Synthesis of MUC1-lipopeptide chimeras

Article abstract of DOI:10.1039/c0cc01360a

An efficient method for the convergent assembly of MUC1-lipopeptide vaccine candidates is described. Chimeras consisting of MUC1 glycopeptides (bearing multiple copies of the T_N and T tumour-associated carbohydrate antigens) tethered to the lipopeptide immunoadjuvant Pam₃CysSer were synthesised in high yields using a fragment-based condensation strategy.

Full text of DOI:10.1039/c0cc01360a

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Synthesis of MUC1–lipopeptide chimerasw
Brendan L. Wilkinson, Lara R. Malins, Candy K. Y. Chun and Richard J. Payne*
Received 12th May 2010, Accepted 25th June 2010
DOI: 10.1039/c0cc01360a
An efficient method for the convergent assembly of MUC1–
lipopeptide vaccine candidates is described. Chimeras consisting
of MUC1 glycopeptides (bearing multiple copies of the TN and T
tumour-associated carbohydrate antigens) tethered to the
lipopeptide immunoadjuvant Pam3CysSer were synthesised in
high yields using a fragment-based condensation strategy.
five potential O-glycosylation sites are derivatised with the cancer
associated TN and T antigens.
Our synthetic strategy can be divided into three independent
stages: solution phase synthesis of the TLR-2 ligand Pam3Cys,
solid-phase synthesis of MUC1 peptides, glycopeptides and a
Pam3CysSer fragment (bearing a C-terminal triethylene glycolic
acid linker) using Fmoc-based techniques, and finally a penta-
fluorophenyl ester-mediated fragment condensation of the
triethylene glycolic acid-derivatised Pam3CysSer lipopeptide to
completely deprotected MUC1 (glyco)peptides in order to
furnish the corresponding target MUC1–lipopeptide chimeras.
The synthesis of Pam3Cys 1 was achieved over five steps starting
from Fmoc-Cys-OAll 29 (Scheme 1). Treatment of 2 with the
optically pure (R)-bromo-1,2-propane diol 310 in the presence of
Cs2CO3 and TBAI11 furnished diol 4 in moderate yield (43%).12
Carbodiimide-promoted esterification of 4 using palmitic acid
resulted in smooth conversion to the dipalmitoylated cysteine
derivative 5 in excellent yield.13 One-pot deprotection of the
Fmoc-carbamate and palmitoylation gave allyl protected Pam3Cys
precursor 6 in 72% yield. Finally, Tsuji–Trost deallylation using
tetrakis(triphenylphosphine)palladium(0) and N-methylaniline as a
scavenger provided Pam3Cys 1 in quantitative yield.
A classic feature of oncogenically transformed cells is the
aberrant glycosylation of cell surface glycoproteins arising from
the dysregulation of carbohydrate processing enzymes.1 In the
case of mucin glycoproteins located on the epithelial cell-surface,
the over-expression of O-linked tumour-associated carbohydrate
antigens (TACAs) such as the TN, T and the corresponding
sialylated derivatives has been directly correlated with tumour
progression and growth.2 These glycans exhibit poor immuno-
genicity, a direct consequence of over-riding self-tolerance by the
immune system. As such, these TACAs cannot be employed as
vaccines alone. Advances in our understanding of the relation-
ship between innate and adaptive immunity have provided new
opportunities in cancer vaccine development, thus stimulating
significant interest in the rational design and preparation of
synthetic MUC1-based glycopeptide vaccines.3 This burgeoning
knowledge base has culminated in the impressive work of
Boons,4 Danishefsky,5 Kunz6 and others.7 In particular, it has
been demonstrated that highly specific antibodies recognising
tumour-associated epitopes can be achieved by utilising self-
adjuvanting, multi-component MUC1 glycopeptide vaccines.
These have been prepared via covalent attachment of segments
of the cancer-associated MUC1 variable number tandem repeat
domain (VNTR) to an immunostimulating adjuvant, with
or without a helper T-cell epitope.3,4 Recently, it was reported
that hyperglycosylation of the MUC1 VNTR sequence
(GVTSAPDTRPAPGSTAPPAH), whereby all five potential
O-glycosylation sites are occupied, is an important feature for
the production of a strong antibody response against the glyco-
peptides.2,5a Hyperglycosylated MUC1 peptides conjugated to
an immunostimulating adjuvant represent an underexplored
class of vaccine candidates. Herein, we report the design and
synthesis of a new class of self-adjuvanting MUC1 vaccine
candidates possessing a full copy of the 20 amino acid MUC1
peptide VNTR domain and the immunostimulating Toll-like
receptor 2 (TLR-2) ligand, Pam3CysSer, first described by Jung
and Bessler.8 These constructs represent the first fully-synthetic
MUC1-based multi-component vaccine candidates in which all
With optically pure Pam3Cys in hand, our attention focused
on the preparation of the lipopeptide component of the
proposed chimeras bearing a short triethylene glycolic acid
spacer14 via solid-phase peptide synthesis (SPPS) (Scheme 2).
Ethylene glycol units are often incorporated to serve as flexible,
polar and immunosilent linkers between the various recognition
elements of a vaccine construct.6 The flexible spacer is thought
to minimise any potential conformational distortion caused by
the lipopeptide fragment, thus ensuring all elements of the
vaccine bind with maximum affinity to their cognate biological
School of Chemistry, University of Sydney, NSW 2006, Australia.
E-mail: richard.payne@chem.usyd.edu.au; Fax: (+61)-2-9351-3329;
Tel: (+61)-2-9351-5877
w Electronic supplementary information (ESI) available: Experimental
procedures and full spectroscopic data for new compounds. See DOI:
10.1039/c0cc01360a
Scheme 1 Synthesis of Pam3Cys 1.
c
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Chem. Commun., 2010, 46, 6249–6251 6249

