Synthesis of Tn/T antigen MUC1 glycopeptide BSA conjugates and their evaluation as vaccines

Article abstract of DOI:10.1002/ejoc.201100304

The tumor-associated mucin MUC1 over-expressed in most epithelial tumor tissues is considered a promising target for immunotherapy. The extracellular part of MUC1 contains a domain of numerous tandem repeats of the amino acid sequence HGVTSAPDTRPAPGSTAPPA, including five potential O-glycosylation sites. In this study, T9 and S15 have been chosen as the positions of glycosylation. The glycopeptides N-terminally equipped with a triethylene glycol spacer were synthesized by microwave-assisted Fmoc solid-phase peptide synthesis. After detachment from the resin and deprotection, the MUC1 glycopeptides were conjugated to bovine serum albumin (BSA). To evaluate the immunological properties, balb/c mice were immunized with these BSA vaccines. Copyright

Full text of DOI:10.1002/ejoc.201100304

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201100304
Synthesis of Tn/T Antigen MUC1 Glycopeptide BSA Conjugates and Their
Evaluation as Vaccines
Hui Cai,[a] Zhi-Hua Huang,[a] Lei Shi,[a] Peng Zou,[b] Yu-Fen Zhao,[a] Horst Kunz,*[c] and
Yan-Mei Li*[a]
Keywords: Carbohydrates / Glycosylation / Glycopeptides / Antitumor agents / Solid-phase synthesis
The tumor-associated mucin MUC1 over-expressed in most
epithelial tumor tissues is considered a promising target for
immunotherapy. The extracellular part of MUC1 contains a
domain of numerous tandem repeats of the amino acid se-
N-terminally equipped with a triethylene glycol spacer were
synthesized by microwave-assisted Fmoc solid-phase pep-
tide synthesis. After detachment from the resin and deprotec-
tion, the MUC1 glycopeptides were conjugated to bovine se-
quence HGVTSAPDTRPAPGSTAPPA, including five poten- rum albumin (BSA). To evaluate the immunological proper-
tial O-glycosylation sites. In this study, T9 and S15 have been
ties, balb/c mice were immunized with these BSA vaccines.
chosen as the positions of glycosylation. The glycopeptides
of these glycopeptides as B-cell epitopes is low. In order to
induce a sufficiently strong immune response, the synthetic
glycopeptides need to be conjugated with an immunostimu-
lating component.[5]
Introduction
MUC1 is a membrane glycoprotein expressed on almost
all types of epithelial tissues. Its tumor-associated form has
been found to be strongly over-expressed in breast, pancre-
atic, and ovarian cancers.[1] In its extracellular part it con-
tains a large number of tandem repeats consisting of the
sequence HGVTSAPDTRPAPGSTAPPA. Each tandem re-
peat includes five potential O-glycosylation sites (T4, S5,
T9, S15, and T16). Glycosylation of MUC1 is essential for
its biological functions.[2] On tumor cells, the glycosylation
pattern of MUC1 is distinctly different to that of normal
cells. Typically, O-glycosylation of the tumor cells is trunc-
ated, resulting in the Thomsen–Friedenreich antigen (T an-
tigen), its precursor (Tn antigen), and their sialylated
forms.[3] Due to aberrant truncated glycosylation, backbone
peptide epitopes of tumor-associated MUC1 are demasked
and exposed together with tumor-associated carbohydrate
antigens.[4] These tumor-associated glycopeptide antigens
are considered potential target structures for attack of the
human immune system. Unfortunately, the immunogenicity
The type and site of glycosylation can distinctly influence
the conformation and the immunogenicity of tumor-associ-
ated MUC1 glycopeptides.[6] For development of a vaccine,
it is necessary to study the relationship between the gly-
cosylation type and immunogenicity. The sequence PDTRP
within the tandem repeat has been identified as an immuno-
dominant motif.[7] It was also found that tumor cell recog-
nizing monoclonal antibodies bind to this PDTRP motif
when it contains the Tn antigen.[8] Therefore, the glycopep-
tides comprising the PDTRP sequence O-glycosylated with
either Tn or T antigen were synthesized in this study as
candidates for MUC1 antitumor vaccines.
