Synthesis of an MUC1 Glycopeptide Dendrimer Based on β-Cyclodextrin by Click Chemistry

Article abstract of DOI:10.1055/s-0036-1590796

Glycopeptide dendrimers are attractive candidates for biomedical applications. Here, an efficient method for preparing multivalent MUC1 glycopeptide dendrimers based on β-cyclodextrin is described. By using copper(I) bromide and thioanisole as a catalyst system, precisely defined heptavalent conjugates were efficiently obtained. Using this heptavalent glycopeptide dendrimer, we observed multivalent effects in recognition and association processes in antibody and epitope interactions, which might have biomedical applications..

Full text of DOI:10.1055/s-0036-1590796

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Synthesis of an MUC1 Glycopeptide Dendrimer Based on β-Cyclo-
dextrin by Click Chemistry
Pu-Guang Chen
Zhi-Hua Huang
Zhan-Yi Sun
Qian-Qian Li
Yong-Xiang Chen
Yu-Fen Zhao
Yan-Mei Li*
Department of Chemistry, Key Laboratory of Bioorganic
Phosphorus Chemistry and Chemical Biology (Ministry of
Education), Tsinghua University, Beijing 100084, P. R. of
China
liym@mail.tsinghua.edu.cn
Published as part of the Cluster Recent Advances in Protein
and Peptide Synthesis
Received: 13.04.2017
Accepted after revision: 22.05.2017
Published online: 06.07.2017
lysine core have been synthesized by means of the Fmoc
solid-phase peptide-synthesis strategy,10 the N-alkylcyste-
ine-assisted sequential-segment-coupling strategy,11 and
the copper-catalyzed azide–alkyne cycloaddition reaction
(CuAAC) strategy.12 A series of multivalent MUC1 glycopep-
tides have constructed on various polymer scaffolds, such
as poly[N-(2-hydroxypropyl)methacrylamide],13 polyami-
doamine,14 poly(N-isopropylacrylamide),15 or poly(tert-bu-
tyl acrylate).16 Other multivalent neoglyconjugates have
been constructed by using GlcNAc-centered glycoclusters,
glycocyclopeptides, or tribranched glycopeptide mimics,
among others.17 Moreover, Spadaro and colleagues conju-
gated multiple units of the MUC1 core sequence with a ca-
lix[4,8]arene platform modified with Toll-like receptor 2 li-
gands, to form self-adjuvant vaccine candidates.18
β-Cyclodextrin (β-CD) is a naturally occurring cyclic oli-
gosaccharide that has proved to be useful as a well-defined
multivalent molecular scaffold.6 Many functional mole-
cules, such as amines, sugars, amino acids, polymers, or
drugs, have been conjugated with β-CD for use in pharma-
ceuticals, supramolecular chemistry, or material science.6,19
However, due to the complexity of peptides and β-CD, only
few multivalent peptide dendrimers based on β-CD have
been developed.20 To the best of our knowledge, no glyco-
peptide dendrimers based on β-CD have been developed.
We successfully synthesized a multivalent MUC1 glycopep-
tide dendrimer based on β-CD by means of click chemistry.
By using CuBr/thioanisole as a catalyst system, precisely de-
fined heptavalent conjugates were effectively obtained
through azide–alkyne cycloaddition reaction. Moreover, by
using this heptavalent glycopeptide dendrimer, we ob-
served multivalent effects in the recognition and interac-
tion process of antibody with its epitopes. We demonstrat-
ed that antibody avidity to a multivalent epitope was al-
DOI: 10.1055/s-0036-1590796; Art ID: st-2017-w0259-c
Abstract Glycopeptide dendrimers are attractive candidates for bio-
medical applications. Here, an efficient method for preparing multiva-
lent MUC1 glycopeptide dendrimers based on β-cyclodextrin is de-
scribed. By using copper(I) bromide and thioanisole as a catalyst
system, precisely defined heptavalent conjugates were efficiently ob-
tained. Using this heptavalent glycopeptide dendrimer, we observed
multivalent effects in recognition and association processes in antibody
and epitope interactions, which might have biomedical applications.
Key words glycopeptide dendrimers, cyclodextrin, copper catalysis,
thioanisole, antibody titer, click chemistry
Multivalent effects occur widely in biological systems,
for example in cell–cell interactions, receptor–ligand inter-
actions, or lectin–glycan interactions.1 Many efforts have
been made to construct multivalent molecules for use as
antiinfective drugs, antiinflammatory drugs, or anticancer
drug carriers or for tissue-engineering applications.2–6 Gly-
copeptide dendrimers, which contain both peptide and car-
bohydrate moieties, have shown great potential for devel-
oping inhibitors or synthetic vaccines.7 Reymond and co-
workers constructed a series of glycosylated peptide den-
drimers to inhibit the biofilm formation of Pseudomonas
aeruginosa, which might be useful as therapeutic agents for
this bacterium.8
MUC1 glycopeptide, which is the variable number tan-
dem repeats (VNTR) of MUC1 glycoprotein in the extracel-
lular domain, has been identified as a potent tumor-associ-
ated antigen for cancer immunotherapy.9 Many groups have
tried to construct multivalent MUC1 glycopeptide architec-
tures with unique geometric characteristics. Several multi-
valent MUC1 glycopeptide dendrimers based on a dendritic
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most five times higher than that to the corresponding
monovalent epitope, a result that might have biomedical
applications.
