Antibody recognition of fluorinated MUC1 glycopeptide antigens

Article abstract of DOI:10.1039/c1cc15139h

The syntheses of various fluorinated MUC1 glycopeptide antigens and their specific binding to serum antibodies from mice immunized with natural and fluorinated TF⁶-MUC1-TTox conjugate vaccines are presented.

Full text of DOI:10.1039/c1cc15139h

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Antibody recognition of fluorinated MUC1 glycopeptide antigenswz
b
b
b
Thomas Oberbillig,ab Christian Mersch, Sarah Wagner and Anja Hoffmann-Roder*
¨
Received 18th August 2011, Accepted 28th September 2011
DOI: 10.1039/c1cc15139h
The syntheses of various fluorinated MUC1 glycopeptide
antigens and their specific binding to serum antibodies from
mice immunized with natural and fluorinated TF6-MUC1-TTox
conjugate vaccines are presented.
positions C20, C60 and C6. With the fluorine atoms in close
proximity to the glycosidic linkages, these glycomimetics9
should not only be more immunogenic than the natural
compounds, but also more metabolically stable.
In addition to the previously synthesized TF antigen analog 1,10
the requisite fluorinated glycosyl amino ester building blocks
3, 9 and 12 have been prepared from Fmoc-(aGalNAc)-
Thr-OtBu (5)11 and its fluorinated analog 612 (Scheme 1).
Therefore, 6-fluorogalactal 712 was converted to 3,4-di-
O-benzyl-6-deoxy-6-fluoro-galactosyl trichloroacetimidate (8)
through a reaction sequence of de-O-acetylation, benzylation,
electrophilic Selectfluors-mediated fluorination and base-
catalyzed addition to trichloroacetonitrile (77%, 4 steps).
The resulting fluorinated glycosyl donor 8 was coupled under
Schmidt conditions (TMSOTf, CH2Cl2)13 to TN derivatives
5 and 6 to give the fluoro-TF antigen analogs 9 and 11 in good
yields, albeit low stereoselectivities. Unfortunately, all attempts
to increase the b-selectivities by varying the reaction conditions
(solvent, promotor, temperature) failed, so far. Moreover, in
some reactions of the 6-fluoro-TN derivative 6, small amounts
of the corresponding 3,4-bisglycosylated product (o15%)
were observed, which can be supressed by protection of the
4-OH prior to glycosylation. After purification of the fluoro-
TF derivatives 9 and 11, their benzylidene acetal protecting
groups were exchanged for acetyl groups. Subsequent acido-
lysis of the tert-butyl esters yielded the desired glycosyl amino
acids 10 and 13, which were used together with their congeners
2 and 4 for the assembly of MUC1 tandem-repeat sequences.
The syntheses of the target MUC1 glycopeptides bearing
N-terminal triethylene glycol units were achieved by Fmoc-
based solid-phase peptide synthesis employing preloaded
Fmoc-Pro-Trt-TentaGel resins (see Scheme 1 and ESIz). In
contrast to the standard procedure, the couplings of the
fluorinated glycosyl amino acids always required longer reaction
times (8 h) and usage of the more reactive reagent cocktail
HATU/HOAt/NMM in NMP.11 Moreover, to minimize the
amount of byproducts, unreacted amino groups were capped
with Ac2O/HOBt/iPr2NEt in NMP after each coupling cycle.
The resulting glycopeptides were then cleaved from the resin with
concomitant removal of the acid-labile side chain protecting
groups using a mixture of TFA, triisopropylsilane (TIS) and
water, followed by deprotection of the glycans via hydrogenolysis
(H2, Pd(OAc)2)12 or transesterification with NaOMe in MeOH.14
Preparative RP-HPLC finally furnished the desired fluorinated
TACA analogs 14–1812,15 in yields of 15–44% based on the
original resin loadings.
The identification of aberrant glycosylation patterns on cell-
surface glycoproteins of malignant cells has spurred research
efforts towards the development of cancer immunotherapy.1
Unusual glycoforms of mucin glycoproteins which are expressed
on most carcinoma represent attractive target structures for
carbohydrate-based anti-cancer vaccines.2 Although various
tumor-associated carbohydrate antigens (TACA) have already
been used for the design of anti-cancer vaccines,3 their targeting
is often hampered by a weak immunogenicity and a T-cell-
independent immune response. Successful strategies to overcome
these obstacles include the conjugation of MUC1-derived
TACA to immunogenic peptides,4 carrier proteins,5 and lipo-
peptides.6 Moreover, enhancement of the immunogenicity
of the TACA by exploring structural variants such as
non-natural glycosidic linkages and modified carbohydrate
epitopes has been studied.7 In this context, the strategic
introduction of fluorine atoms into the glycan moiety seems
to be a particularly promising strategy to improve the immuno-
genicity of carbohydrate-based antigens.8 Furthermore, fluoro-
sugars show enhanced chemical and metabolic stabilities which
make fluorinated glycostructures attractive targets for medical
applications including immunotherapy. However, owing to
their challenging multi-step syntheses and lengthy chromato-
graphic separations or separation procedures, only few examples
of complex fluorinated TACA for immunotherapeutic purposes
have been reported, so far.8
Herein, the syntheses and antibody binding properties of novel
fluorinated MUC1 glycopeptide antigens are presented. All of
these TACA feature at Thr6 of the 20 amino acid MUC1 tandem-
repeat sequence a variably fluorinated Thomsen-Friedenreich
antigen side-chain carrying up to three fluorine atoms at
a Institut fur Mikrotechnik Mainz GmbH, Carl-Zeiss-Strasse 18-20,
¨
55129 Mainz, Germany
b 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 ‘Emerging Investigators 2012’
themed issue.
z Electronic supplementary information (ESI) available: Experimental
procedures, characterization data and selected spectra of new compounds.
See DOI: 10.1039/c1cc15139h
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 1487–1489 1487

