Synthesis of a MUC1-glycopeptide-BSA conjugate vaccine bearing the 3′-deoxy-3′-fluoro-Thomsen-Friedenreich antigen

Article abstract of DOI:10.1039/c1cc13184b

A novel MUC1-glycopeptide-BSA conjugate vaccine with a specifically fluorinated Thomsen-Friedenreich antigen side chain at Thr6 was prepared. Preliminary immunological experiments reveal specific binding of the tumor-associated glycopeptide antigen analog by anti-MUC1-mouse antibodies.

Full text of DOI:10.1039/c1cc13184b

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COMMUNICATION
Synthesis of a MUC1-glycopeptide–BSA conjugate vaccine bearing the
30-deoxy-30-fluoro-Thomsen–Friedenreich antigenwz
¨
Anja Hoffmann-Roder* and Manuel Johannesy
Received 30th May 2011, Accepted 19th July 2011
DOI: 10.1039/c1cc13184b
A novel MUC1-glycopeptide–BSA conjugate vaccine with a
specifically fluorinated Thomsen–Friedenreich antigen side chain
at Thr6 was prepared. Preliminary immunological experiments
reveal specific binding of the tumor-associated glycopeptide
antigen analog by anti-MUC1-mouse antibodies.
MUC1-glycopeptide vaccines,4j we now report on the successful
preparation of a first 30-fluoro-TF antigen–MUC1–bovine
serum albumin (BSA) conjugate. Because tumor-associated
glycopeptides are T cell-independent self-antigens and as such
tolerated by the immune system, their conjugation to suitable
immunogenic carrier proteins, e.g. BSA, is needed to override
this tolerance. Thereby, the carrier protein enhances the presen-
tation of tumor-associated antigens to the immune system and
provides peptide fragments that can activate TH cells.
Mucin glycoproteins are abundantly expressed on the surface of
epithelial cells where they mediate crucial cellular interactions
in the course of embryogenesis, organogenesis, carcinogenesis, and
cancer metastasis.1 Unusual glycoforms of mucins with specifi-
cally altered O-glycan chains expressed on carcinomas have
been exploited as diagnostic biomarkers and are promising target
structures in the quest for effective carbohydrate-based cancer
vaccines and immunotherapeutics.2 In this regard, the tumor-
associated mucin MUC13 has attracted much attention, and
synthetic glycopeptides of its tandem-repeat (TR) domain with
cancer-related saccharides such as the TN antigen, its sialylated
congener (STN), and the Thomsen–Friedenreich (TF) antigen
were shown to elicit highly specific immune responses in mice.4
An interesting concept for improving the immunological
properties of tumor-associated antigens relies on the use of
fluorine-substituted mimics. Thus, with a view to the absence
of fluorine in most organisms and the high bond strength to
carbon,5 fluorination might enhance both the immunogenicity
and the bioavailability of the antigen significantly. However,
fluorine substitution has only recently been investigated as a tool
in vaccine development,4j,6 despite its frequent use in medicinal
chemistry.7 To date, many bioactive, fluorinated carbohydrates
have been prepared ranging from simple monosaccharides to
complex glycostructures, although the stereoselective assembly
of the latter still remains a synthetic challenge.8
A key step in the synthesis of the 30-fluoro-TF-antigen
glycosyl amino acid building block 9 is the coupling of a
3-fluoro-galactosyl bromide 68d to the TN-antigen–threonine
conjugate 7.9 Analogously to the synthesis of Brimacombe
et al.,10 compound 6 was prepared in ten steps starting from
glucose 1 (Scheme 1). Thus, 1 was converted into 1,2;5,6-di-O-
isopropylidene-glucofuranose in acetone at 10 1C and oxidized
at C3 with pyridinium dichromate (PDC)–acetic anhydride
(Ac2O) under reflux (46%, two steps).11 To set up the correct
stereochemistry, both stereocenters at C3 and C4 needed to be
inverted. Therefore, ketone 2 was transformed into the corres-
ponding hydrate and acetylated with Ac2O–pyridine to furnish
after b-elimination enol acetate precursor 3 (59%, two steps).
The latter was then stereoselectively hydrogenated from the
less hindered top side by Pd/C in MeOH12 to provide 1,2;5,6-
di-O-isopropylidene-3-O-acetyl-D-gulofuranose. Deprotection
of the remaining acetate group finally furnished precursor 4
for the subsequent nucleophilic deoxy-fluorination reaction in
a total yield of 19% over six steps. Thus, upon treatment of 4
with diethylaminosulfur trifluoride (DAST) and N,N-dimethyl-
aminopyridine (DMAP),13 smooth conversion to the desired
3-deoxy-3-fluoro-D-galactofuranose was observed. Acidolysis
of the isopropylidene acetals then liberated 3-deoxy-3-fluoro-
D-galactopyranose 5 which was peracetylated and converted to
the anomeric bromide 6 in 69% yield over two steps.
As part of a research programme devoted to the synthesis
and immunological evaluation of selectively fluorinated
The effectiveness of glycosyl bromides in synthesizing the
TF antigen and its fluorinated derivatives has been demonstrated
before,14,8d,8h and consequently, compound 6 was subjected to
a b-selective Helferich-type glycosylation with TN antigen
acceptor 7. Thus, the hitherto unknown 30-deoxy-30-fluoro-TF
antigen analog 8 was stereoselectively formed in 67% yield by
a Hg(CN)2-promoted glycosylation of 7 at 80 1C under micro-
wave irradiation. Subsequent protecting group manipulations,
i.e., cleavage of the benzylidene acetal followed by acetylation
and acidolysis of the tert-butyl ester finally provided the target
Institut fur Organische Chemie, Johannes Gutenberg-Universitat
¨
¨
Mainz, Duesbergweg 10-14, D-55128 Mainz, Germany.
E-mail: hroeder@uni-mainz.de; Fax: +49 6231 3926066;
Tel: +49 6131 3922417
w This article is part of the ChemComm ‘Glycochemistry and glyco-
biology’ web themed issue.
z Electronic supplementary information (ESI) available: Experimental
procedures, characterization data and selected spectra of new compounds.
See DOI: 10.1039/c1cc13184b
y Current address: Institut fur Pharmazeutische Wissenschaften,
¨
Wolfgang-Pauli-Str. 10, ETH-Honggerberg HCI, CH-8093 Zurich,
Switzerland.
¨
¨
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 9903–9905 9903