Azidation of β-CD on its upper rim was achieved by the
previously reported method.24 The β-CD was first substitut-
ed with iodine to afford per-6-iodo-β-CD and then directly
substituted with NaN3 to give per-6-azido-β-CD (see Sup-
porting Information; Scheme S1). Because of the poor solu-
bility of azido-modified β-CD, CuSO4 and sodium ascorbate,
the standard catalyst system for CuAAC, is unsuitable for
this conjugation. CuI/triethylamine was also found to be an
inefficient catalyst system for peptide conjugation with β-
CD, as the heptavalent β-CD-peptide conjugates could only
be obtained with 80% purity.20c Previously, CuBr/thioanisole
has been identified as a rapid and highly efficient catalyst
system for click chemistry of water-insoluble substrates.25
We therefore attempted to perform the click reaction of the
nonglycosylated peptide 3 with per-6-azido-β-CD by using
CuBr/thioanisole as a catalyst system in DMF at 65 °C. RP-
HPLC with ESI-MS detection showed that the conjugation
reaction was nearly complete within two hours, and incom-
pletely converted compounds such as hexavalent, pentava-
lent, or tetravalent glycopeptide dendrimers were not ob-
tained (Schemes 2a, 2b, and 2d). The target molecule 1 was
obtained by RP-HPLC in high purity and further lyo-
philized.26 For glycopeptide 4, the same catalyst system was
used and the target β-CD-based glycopeptide dendrimer 2
was obtained with high purity by RP-HPLC after ten hours
of reaction (Schemes 2a, 2c, and 2e).27
MUC1 glycopeptide consists of 20 amino acid residues
associated with various tumor-associated carbohydrate an-
tigens such as Tn, T, STn, or ST at Ser/Thr residues.21 It had
been demonstrated that the peptide sequence PDTRP de-
rived from the MUC1 glycopeptide is highly immunogen-
ic.9b,21 Glycosylation on the Thr residue of the PDTRP do-
main can stabilize the β-turn conformation and improve
the immunogenicity of the MUC1 glycopeptide.22 We used
the glycosylated PDTRP domain as a model glycopeptide to
construct the glycopeptide dendrimer. To suppress the for-
mation of diketopiperazine during the solid-phase peptide
synthesis (SPPS), we extended the PDTRP sequence with al-
anine at the C-terminus. First, we synthesized native and
Tn-antigen-glycosylated PDTRPA peptides by Fmoc SPPS. A
bifunctional poly(ethylene glycol)-based linker, which con-
tained a carboxy group and an alkynyl group, was attached
to the N-terminus of each of the two peptides. After cleav-
age from the resins, the crude peptides were purified by re-
verse-phase HPLC (RP-HPLC). The GalNAc protective groups
were subsequently removed by treatment with 1% NaOMe
in MeOH to give compounds 3 and 4 (Scheme 1).23
To investigate differences in the recognition and binding
by antibodies of multivalent antigens and monovalent anti-
gens, we synthesized a pair of two-component vaccines to
provide MUC1-glycopeptide-specific antibodies (Scheme
3). A two-component vaccine consisting of a B-cell epitope
and a T-helper-cell epitope effectively activates the immune
system and elicits a high titer of antigen-specific antibod-
ies.28 Tn-antigen-glycosylated and nonglycosylated PDTRPA
peptides were extended with a T-helper cell epitope, con-
sisting of 21 amino-acid residues derived from tetanus tox-
in, which has been shown to be a potent T-helper cell epi-
tope for synthetic chemical vaccines,28b,29 to form a pair of
two-component vaccines (compounds 5 and 6; Scheme 3a)
Female Balb/c mice were immunized with the two-compo-
nent vaccines without an adjuvant through intraperitoneal
injection at days 0, 14, 28, 42, and 56. At day 63, the mice
were sacrificed, and the serum was collected (Scheme 3b).
Through enzyme-linked immunosorbent assay (ELISA),
the antibody titers of the collected serum were measured
for the peptide dendrimer 1, glycopeptide dendrimer 2, na-
tive PDTRPA peptide 3, and glycosylated PDTRPA peptide 4
as coating epitopes, respectively. For nonglycosylated-pep-
tide-specific antibody, the mean antibody titers of native
PDTRPA peptide 3 and glycosylated PDTRPA peptide 4 were
20800 and 11200, respectively (Scheme 3c), which indicat-
ed that the two-component vaccines containing nonglyco-
sylated MUC1 peptide induced a particular antibody for
specific binding with native PDTRPA peptide 3. Moreover,
the mean antibody titers for peptide dendrimer 1 and na-
Scheme 1 Synthesis of alkyne-spacer-modified peptide 3 and glyco-
peptide 4. Detailed conditions and RP-HPLC and ESI-MS analyses are
given in the Supporting Information.
