed and non-fixed cells. This can be attributed to the
‘incorrect’ peptide sequence, which is shifted by one
amino acid (328À340OVA), rather than to the additional
groups. Obviously, a similar trend was observed for the
neoglycopeptide 1, which was presented to the TCR less
efficiently than the reference fragment peptide 327À339OVA
but similarly to peptide 6 (Fig. 3a). Due to the low effi-
ciency of presentation of neoglycopeptide 1, the
sequence 327À339OVA was chosen for the synthesis of
neoglycopeptides 2 and 3, bearing one or two B-epi-
topes, respectively. Identical proliferation tests were
then performed with neoglycopeptides 2 and 3 (Fig. 3b
and c, respectively). In both cases, the neoglycopeptides
could be presented to the TCR also in the absence of
internalization and processing, indicating that binding
from outside the cells could occur and that the presence
of the sugar would not impede TCR–MHC–peptide
recognition, as previously observed by Grey22b and
Unanue.28
of induction of immune response and antibody produc-
tion to the natural tumour associated antigen Tn, after
immunization with neoglycopeptide 3 in the presence or
absence of exogenous adjuvant. In addition, in order to
evaluate the ability of C-glycosidic analogue of N-acetyl
galactosamine to simulate the natural counterpart, pro-
tection experiments to the highly aggressive TA3-Ha
adenocarcinoma will be performed.
Experimental
General
All solvents were dried over molecular sieves, for at
least 24 h prior to use. Thin-layer chromatography
(TLC) was performed on Silica Gel 60F
plates
254
(Merck) with detection with UV light when possible, or
charring with a solution containing concd H2SO4/
EtOH/H2O in a ratio of 5/45/45. Flash column chro-
matography was performed on silica gel 230–400 mesh
(Merck).
All resins and protected amino acids were purchased
from Calbiochem-Novabiochem AG (CH). Na-Fmoc-
protected amino acids had the following protecting
groups for their side chains: pentamethylchromatin
(Pmc) for arginine; tert-butyl for glutamic acid; triphe-
nylmethyl (Trt) for histidine and asparagine.
Interestingly, neoglycopeptide 3, bearing two sugar
moieties, was presented by non-fixed cells with a higher
efficiency than the 327À339OVA peptide at low con-
centrations (Fig. 3c). This suggested that the glycidic
moieties might somehow facilitate either the inter-
nalization of the peptide or the activation of T cells may
be due to increased costimulation by DC. Activation of
DC results in the upregulation of MHC and costimula-
tory molecules which are necessary for the correct sti-
mulation of T cells. Cross-linking of surface receptors
can lead to DC activation. Thus, if the sugar would
induce the cross-linking of a putative receptor expressed
on the surface of DC, we would expect that only neo-
glycopeptide 3, bearing the two sugar moieties, would
induce the upregulation of costimulatory molecules on
DC. Indeed, as shown in Figure 4, the B7.2 molecule (a
prototype of costimulatory molecules expressed by DC)
was upregulated only in the presence of neoglycopeptide
3. A possible artefactual effect due to lipopolysaccharide
(LPS) contamination could be excluded because den-
dritic cells for this experiment were derived from bone-
marrow of B10ScCr mice which lack the expression of
TLR4, the transducer of cell activation induced by
LPS.29
1H and 13C NMR spectra were recorded at 300 MHz on
a Brucker AC 300 instrument using CDCl3 as solvent
unless otherwise stated. Chemical shifts are reported in
ppm downfield from TMS as an internal standard. Mass
In conclusion, conjugation of one or two sugar moieties
to the peptidic OVA T-cell epitope does not interfere
with the capacity of dendritic cells to present the anti-
genic peptide to OVA specific T cells. By contrast, the
presence of two sugar moieties acts as an adjuvant on
DC by upregulating the expression of B7.2 molecule
and increasing the presentation of the OVA peptide to T
cells. This suggests that the N-acetyl galactosamine
analogue not only can be used as a B cell epitope, but in
multiple copies, it can target the vaccine to dendritic
cells by binding and cross-linking its surface receptor.