During the past years a number of potential antitumor
vaccines consisting of tumor-associated MUC1 glycopep-
tides and different immunostimulants, such as BSA, tetanus
toxin, or T cell epitope peptides from Ovalbumin (OVA),
have been synthesized.[9] Most of the glycopeptides were
glycosylated at the T4, T9, and T16 sites of the tandem
repeat sequence. Taking into account this feature and the
fact that the glycosylation of serine or threonine has dif-
ferent implications in the backbone conformation of the
underlying amino acid,[10] we here focus on glycopeptides
with glycosylation at sites S15 and T9 of the tandem repeat
sequence. A series of glycopeptide antigens that contain the
complete tandem repeat sequence and that bear the Tn and
T antigen side chains was synthesized by solid-phase glyco-
peptides synthesis. These synthetic glycopeptides were con-
jugated to the carrier protein bovine serum albumin (BSA)
through a triethylene glycol spacer.[5a]
[a] Department of Chemistry, Key Laboratory of Bioorganic Phos-
phorus Chemistry & Chemical Biology (Ministry of Educa-
tion), Tsinghua University,
Beijing 100084, P. R. China
Fax: +0086-10-62781695
E-mail: liym@mail.tsinghua.edu.cn
[b] School of Life Sciences, Tsinghua University,
Beijing 100084, P. R. China
[c] Institute of Organic Chemistry, Johannes Gutenberg University
of Mainz,
Duesbergweg 10-14, 55128 Mainz, Germany
Fax: +49-6131-392 4786
E-mail: hokunz@uni-mainz.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201100304.
Eur. J. Org. Chem. 2011, 3685–3689
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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H. Cai, Z.-H. Huang, L. Shi, P. Zou, Y.-F. Zhao, H. Kunz, Y.-M. Li
SHORT COMMUNICATION
Results and Discussion
The Fmoc-protected Tn and T antigen threonine and
serine building blocks bearing O-acetyl protection within
the glycans were synthesized according to reported meth-
ods.[11] d-Galactose was transformed into 2-azidogalactosyl
bromide 3 as a glycosyl donor in three steps (Scheme 1).
Amino acid glycosyl acceptor 4 was obtained from serine
or threonine after Fmoc protection of the amino group and
introduction of the tert-butyl ester. After glycosylation ac-
cording to modified Koenigs–Knorr methodology, the O-
glycosyl amino acid was transformed into fully protected
Tn antigen conjugate 5 through reduction of the azido
group and acetylation. Removal of the tert-butyl ester by
using TFA afforded Tn antigen building blocks 1 and 2,
which can be applied to solid-phase glycopeptide synthesis.
Scheme 2. Synthesis of T building blocks. Reagents and conditions:
(a) NaOMe/MeOH, pH 9.0, 75%; (b) benzaldehyde–dimethylace-
tal, cat. pTsOH, 80%; (c) Hg(CN)2, 65%; (d) 1. 80% AcOH/H2O,
90%; 2. Ac2O, pyridine, 95%; 3. TFA, anisole, 95%.
unreacted amino groups were capped using acetic anhy-
dride/HOBt/DIPEA in DMF. Removal of the Fmoc group
was performed with a solution of 20% piperidine/0.1 m
HOBt in DMF. In order to later conjugate the glycopep-
tides with the carrier protein, each peptide was extended
with a triethylene glycol spacer. After completion of the
peptide synthesis, crude glycopeptides 10–16 were detached
from the resin using trifluoroacetic acid (TFA)/(triiso-
propylsilane) TIS/water (15:0.9:0.9), while all acid-sensitive
protecting groups of the amino acid side chains were re-
moved concomitantly. However, the hydroxy groups of the
carbohydrate portion remained O-acetylated. Obtained gly-
copeptides 10–16 were purified by preparative HPLC on a
C-18 column and isolated in yields of 55–65%. The Fmoc
group and the O-acetyl groups of the carbohydrate were
removed by treatment with a MeONa/MeOH solution
Scheme 1. Synthesis of Tn building block. R = H (Ser), CH3 (Thr).
Reagents and conditions: (a) 1. Ac2O, pyridine; 2. 30% HBr/
AcOH; 3. Zn, CuSO4; 85% (3 steps); (b) CAN, NaN3, 42%;
(c) LiBr, 92%; (d) FmocOSu, NaHCO3, quant.; (e) tBuOH, DCC,
cat. CuCl, 67–70%; (f) AgClO4, AgCO3, 55–70%; (g) Zn, AcOH,
Ac2O, 69–77%; (h) TFA, anisole, 99%.