β-CD is a unique multivalent molecular scaffold with
well-defined geometrical characteristics, so that many β-
CD-based conjugates have been developed for various ap-
plications through selective functionalizations of β-CD.
Most functionalizations of β-CDs were based on the hy-
droxy groups on the upper rim (the 6-OHs), due to the dif-
ferent chemical reactivities of the 2-OH, 3-OH, and 6-OH
groups.
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tive PDTRPA peptide 3 were 57600 and 11200, respectively
(Scheme 3c). This indicates that the antibody titer for the
peptide dendrimer was five time higher than that for the
monovalent peptide, implying that the multivalent effect
has an important roles in antibody–epitope recognition and
interaction. For the glycosylated peptide specific antibody,
the mean antibody titers for compounds 1–4 were 51200,
179200, 12000, and 70400, respectively (Scheme 3d). The
results proved that two-component vaccines consisting of
glycosylated mucin peptide specifically induced an anti-
body that bound preferentially with the glycosylated PDTR-
PA peptide 4. What is more, the mean antibody titer of gly-
copeptide dendrimer 2 was about 2.5 times higher than
that is of the glycosylated PDTRPA peptide 4, which is con-
sistent with data for a nonglycosylated-peptide-specific an-
tibody, and confirmed the existence of a multivalent effect
in the antibody–epitope recognition and association pro-
cess (Scheme 3c). The multivalent effect in antibody recog-
nition and binding with epitopes might have biomedical
applications, for example in antibody detection, serum
analysis, or disease diagnosis.
Scheme 3 (a) The synthesis of the two-component vaccines 5 and 6.
Detail information is given in Supporting Information. (b) Immunization
schedule for the synthetic vaccines. Compounds 5 and 6 were injected
intraperitoneally into Balb/c mice at days 0, 14, 28, 42, and 56. The se-
rum was collected at day 63. The antibody titers of compounds 5 (c)
and 6 (d) were measured by ELISA. The coating epitope were peptide
dendrimer 1, glycopeptide dendrimer 2, nonderivatized PDTRPA pep-
tide 3, and glycosylated PDTRPA peptide 4. The antibody titer is defined
as the highest dilution with 0.1 optical absorption or with an absor-
bance greater than that of negative control sera.9c
Scheme 2 (a) Syntheses of peptide dendrimer 1 and glycopeptide den-
drimer 2. The compound 3 was conjugated with per-6-azido-β-CD by
using CuBr/thioanisole as a catalyst in DMF at 65 °C for 2 h, whereas
compound 4 was conjugated with per-6-azido-β-CD by using CuBr/
thioanisole in DMF at 65 °C for 10 h. More details are provided in the
Supporting Information. RP-HPLC (b) and ESI-MS analyses (d) of peptide
dendrimer 1. RP-HPLC (c) and ESI-MS analyses (e) of glycopeptide den-
drimer 2.
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In conclusion, by using CuBr and thioanisole as a cata-
lyst system, we successfully and efficiently synthesized a
heptavalent MUC1 glycopeptide dendrimer based on β-CD,
with high purity. To our limited knowledge, this is the first
glycopeptide dendrimer based on β-CD. By using this hep-
tavalent glycopeptide dendrimer, we observed that anti-
body avidity to a multivalent epitope was about five times
higher than that to a monovalent epitope. This difference in
antibody avidity indicated a multivalent effect in antibody
and epitope interactions, which might have applications in
antibody detection, serum analysis, and disease diagnosis.
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Funding Information
This work is funded by the Major State Basic Research Development
Program of China (2013CB910700) and the National Natural Science
Foundation of China (21332006 and 21672126)
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Acknowledgment
We thank Professor Hua Fu for helpful suggestions and discussions.
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Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0036-1590796.
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HRMS: m/z [M + H]+ calcd for C301H476N84O126: 7284.3422; found
(MALDI-TOF) 7285.5317; (ESI ) [M + 4H]4+ 1823.0, [M + 5H]5+
1458.4, [M + 6H]6+ 1215.7, [M + 7H]7+ 1042.1.
(27) Compound 2
Glycopeptide dendrimer 2 was obtained by a similar procedure
to that of peptide dendrimer 1, except that the reaction time
was 10 h: yield: 9.4 mg (1.1 μmol, 59%).
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6H]6+ 1453.1, [M + 7H]7+ 1245.5.
To a solution of thioanisole (20 μL 166 mol) in DMF (200 μL) was
added CuBr (2.1mg, 15.0 μmol) to form a dark-green solution
when the CuBr was fully dissolved. A solution of peptide 3 (13
mg; 15.2 μmol) in DMF (0.8 mL) was added to a solution of per-
6-azido-β-cyclodextrin (2 mg; 1.5 μmol) in DMF (0.5 mL), and
the soln was deoxygenated with N2 gas. The dark-green catalyst
solution was then carefully added to the peptide solution and
the mixture was stirred at 65 °C for 2 h. The DMF was removed
in vacuo, and the crude product was dissolved in 1:1 H2O–
MeCN (3 mL), purified by RP-HPLC (C18 column), and lyo-
philized to give a white powder; yield: 7.9 mg (1.1 μmol, 73%).
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© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–E