Future perspectives of this work include in vivo studies
Figure 4. Upregulation of B7.2 molecule after incubation of DC with
compounds 9, 8, 2, 3, respectively. Only neoglycopeptide 3 induced
significant upregulation of B7.2. The percentage of B7.2 high cells
corresponding to the M1 statistical area is reported.

L. Cipolla et al. / Bioorg. Med. Chem. 10 (2002) 1639–1646
1643
spectra were recorded on a MALDI2 Kompact Kratos
instrument, using gentisic acid (DHB) or a-cyano-4-
hydroxycinnamic acid (CHCA) as matrices.
white solid. Mp 95–97 ꢀC; 1H NMR: d 8.29 (s, 1H; NH),
7.80–7.27 (m, 8H; CHarom), 4.72 (s, 2H; H-2), 4.47 (d,
J=6.9 Hz, 2H; CH2-Fmoc), 4.22 (t, J=6.9 Hz, 1H;
CHFmoc), 2.82 (s, 4H; CH2CON); MALDI-TOF-MS:
calcd for C21H18N2O7 410.4, found m/z (M+Na)+
433.6, (M+K)+ 449.7.
Colorimetric assays. TNBS (2,4,6-trinitrobenzenesulfonic
30
acid) test. Two standard solution are prepared as fol-
lows. Solution A: a 10% solution of diisopropylethyl-
amine (DIPEA) in dry DMF; solution B: a 1% solution
of TNBS in dry DMF. A small portion of the resin,
previously washed with ethanol, is suspended in a mix-
ture of equal amounts of the two solutions. After a few
minutes, the presence of unreacted amino groups will be
detected by the appeareance of a red colour on the
beads.
Peptide 6. Synthesis. The synthesis was carried out using
1 g of a Wang resin (100–200 mesh, maximum loading
0.76 mmol/g). The first amino acid (Na-Fmoc-Gly-OH)
was coupled to the resin upon activation of the carboxylic
group as 1-benzotriazolyl ester prepared in situ by reac-
tion of the amino acid (684 mg, 2.3 mmol, 3 equiv), 1-
hydroxybenzotriazole (HOBt, 311 mg, 2.3 mmol, 3 equiv)
catalytic 4-dimethylaminopyridine (DMAP, 56 mg,
0.46 mmol, 0.2 equiv), 1,3-diisopropylcarbodiimide
(DIC, 358 mL, 2.3 mmol, 3 equiv) in dry DMF (15 mL).
After 30min, the solution was added to the resin, which
was mechanically stirred for 16 h. Resin was washed
with DMF and dichloromethane between each coupling
and deprotection step. After the first coupling, a cap-
ping step of possibly unreacted hydroxyl functions on
the resin was performed; the resin was reacted with a
10% solution of acetic anhydride in dry DMF for 2 h
(10mL); afterwards, the resin was washed as usual. Na-
Fmoc deprotection of the peptide resin was performed
by treatment with 20% piperidine in DMF (3Â10min).
All subsequent Na-Fmoc amino acids (2.3 mmol, 3
equiv) were coupled after preactivation with HOBt
(311 mg, 2.3 mmol, 3 equiv), DIC (358 mL, 2.3 mmol, 3
equiv), DIPEA (400 mL, 2.3 mmol, 3 equiv), in dry
DMF (15 mL) for 30min. As the last step on solid-
phase 9-fluorenylmethoxycarbonyl-aminooxyacetic acid
succinic ester 5 (944 mg, 2.3 mmol, 3 equiv) was added
to the peptide resin dissolved in dry DMF (10mL),
using DIPEA (400 mL, 2.3 mmol, 3 equiv), as the base.
The completeness of all reactions was monitored by
TNBS test.