The Tn antigen was converted into the T antigen by β- (pH 10.0) over 12 h. Free glycopeptides 17–22 were isolated
galactosylation of the GalNAc 3-hydroxy group. After care- after preparative HPLC in yields of 65–70%.
ful removal of the O-acetyl groups of 5[11b] and protection
The terminal amino groups of 17–22 and 10 were treated
of the 4- and 6-hydroxy groups as benzylidene acetal, the with diethyl squarate in EtOH/H2O (1:1) by adjusting the
free 3-hydroxy group of glycosyl acceptor 6 was glycosyl- solution to pH 8 with a saturated aqueous Na2CO3 solu-
ated with tetra-O-acetyl-galactosyl bromide under pro- tion. Obtained squaric acid monoamides 23–29 of the gly-
motion by mercury(II) cyanide to give T antigen conjugate copeptides were dissolved with BSA in a buffer solution at
7 in high yield and selectivity[12] (Scheme 2). Acidolytic re- pH 9.5 to afford BSA-conjugated glycopeptides vaccines
moval, acetylation, and acidolysis of the tert-butyl ester af- 30–36. All the conjugated vaccines were purified by ultrafil-
forded Fmoc T antigen building blocks 8 and 9[11,13] appli- tration against deionized water using a membrane of
cable to solid-phase glycopeptide synthesis.
12 kDa. The loading rates of the glycopeptide–BSA conju-
The microwave-assisted Fmoc solid-phase peptide syn- gates were determined by MALDI-TOF mass spectrometry
thesis of the MUC1 tandem repeat glycopeptides was per- (Figure 1, Table 1). On average, 8–12 molecules of the gly-
formed according to a known procedure[14] starting from a copeptides were found linked to one BSA protein molecule.
2-chlorotrityl resin preloaded with alanine (Scheme 3). The
In order to evaluate the immunological properties of the
Fmoc amino acid (6 equiv.) was activated with HBTU/ glycopeptide–BSA conjugates, each synthetic vaccine con-
HOBt[15] in DMF in the presence of DIPEA. Fmoc pro- taining 10 μg of the glycopeptide in combination with com-
tected Tn and T antigen building blocks 1, 2 or 8, 9 plete Freund’s adjuvant (CFA) were injected into four balb/
(2.0 equiv.), respectively, were activated by the more reactive c mice. After an interval of two weeks, the second and third
reagent HATU/HOAt[16] in NMP by using N-methyl- immunizations were performed with incomplete Freund’s
morpholine (NMM) as the base.[9f] After each coupling, the adjuvant (IFA). One week after the third immunization,
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3685–3689

Synthesis of Tn/T Antigen MUC1 Glycopeptide BSA Conjugates
Scheme 3. Solid-phase synthesis of the Tn/T-antigen-MUC1 glycopeptides and their conjugation to the carrier protein BSA: SPPS =
solid-phase peptide synthesis, Fmoc = fluorenyl-9-methoxycarbonyl, HBTU = O-benzotriazol-1-yl-N,N,NЈ,NЈ-tetremethyluronium hexa-
fluorophosphate, HOBt = 1-hydroxybenzotriazole. DIPEA = diisopropylethylamine, HATU = O-(7-azabenzotriazole-1-yl)-N,N,NЈ,NЈ-
tetramethyluronium hexafluorophosphate, HOAt = N-hydroxy-7-azabenzotriazole, NMM = N-methylmorpholine, TFA = trifluoroacetic
acid, TIS = triisopropylsilane, BSA = bovine serum albumin.
Eur. J. Org. Chem. 2011, 3685–3689
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H. Cai, Z.-H. Huang, L. Shi, P. Zou, Y.-F. Zhao, H. Kunz, Y.-M. Li
SHORT COMMUNICATION
Figure 2. ELISA of sera isolated from mice immunized with 30–36
after third immunization, coating of the microtiter plate with 10
and 17–22.
particularly at T9, which belong to the PDTRP immunodo-
minant motif,[7,8] and in this case similar responses were
obtained with both the T and Tn antigens. However, glyco-
sylation at S15 is not a requisite to get a high immune re-
sponse, as only good values were obtained when T9 is also
glycosylated.