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11 (Scheme 3). Resin bound pentapeptide 12 was assembled via
Fmoc-strategy SPPS. At this stage, the synthesis diverged at the
first O-glycosylation site. Synthesis of the solid supported
unglycosylated peptide 10a was achieved by iterative Fmoc-
strategy SPPS using PyBOP and NMM. In the case of the resin
bound glycopeptides 10b and 10c, coupling of the glycosylamino
acids 13–1616,17 was achieved using only 1.2 equivalents of the
precious building blocks and HATU as the coupling reagent.
These conditions were employed so as to prevent excess waste of
precious glycosyl amino acid building blocks whilst maintaining
high coupling yields. Upon complete assembly of peptides 17a–c,
the resins were treated with 10% piperidine in DMF to remove
the N-terminal Fmoc-carbamate moiety, followed by treatment
with TFA/TIS/thioanisole/H2O (85 : 5 : 5 : 5 v/v/v/v) to facilitate
side chain deprotection and cleavage from the resin. At this stage
peptide 10a was purified by HPLC and isolated in 45% yield
(based on the original resin loading). De-O-acetylation of glyco-
peptides was achieved using aqueous hydrazine and afforded
target glycopeptides 10b and 10c, in 22% and 14% yields
respectively after HPLC purification.
Scheme 2 SPPS of lipopeptide component 7.
targets.6 The desired lipopeptide 7 was assembled on 2-chloro-
trityl chloride resin which was first loaded with Fmoc-protected
triethylene glycolic acid 8.15 The solid-supported linker was
subsequently deprotected with 10% piperidine in DMF, and
coupled to Fmoc-Ser(OtBu)-OH using benzotriazol-1-yl-oxy-
tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP)
and N-methylmorpholine (NMM) to afford resin bound 9.
Following Fmoc deprotection, Pam3Cys was installed by
treatment with 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl
uronium hexafluorophosphate (HATU) and NMM in DMF
for 20 h. Following cleavage and purification by flash column
chromatography, the desired lipopeptide 7 was isolated in
95% yield.
Having successfully synthesised the desired peptide and
glycopeptide fragments by SPPS, we next explored possible
convergent conjugation methods to lipopeptide 7. Recently,
Kunz and co-workers reported the HATU-promoted frag-
ment condensation of a protected lipopeptide with deprotected
MUC1 glycopeptides in solution which provided conjugates in
20–25% yields.6c In addition, Boons and co-workers have
demonstrated that glycopeptides can be conjugated to
lipopeptide thioesters using native chemical ligation.4
However, owing to the hydrophobicity of the lipopeptide frag-
ment, it was necessary to conduct these reactions in liposomes.
Having successfully prepared lipopeptide fragment 7, we
embarked on the synthesis of peptide 10a and glycopeptides
10b and 10c bearing multiple copies of the TN and T TACAs,
respectively. The synthesis of these targets was conducted from
2-chlorotrityl chloride resin preloaded with Fmoc-His(Trt)-OH
Scheme 3 SPPS of peptide 10a and glycopeptides 10b and 10c.
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Scheme 4 Synthesis of MUC1–lipopeptide chimeras 18a–c via pentafluorophenyl ester-mediated fragment condensation.
We were interested in investigating an alternative fragment
condensation approach for the high yielding preparation of
MUC1–lipopeptide chimeras to serve as cancer vaccine candi-
dates. To this end, we decided to explore the utility of penta-
fluorophenyl esters as N-acylation donors to prepare these
constructs.18 In a preliminary reaction, the free carboxylate
Notes and references
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group was pre-activated by treatment of lipopeptide
7
with N,N0-diisopropylcarbodiimide and pentafluorophenol.19
Formation of the desired active ester could be monitored by
TLC analysis and proceeded to completion within 1 h. The
pentafluorophenyl ester was subsequently condensed with a
slight excess (1.2 equivalents) of the unglycosylated MUC1
eicosopeptide in the presence of 1-hydroxybenzotriazole (HOBt)
and DIPEA and the reaction monitored by LC-MS (Scheme 4).
After complete consumption of lipopeptide 7 the side chain tert-
butyl protecting group was removed in situ using an acidic
cocktail. Purification by preparative HPLC furnished the target
MUC1–lipopeptide chimera 18a in excellent yield (90%).
Having established a simple and efficient method for the
fragment-based condensation reaction of the unglycosylated
MUC1 peptide 10a, we shifted our attention towards the pre-
paration of chimeras bearing multiple copies of the TACAs. To
this end, homogeneous MUC1 glycopeptides bearing five copies
of the TN 10b and T antigen 10c, respectively, were subjected
to the above conditions. Gratifyingly, MUC1 glycopeptide–
lipopeptide constructs 18b and 18c were furnished in excellent
yields (79% and 72%, respectively) after HPLC purification.
In summary, we have successfully exploited a convergent
fragment condensation approach for the high yielding
synthesis of chimeras for use as cancer vaccine candidates.
Specifically, a pentafluorophenyl ester-mediated condensation
allowed for the rapid construction of a number of MUC1
lipopeptide chimeras incorporating the full length MUC1
tandem repeat sequence and the TLR-2 ligand Pam3Cys.
These constructs represent the first fully synthetic glycopeptide
MUC1-based vaccine candidates carrying the full length
tandem repeat domain where all five potential O-glycosylation
sites are occupied with either the TN or T TACAs.
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19 The pentafluorophenyl ester could not be isolated by silica or
reverse phase chromatography without significant hydrolysis.
Such constructs should serve as useful leads for immuno-
logical studies which will be reported in due course. In
addition, it is anticipated that the synthetic methodology
described will be broadly applicable to the assembly of larger
peptides and proteins in the future.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 6249–6251 6251