31
Bromophenol blue test.
A few drops of a 50 mm
solution of bromophenol blue in dry DMF is added to a
small portion of resin previously washed with DMF; a
blue color of the beads indicates the presence of
unreacted amino groups.
Purification of peptides was performed by preparative
reversed-phase HPLC on a Waters HPLC system
equipped with a Merck LiChrosper 100 RP18 column
(250Â10mm; 10 mm), and eluting with a flow rate of
7 mL/min with eluent A (0.1% TFA in water) and elu-
ent B (0.1% TFA in 90% aq acetonitrile). Analytical
HPLC purity checks were performed using a Merck
LiChrosper 100 RP18 column (250Â4 mm; 5 mm), elut-
ing at a flow rate of 1 mL/min and with the same eluent
system. Detection was at 215 and 280nm with an
absorbance detector Waters 2487 dual l.
9-Fluorenylmethoxycarbonyl-aminooxyacetic acid (4).
Aminooxy acetic acid hydrochloride (500 mg,
4.57 mmol) was dissolved in an aqueous solution of
Na2CO3 (1.2 g in 20mL); the solution was cooled to
0 ꢀC with an ice-bath and then 9-fluorenylmethyl
chloroformate (Fmoc-Cl) (1.30g, 5.02 mmol) in dioxane
(10mL) was added dropwise. The reaction mixture was
stirred overnight; the reaction mixture was concentrated,
acidified with 5% aqueous HCl and the product pre-
cipitated from the solution. 1.1 g (77% yield) of pure 4
were obtained as a white solid. Mp 129–131 ꢀC; 1H
NMR (CD3OD): d 7.80–7.29 (m, 8H; CHarom), 4.41 (d,
J=7.0Hz, 2H; CH 2-Fmoc), 4.34 (s, 2H; H-2), 4.18 (t,
J=7.0Hz, 1H; CH Fmoc); MALDI-TOF-MS: calcd for
C17H15NO5 313.3, found m/z (M+Na)+ 336.4,
(M+K)+ 352.0.
Cleavage. Peptide 6 was cleaved from the resin by
incubation with a 95:2.5:2.5 mixture of trifluoroacetic
acid (TFA)/triisopropylsilane32/H2O (10mL). After 2 h,
the resin was removed by filtration and washed carefully
with the cleavage mixture (2Â5 mL). The combined fil-
trates were concentrated, then addition of cold diethyl
ether (10mL) allowed the precipitation of the crude
peptide 6, which finally was filtered and dried.
Purification. Purification of the crude by preparative-
scale HPLC (eluent: linear gradient 10!50% eluent B
in eluent A over 40min), afforded peptide 6 in 10%
yield. The purity was checked by analytical HPLC (elu-
ent: linear gradient 0!100% eluent B in eluent A; ana-
lysis time 30min, tR=12.7 min). MALDI-TOF-MS:
calcd 1504, found m/z (M+H)+ 1505.