Figure 1. MALDI-TOF MS of (a) BSA and (b) vaccine 23.
Table 1. MALDI-TOF mass analysis of the squaric acid mono-
amides of glycopeptides 23–29 and their BSA conjugates 30–36.
Conclusions
Squaric acid
Detected
BSA
Detected
Loading of the
glycopeptide
monoamides molecular conjugates molecular
MUC1 glycopeptides containing a full-length tandem re-
peat sequence and bearing the Tn and/or T antigen at the
glycosylation sites threonine 9 and serine 15 were synthe-
sized by microwave-assisted solid-phase glycopeptides syn-
thesis. These fully synthetic glycopeptides antigens were
conjugated with BSA through a triethylene glycol spacer by
using diethyl ester squarate as the coupling reagent. The
obtained MUC1 glycopeptide vaccines were characterized
by MALDI-TOF mass spectrometry. These BSA conjugates
carried on average 8–12 glycopeptides per molecule protein.
The MUC1-BSA vaccines were used for immunization of
balb/c mice. The induced strong immune responses were
evaluated by ELISA.
glycopeptide
weight
weight
23
24
25
26
27
28
29
2214.07
2417.85
2579.18
2417.11
2579.13
2620.22
2782.32
30
31
32
33
34
35
36
85585.87
90313.9
91733.9
87402.1
96591.6
87188.9
88313.2
9
10
10
9
12
8
8
blood was drawn from the tail vein of each mouse, and the
isolated sera were used for analysis of the induced antibod-
ies by enzyme-linked immunoabsorbent assay (ELISA). In
the ELISA tests, the microtiter plates were coated with un-
glycosylated peptide 10 and deprotected glycopeptides 17–
22. The sera were titrated at increasing dilution in a phos-
phate buffered saline (PBS) solution. The induced antibody
concentration was detected photometrically by using a sec-
Experimental Section
General Methods: Reverse-phase HPLC separation was carried out
ondary anti-mouse antibody conjugated to horseradish per- with Waters 2487 (600E) equipment. Elution was performed with
solution A (80% acetonitrile/water with 0.6% trifluoroacetic acid)
or with solution B (100% water with 0.6% trifluoroacetic acid).
Detection of the signal was achieved with a UV detector at a wave-
length of 215 nm. Separation of the glycopeptides was performed
by preparative HPLC using a C-18 column (10ϫ250 mm) at a flow
rate of 7 mLmin–1. Ultrafiltration purification of the BSA conju-
gates was performed by using a dialysis tube (12 kDa, Sigma).
MALDI-TOF MS spectra were recorded by using α-cyano-4-hy-
droxycinnamic acid (CHCA) or 2,5-dihydroxybenzoic acid (DHB)
with an Applied Biosystems 4700 Proteomics Analyzer 283.
oxidase (HRP, Figure 2). The immune response values cor-
responding to BSA conjugates of the glycopeptides can be
classified into three groups. Unglycosylated peptide 30 and
peptide 31, glycosylated with Tn antigen at S15, elicited a
lower immune response compared to the other glycopep-
tides vaccines. The second group showed a medium re-
sponse and it comprises glycopeptides 32 and 36, which car-
rying T or Tn antigens at S15. The higher immune re-
sponses were obtained with BSA conjugates 33, 34, and 35,
which present in common the feature that they are glycosyl-
ated with T or Tn at T9.
General Procedure for the Synthesis of MUC1 Glycopeptides 10–16:
Commercially available NH2-Ala-2-chloro trityl resin was used for
all the solid-phase syntheses of the glycopeptides. The loading of
the resin in these synthesis was 0.13 mmol/g, and the scale used
In conclusion and to rationalize these results, it is impor-
tant to notice that in order to get a high immune response,
it is necessary for the peptide sequence to be glycosylated, for each glycopeptide was 0.1 mmol. All cycles in the solid-phase
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Eur. J. Org. Chem. 2011, 3685–3689

Synthesis of Tn/T Antigen MUC1 Glycopeptide BSA Conjugates
synthesis were executed in a microwave peptide synthesizer (Lib-
erty, CEM Corporation). All Fmoc amino acid building blocks and
the triethylene glycol linker were coupled by using HBTU/HOBt.