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Scheme 1 Preparation of the requisite fluorinated glycosyl amino acid building blocks 2, 4, 10, 13 and their use in solid-phase glycopeptide
synthesis for the assembly of fluorinated mucin-type glycopeptide analogs 14–18. For details of the syntheses, see ref. 8c, 10, 12, and ESI.z
To investigate if the position and number of fluorine sub-
stituents at the glycan moieties affect the antibody binding
properties, neutralization experiments of the glycopeptides
14–18 with anti-MUC1-serum antibodies were performed.
For instance, four different concentrations of mouse antiserum
obtained from immunization with a synthetic MUC1-TTox
conjugate vaccine carrying the natural TF antigen glycan
at Thr68c were incubated with both the natural glycopeptide
antigen and its fluorinated analogs 14–18 for 1 h at 37 1C
(Fig. 1). The solutions were then transferred to an ELISA
plate coated with the natural (non-fluorinated) MUC1 glyco-
peptide-BSA conjugate and a biotinylated secondary antibody
was added. Subsequent treatment with streptavidin-horseradish
peroxidase (HPO) allowed visualization of the residual anti-
bodies’ binding affinities.5a,5d
Fig. 1 shows that the binding of the untreated serum
(positive control) was neutralized significantly by addition of
the glycopeptide antigens. Similar results were obtained in the
neutralization experiments performed with antisera from mice
immunized with a 20-fluoro-TF6-MUC1-TTox16 (Fig. 2) and a
6,60-difluoro-TF6-MUC1-TTox vaccine,8c respectively (Fig. 3).
Thus, for all fluorinated MUC1 antigens 14–18 strong
binding of both antisera was observed, irrespective of the
number and position of the fluorine atoms. However, a
structural mismatch between the glycopeptide ligand and the
cognate antigen can in some cases affect the binding affinity.
For example, the antibodies elicited by the difluorinated
Fig. 1 Neutralization experiments using mouse antiserum raised by
immunization with a TF-MUC1-TTox vaccine8c and mucin-type
glycopeptide analogs 15–18.
Fig. 2 Neutralization experiments using mouse antiserum raised by
immunization with a 20F-TF-MUC1-TTox vaccine16 and mucin-type
glycopeptide analogs 14–18 and a related MUC4 glycopeptide.17
c
1488 Chem. Commun., 2012, 48, 1487–1489
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Fig. 3 Neutralization experiments using mouse antiserum raised by
immunization with a 6F,60F-TF-MUC1-TTox vaccine8c and mucin-
type glycopeptide analogs 14–18.
vaccine bound slightly weaker to glycopeptides 16 and 18,
whose glycan chains lack the fluorine atom in position 6
(Fig. 3). Likewise, the neutralization of the antiserum induced
by the 20-fluoro-TF6-MUC1-TTox vaccine was less effective
with glycopeptide 17, i.e., in the absence of the 20-fluoro
substituent (Fig. 2). Nonetheless, the serum antibodies differ-
entiate the modified glycan structures only slightly and their
binding affinities seem mainly determined by the glycopeptide
backbone. Thus, a related MUC4 glycopeptide5a,17 with a
deviating peptide sequence was not recognized at all (Fig. 3).
This suggests that the majority of antibodies raised by both the
natural and the fluorinated glycoconjugate vaccine were spe-
cific to the whole MUC1 glycopeptide antigen rather than to
its partial structures such as the (fluorinated) carbohydrate
part—a pre-requisite for the design of glycomimetic-based
vaccines.
In summary, we have prepared various modified MUC1
glycopeptide antigens with fluorinated Thomsen-Friedenreich
side chains and evaluated their specific binding to mouse
antisera by means of ELISA experiments. The fact that the
antisera derived from natural and fluorinated TF6-MUC1-
TTox vaccines showed very little differences in binding to the
fluorinated glycopeptide antigens 14–18 supports the idea of
using strategically fluorinated TACA for the design of specific
carbohydrate-based vaccines with enhanced immunogenicity
and metabolic stability. Moreover, owing to the subtle differ-
ences in antibody affinity, glycopeptide antigens 14–18 are
useful tools for analyzing humoral immune responses against
MUC1 antigens with different glycan structures.
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This work was supported by the Deutsche Forschungsge-
10 M. Johannes, T. Oberbillig and A. Hoffmann-Roder, Org. Biomol.
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meinschaft and the Institut fur Mikrotechnik Mainz. We
¨
thank Prof. Dr H. Kunz and Prof. Dr E. Schmitt (University
of Mainz) for fruitful discussions.
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17 C. Brocke and H. Kunz, Synthesis, 2004, 525.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 1487–1489 1489