9-Fluorenylmethoxycarbonyl-aminooxyacetic acid succi-
nic ester (5). Compound 4 (5.29 g, 16.9 mmol) was dis-
solved in a 1:1 mixture of ethyl acetate/dioxane, and the
solution cooled to 0 ꢀC with an ice-bath; N-hyd-
roxysuccinimide (2.16 g, 18.8 mmol) and dicyclohexyl-
carbodiimide (3.88 g, 18.8 mmol) were sequenctially
added to the solution. After 24 h, the precipitate was fil-
tered off, and the solvent evaporated to dryness under
reduced pressure. The residue was then dissolved in
ethyl acetate, washed with 5% aq NaHCO3, the organic
layer dried over Na2SO4, filtered and evaporated. The
active ester 5 was obtained in 93% yield (6.42 g) as a
Neoglycopeptide 1. Peptide 6 (50 mg, 0.033 mmol) was
dissolved in a buffer solution at pH=4.5 (1.5 mL) pre-
pared adjusting a 0.1 m solution of sodium acetate to
the desired pH with glacial acetic acid. The C-glycoside
920 (11 mg, 0.042 mmol) dissolved in water was then

1644
L. Cipolla et al. / Bioorg. Med. Chem. 10 (2002) 1639–1646
added to the aminooxy peptide. The reaction mixture
was allowed to stir for 90min, and monitored by
reversed-phase analytical HPLC (eluent: linear gradient
10!50% eluent B in eluent A over 40 min; tR=15.2 and
15.5 min). The solution was then freeze-dried and the
crude purified by preparative-scale HPLC (eluent: linear
gradient 15!25% eluent B in eluent A over 40min). 19 mg
of neoglycopeptide 1 were obtained, corresponding to
33% yield, as a mixture of E/Z isomeric forms. MALDI-
TOF-MS: calcd 1748 found m/z (MÀH2O+Na)+ 1752;
amino acid analysis: Ala 4.02 (4), Arg 0.99 (1), Asn 1.01
(1), Gly 2.01 (2), Glu 1.98 (2), His 2.03 (2), Ile 0.98 (1),
Val 1.00 (1).
Neoglycopeptide 2. Peptide 7 (4.0 mg, 0.003 mmol) was
dissolved in a buffer solution at pH 4.5 (300 mL). The C-
glycoside 9 (1.0 mg, 0.004 mmol, 1.3 equiv) dissolved in
water (300 mL) was then added to the aminooxy peptide.
The reaction mixture was stirred for 30min, and mon-
itored by reversed-phase analytical HPLC (eluent: linear
gradient 10!40% eluent B in eluent A over 50 min;
tR=19.1 and 19.6 min). The solution was then freeze-
dried and the crude purified by preparative HPLC (elu-
ent: linear gradient 10!40% eluent B in eluent A over
50min). 3.4 mg of neoglycopeptide 2 (71% yield) were
obtained as a mixture of E/Z isomeric forms. MALDI-
TOF-MS: calcd 1605, found m/z (M+Na)+ 1629;
amino acid analysis: Ala 5.03 (5), Asn 0.99 (1), Gly 1.02
(1), Glu 1.99 (2), His 2.01 (2), Ile 0.98 (1), Val 1.02 (1).
Peptide 7. Synthesis. The synthesis was carried out on
215 mg of HMPA-Novagel1 resin (100–200 mesh, max-
imum loading 0.67 mmol/g). The first amino acid (Na-
Fmoc-Gly-OH, 128 mg, 0.43 mmol, 3 equiv) was coupled
to the resin upon activation with 1-(mesitylsulfonyl)-3-
nitro-1,2,4-triazole (MSNT, 127 mg, 0.43 mmol, 3 equiv)
in the presence of 1-methylimidazole (26 mL, 0.32 mmol,
2.25 equiv) in dry CH2Cl2 (5 mL) for 45 min.33 The
coupling was repeated a second time with the same
quantities of reactants and reaction time. Resin was
washed with dichloromethane and DMF. After the first
coupling a capping step of possibly unreacted hydroxyl
functions on the resin was performed; the resin was
reacted with a 10% solution of acetic anhydride in dry
DMF for 2 h (5 mL); afterwards the resin was washed
with DMF. Na-Fmoc deprotection of the peptide resin
was performed by treatment with 20% piperidine in
DMF (2+16 min). Resin was washed with DMF
(6Â2 min) between each coupling and deprotection step.
All subsequent Na-Fmoc amino acids (0.43 mmol, 3
equiv) were coupled with O-(benzotriazol-1-yl)-
Peptide 8. Synthesis. The synthesis was carried out on
150mg of HMPA-Novagel 1 resin (100–200 mesh,
maximum loading 0.67 mmol/g). The first amino acid
(Na-Fmoc-Gly-OH, 0.30 mmol, 89 mg, 3 equiv) was
coupled to the resin upon activation with MSNT (89 mg,
0.30 mmol, 3 equiv) in the presence of 1-methylimidazole
(18 mL, 0.22 mmol, 2.25 equiv) in dry CH2Cl2 (4 mL) for
45 min. The coupling was repeated a second time with
the same quantities of reactants and reaction time.