Typically, coupling reactions of amino acids were conducted under
microwave irradiation for 15 min with a power of 18 W at a tem-
perature of 50 °C. The coupling was repeated two times for the
coupling of Fmoc-Arg (Pbf)-OH. The coupling of Fmoc-His(Trt)-
OH was performed at 40 °C for 25 min and also repeated two times.
The amino acids (6 equiv.), HBTU (6 equiv.), and HOBt (6 equiv.)
were dissolved in DMF, and DIPEA (12 equiv.) was dissolved in
NMP. These solutions were added automatically. The protected Tn
and T building blocks were coupled by using HATU/HOAt/NMM.
The glycosyl amino acids building blocks (2.0 equiv.), HATU
(2.5 equiv.), HOAt (2.5 equiv.), and NMM (5.0 equiv.) were dis-
solved in NMP, and these solutions were added manually. After the
coupling, the unreacted free amino groups were capped by acety-
lation using 0.013 m HOBt in Ac2O/DIPEA/DMF (4.75:2.25:93.0,
v/v/v). The capping was performed under microwave irradiation at
50 °C for 180 s with a power of 20 W. A solution of 20% piperidine/
0.1 m HOBt in DMF was used for Fmoc removal, which was con-
ducted under microwave irradiation at 50 °C for 90 s and 300 s with
a power of 24 W. After coupling all the building blocks, the resin
was transferred from the peptide synthesizer into a reactor flask
and treated with a mixture of TFA/TIS/H2O (15:0.9:0.9, v/v/v) for
2 h in order to detach the glycopeptide from the resin.
Acknowledgments
This work was supported by the National Natural Science Founda-
tion of China (20825206 and 21028004) and by the Sino-German
Center for Research Promotion (GZ561).
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Deacetylation of Glycopeptides 11–16: Glycopeptides 11–16 were
dissolved in methanol. Then, 1% MeONa/MeOH solution was
added carefully until pH 10.0 was reached. After 12 h, the solution
was neutralized by acetic acid, and crude products 17–22 were puri-
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General Procedure for the Synthesis of Glycopeptides Squaric Acid
Monoamides 23–29: Deprotected glycopeptides 17–22 and 10 were
dissolved in EtOH/H2O (1:1). 3,4-Diethoxy-3-cyclobutene-1,2-di-
one (1.0 equiv.) was added. Then, a solution of sat. Na2CO3 (5 μL)
was added in intervals of 5 min, until pH 8 was reached. After stir-
ring for 1.5 h at room temperature (controlled by RP-HPLC), the
reaction mixture was neutralized by adding HOAc. The solvent was
removed in vacuo, and the water was removed by lyophilization.
The products were purified by RP-HPLC.
General Procedure for the Conjugation of 23–29 with BSA: Glyco-
peptides squaric acid monoamides 23–29 and BSA were dissolved
in 0.07 m Na2B4O7 (600 μL) and 0.035 m KHCO3 (600 μL) buffer
solution. The glycopeptides and BSA mixture was kept 24 h at
room temperature. The glycopeptide–BSA conjugate was dialyzed
with deionized water for 48 h. Then the water was removed by ly-
ophilization.
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Enzyme-Linked Immunoabsorbent Assay (ELISA): 96-Well ELISA
plates were coated with either unglycosylated peptide 10 or MUC1
glycopeptides 17–22 (5 μL/mL) dissolved in 0.1 m NaHCO3
(pH 9.6, adjusted by NaOH) and incubated overnight at 4 °C. The
plates were washed with 0.05% Tween PBS buffer and then blocked
with PBS containing 0.25% gelatine for 2–3 h at room temperature.
After washing with 0.05% Tween PBS buffer, the mouse sera di-
luted in 0.25% gelatine PBS were added to the plates at corre-
sponding dilutions and incubated for 90 min at 37 °C. The plates
were washed and incubated with horseradish peroxidase conju-
gated to rabbit anti-mouse IgG Ab for 2 h at 37 °C. Then the plates
were washed and the substrate solution OPD and H2O2 was added.
After 10–20 min, the absorbance was measured at 450 nm.
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Supporting Information (see footnote on the first page of this arti-
Received: March 3, 2011
cle): Copies of mass spectra of the compounds.
Published Online: May 2, 2011
Eur. J. Org. Chem. 2011, 3685–3689
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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