Scheme 1 Preparation of the 30-deoxy-30-fluoro-Thomsen–Friedenreich antigen building block for solid-phase glycopeptide synthesis.
PDC = pyridinium dichromate, DAST = diethylaminosulfur trifluoride, DMAP = N,N-dimethylaminopyridine, TFA = trifluoroacetic acid.
30-fluoro-TF antigen building block 9 in 72% yield over
three steps.
buffer at pH 9.5, providing the desired vaccine conjugate 11
after dialysis (membrane 30 kDa). The antigen loading level of
11 was determined by MALDI-TOF mass spectrometry and
indicated on average five molecules of glycopeptide per molecule
of BSA (Fig. 1).
Next, the corresponding MUC1-TR-glycopeptide antigen
bearing the 30-fluoro-TF analog at Thr6 was assembled in
an automated synthesizer by Fmoc-SPPS employing trityl-
TentaGel resin preloaded with Fmoc-Pro-OH, as previously
described (Scheme 2 and ESIz).8h Iterative couplings of the
Fmoc-protected amino acids were performed with HBTU/
HOBt and diisopropylethylamine (DIPEA) in DMF, while
the coupling of the glycosyl amino acid 9 required the use
of the more reactive HATU/HOAt cocktail and N-methyl-
morpholine (NMM) in N-methyl-pyrrolidone (NMP). At the
end of the solid-phase synthesis, a triethylene glycol spacer4a
was attached and the glycopeptide was released from the resin
with simultaneous cleavage of the side chain protecting groups
using an acidic cocktail of TFA–triisopropylsilane (TIS)–water
(10 : 1 : 1). After deacetylation with NaOMe in MeOH at pH
9.0, glycopeptide 1015 was isolated in 50% yield by preparative
RP-HPLC and subjected to conjugation to diethyl squarate in
aqueous solution (pH 8).16 Linking the resulting squarate
monoamide to BSA was then achieved in aqueous phosphate
The binding of mouse anti-MUC1-serum antibodies, raised
by immunization with the structurally related synthetic vaccines
6TF–MUC1–TTox and 60,6-difluoro-6TF–MUC1–TTox,4j to
the fluorinated glycopeptide antigen 10 was investigated.
Therefore, four different dilutions of each serum were incubated
for 1 h at 37 1C with 100 mL of the antigen 10, and then
transferred to ELISA plates coated with the parent antigen–BSA
conjugates (Fig. 2 and 3). Upon addition of a biotinylated
secondary anti-mouse antibody and treatment with streptavidin–
horseradish peroxidase (HPO), the residual binding affinities
of the serum antibodies to the antigen–BSA coatings were
photometrically detected at l = 410 nm.4c,h The data in Fig. 2
indicates that the binding of the untreated serum (positive control)
was neutralized considerably when incubated with either the
6TF–MUC1(20)-glycopeptide, the antigen contained in the
vaccine, or its 30-fluorinated derivative 10. Similarly, the binding
Scheme 2 Solid-phase peptide synthesis (SPPS) of the 30-fluoro-6TF–MUC1 glycopeptide and its conjugation to BSA. NMP = N-methylpyrrolidone,
HBTU = O-(1H-benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate, HOBt = N-hydroxybenzotriazole, DIPEA = diisopropyl-
ethylamine, HATU = O-(7-aza-benzotriazole-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate, HOAt = N-hydroxy-7-azabenzotriazole,
NMM = N-methylmorpholine, TIS = triisopropylsilane, BSA = bovine serum albumin.
c
9904 Chem. Commun., 2011, 47, 9903–9905
This journal is The Royal Society of Chemistry 2011

We thank the Deutsche Forschungsgemeinschaft (DFG)
and the Studienstiftung des Deutschen Volkes for their gener-
ous support and E. Schmitt and S. Wagner for help with the
ELISA tests.
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c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 9903–9905 9905