Resin was washed with dichloromethane and DMF.
After the first coupling a capping step with a 10%
solution of acetic anhydride in dry DMF for 2 h (5 mL)
was performed; afterwards the resin was washed with
DMF. Na-Fmoc deprotection of the peptide resin was
accomplished by treatment with 20% piperidine in
DMF (2+16 min). Resin was washed with DMF
(6Â2 min) between each coupling and deprotection step.
All subsequent Na-Fmoc amino acids (0.30 mmol, 3
equiv) were coupled with HBTU (106 mg, 0.28 mmol,
2.8 equiv), and NEM (57 mL, 0.45 mmol, 4.5 equiv). The
coupling with Na-Fmoc protected aminooxyacetic acid
4 to both the a and e amino groups of terminal lysine
was carried out as with the other amino acids, doubling
all the reactants (6 equiv of 4, 9 equiv of NEM, 5.6
equiv of HBTU). The two N-terminal Fmoc protecting
groups were cleaved by treatment with 20% piperidine,
the resin was washed once more with DMF (6Â). The
completeness of all reactions was monitored by the
bromophenol blue test.
N,N,N0,N0-tetramethyluronium
hexafluorophosphate
(HBTU, 153 mg, 0.40 mmol, 2.8 equiv), and N-ethylmor-
pholine (NEM, 82 mL, 0.65 mmol, 4.5 equiv) The final
coupling with N-Fmoc protected aminooxyacetic acid 4
was carried out as with the other amino acids. The N-
terminal Fmoc protecting group was cleaved by treat-
ment with 20% piperidine, the resin was washed once
more with DMF (6Â). The completeness of all reactions
was monitored by the bromophenol blue test.
Cleavage. Peptide 7 was cleaved from the resin by
incubation with a 95:2.5:2.5 mixture of TFA/triisopro-
pylsilane/H2O (5 mL). After 2 h, the resin was removed
by filtration and washed carefully with the cleavage
mixture (2Â5 mL). The combined filtrates were con-
centrated, then addition of cold diethyl ether (10mL)
allowed the precipitation of the crude peptide 7, which
finally was filtered and dried.
Cleavage. Peptide 8 was cleaved from the resin by
incubation with a 95:2.5:2.5 mixture of TFA/triisopro-
pylsilane/H2O (5 mL). After 2 h, the resin was removed
by filtration and washed carefully with the cleavage
mixture (2Â5 mL). The combined filtrates were con-
centrated, then addition of cold diethyl ether (10mL)
allowed the precipitation of the crude peptide 8, which
was finally filtered and dried.
Purification. Purification of the crude by preparative-
scale HPLC (eluent: linear gradient 10!40% eluent B
in eluent A over 50min), afforded peptide 7 in 22%
yield. The purity was checked by analytical HPLC (elu-
ent: linear gradient 10!40% eluent B in eluent A; ana-
lysis time 50min; tR=16.3 min). MALDI-TOF-MS:
calcd 1362, found m/z (M+Na)+ 1386; (M+K)+ 1401.
Purification. Purification of the crude by preparative-
scale HPLC (eluent: linear gradient 10!40% eluent B
in eluent A over 50min), afforded peptide 8 in 9% yield.
The purity was checked by analytical HPLC (eluent:
linear gradient 10!40% eluent B in eluent A; analysis
time 50min, tR=12.3 min). MALDI-TOF-MS: calcd

L. Cipolla et al. / Bioorg. Med. Chem. 10 (2002) 1639–1646
1645
1563, found m/z (M+H)+1565; (M+Na)+ 1587;
Phenotypical activation of DC. Bone marrow derived
DC (BMDC) were generated from single cell suspension
of marrow from femurs of C57BL/10ScCr mice, which
lack the gene expressing for TLR4 . BM cells were split
every 5 days with PBS to detach only loosely adherent
cells. After 15–20days of culture in DC medium,
homogeneity of DC was evaluated by cytofluorimetry.
Only cultures containing more than 90% cells expres-
sing intermediate levels and less than 10% cells expres-
sing high levels of MHC class II (I-Ab) and B7.2 were
used for the experiments. BMDC were incubated with
concentrations of peptides ranging between 0.1 and 10
mm for 24 h in IMDM medium containing 30% GM-
CSF conditioned medium.35 Cells were detached in PBS
2 mm EDTA, stained with anti B7.2 antibody (CD86/
B7.2: GL1, Pharmingen, BD) and analyzed by cyto-
fluorimetry (FACS) using the Cell Quest software (BD).
(M+K)+ 1604.
Neoglycopeptide 3. Peptide 8 (3.0 mg, 0.002 mmol) was
dissolved in a buffer solution at pH 4.5 (300 mL). The C-
glycoside 9 (1.4 mg, 0.005 mmol, 2.6 equiv) dissolved in
water (300 mL) was then added to the aminooxy peptide.
The reaction mixture was stirred for 40min, and mon-
itored by reversed-phase analytical HPLC (eluent: linear
gradient 10!40% eluent B in eluent A over 50 min;
tR=21.0and 25.5 min). The solution was then freeze-
dried and the crude purified by preparative HPLC (elu-
ent: linear gradient 10!40% eluent B in eluent A over
50min). 2.5 mg of neoglycopeptide 3 (62% yield) were
obtained as a mixture of E/Z isomeric forms. MALDI-
TOF-MS: calcd 2050 found m/z (M+H)+ 2052; amino
acid analysis: Ala 4.99 (5), Asn 1.00 (1), Gly 1.03 (1),
Glu 2.02 (2), His 1.98 (2), Ile 1.01 (1), Lys 1.02 (1), Val
0.99 (1).
Acknowledgements
Determination of T-cell hybridoma response. The
response of T-cell hybridoma BO 97.10, specific for the
peptide 327À339OVA in association with the molecule
MHC of class II I-Ab was measured as secreted IL-2
upon incubation of the hybridoma with antigen pre-
senting cells (APC). A growth factor-dependent long
term culture dendritic cells (DC) line was used (D1) as a
source of APC.34 Secreted IL-2 was determined by a
standard assay with a IL-2 dependent T-cell clone
CTLL. Hybridomas BO 97.10were suspended in
IMDM 10% fetal bovine serum (GIBCO), 100 IU/mL
penicillin, 100 mg/mL streptomycin, 2 mM l-glutamine
(purchased by Sigma) and 50 mm 2-mercaptoethanol
(1Â106 cells/mL). D1 cells were suspended in IMDM
5% fetal calf serum (2Â105 cells/mL) or in 0.001% glu-
taraldehyde in PBS for 1 min to fix the cells. D1 cells
were co-cultured (1Â104 cells/well) with BO 97.10
(5Â104 cells/well) and with different concentrations of
antigen (whole ovalbumin, 327À339OVA peptide, pep-
tides 6–8 and neoglycopeptides 1–3) in 96-well tissue
culture plates (Corning). After 24 h, supernatants were
collected and added to murine CTLL line (1Â104 CTLL
in IMDM 5% FCS). The CTLL cultures were incubated
for 24 h, and then pulsed with 1 mCi [3H]thymidine/well
for an additional 6 h (1 mCi/mL Amersham). [3H]thymi-
dine incorporation was measured by using a cell har-
vester (Wallac TOMTEC).
We thank the Ministero dell’Universita e della Ricerca
Scientifica e Tecnologica (MURST) for the financial
support.
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