Design, Synthesis, and Immunological Evaluation of a Multicomponent Construct Based on a Glycotripeptoid Core Comprising B and T Cell Epitopes and a Toll-like Receptor 7 Agonist That Elicits Potent Immune Responses

Article abstract of DOI:10.1021/acs.jmedchem.8b00960

We present here for the first time the synthesis and immunological evaluation of a fully synthetic three-component anticancer vaccine candidate that consists of a β-glycotripeptoid core mimicking a cluster of Tn at the surface of tumor cells (B epitope), conjugated to the OVA 323-339 peptide (T-cell epitope) and a Toll-like receptor 7 (TLR7) agonist for potent adjuvanticity. The immunological evaluation of this construct and of precursor components demonstrated the synergistic activity of the components within the conjugate to stimulate innate and adaptive immune cells (DCs, T-helper, and B-cells). Surprisingly, immunization of mice with the tricomponent GalNAc-based construct elicited a low level of anti-Tn IgG but elicited a very high level of antibodies that recognize the TLR7 agonist. This finding could represent a potential vaccine therapeutic approach for the treatment of some autoimmune diseases such as lupus.

Full text of DOI:10.1021/acs.jmedchem.8b00960

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Design, Synthesis, and Immunological Evaluation of a
Multicomponent Construct Based on a Glycotripeptoid Core
Comprising B and T Cell Epitopes and a Toll-like Receptor 7 Agonist
That Elicits Potent Immune Responses
†
†
‡,§
†
‡,§
Thomas Szekely, Olivier Roy, Edith Deriaud, Aurelie Job, Richard Lo-Man, Claude Leclerc,*,‡,§
́
́
and Claude Taillefumier*,†
†
́
Universite Clermont Auvergne, CNRS, SIGMA Clermont, ICCF, F-63000 Clermont-Ferrand, France
‡
́
́
́
Unite Regulation Immunitaire et Vaccinologie, Equipe Labellisee Ligue Contre le Cancer, Institut Pasteur, 75015 Paris, France
§INSERM U1041, 75724 Paris Cedex 15, France
S
* Supporting Information
ABSTRACT: We present here for the first time the synthesis
and immunological evaluation of a fully synthetic three-
component anticancer vaccine candidate that consists of a β-
glycotripeptoid core mimicking a cluster of Tn at the surface
of tumor cells (B epitope), conjugated to the OVA 323−339
peptide (T-cell epitope) and a Toll-like receptor 7 (TLR7)
agonist for potent adjuvanticity. The immunological evalua-
tion of this construct and of precursor components
demonstrated the synergistic activity of the components
within the conjugate to stimulate innate and adaptive immune
cells (DCs, T-helper, and B-cells). Surprisingly, immunization
of mice with the tricomponent GalNAc-based construct
elicited a low level of anti-Tn IgG but elicited a very high level
of antibodies that recognize the TLR7 agonist. This finding could represent a potential vaccine therapeutic approach for the
treatment of some autoimmune diseases such as lupus.
intensive research has focused on the design and synthesis of a
first generation of vaccine prototypes in which TACAs were
conjugated to a foreign carrier protein (e.g., KLH or BSA) and
used with a nonspecific immunoadjuvant to enhance helper T
cell responses.4 The notable contributions of Livingston and
Danishefsky are key milestones in this research area.
Particularly, based on the demonstration that Tn antigens
are exposed on adjacent Ser/Thr residues in repeating clusters
of 3−5, monovalent clustered vaccine candidates, wherein a
mucin-derived glycopeptide-KLH conjugate contained three
Tn antigens, have been developed.5 These constructs,
coadministrated with the QS-21 adjuvant, advanced to phase
I clinical trials against prostate cancer and proved to elicit
significant antibody responses in patients.6 Nevertheless, in
most cases, the structural heterogeneity of this generation of
vaccine candidates and the potential immunogenicity of carrier
proteins and linkers7 resulted in suboptimal antibody
responses against the TACA epitope, a critical issue for
therapeutic efficacy of these cancer vaccines.
INTRODUCTION
■
Anticancer immunotherapy represents a powerful new strategy
in medicine. In particular, the administration of cancer
vaccines1 to harness the capacity of patient’s immune system
to eliminate tumor cells could represent an efficient alternative
to usual antitumor treatments which are often unselective,
accompanied by many side effects, and which may have
difficulties in eradicating metastatic cells.2
Malignant transformations of normal cells are characterized
by the overexpression of altered mucin-type glycoproteins or
glycolipids. Aberrant glycosylations have been shown to
contribute to the development of clustered tumor-associated
carbohydrate antigens (TACAs),3 such as (1) the glycoprotein
antigens including the Tn (T nouvelle), Thomsen−Frieden-
reich (TF), and sialyl Tn (sTn) linked to serine or threonine
residues and (2) glycolipid antigens including the gangliosides,
the globo class, and the blood group determinants. TACAs
represent attractive targets for antibody-mediated anticancer
vaccines; however, as they are regarded as “autoantigens” or
“self-antigens”, they cannot alone activate helper T cells.
Therefore, they exhibit weak immunogenicity, leading to
difficulties in providing high affinity IgG to eliminate TACA-
expressing human tumor cells. To circumvent these issues,
Received: June 15, 2018
© XXXX American Chemical Society
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Figure 1. Strategy for the synthesis of the three-component carbohydrate-based constructs 35 and 36.
With the aim of improving the presentation of the
carbohydrate antigens in the absence of a carrier protein, a
number of laboratories have designed fully synthetic multi-
component vaccines.8−11 Considerable attention has been paid
to the choice of scaffolds for the presentation of clustered
antigenic units. Calix[4]arene,12 cyclic decapeptides
(RAFTs),13,14 or dendrimeric polylysine fragments extended
with glycopeptidic arms (MAG-Tn3) have been chosen to
develop multivalent vaccines.15 The last, designed by the
groups of Leclerc and Bay, exhibited four arms, composed of a
CD4+ peptide T-helper epitope and a trimeric Tn cluster. In
the presence of a mild adjuvant, this construction elicited high
level of anti-Tn IgG antibodies which have the ability to
mediate the killing of Tn-positive tumor cells in mice15 and
non-human primates.16 They also demonstrated that targeting
macrophage galactose-type C-type lectins (MGL) by Tn
tumor-associated antigens constitutes a promising approach
for enhancing vaccine effectiveness.17
Moreover, advances in the knowledge of the coordination of
innate and adaptive immune systems also offer new avenues for
the design of anticancer vaccines. Indeed, the innate system
detects large classes of highly conserved molecules that are
integral parts of pathogens or abnormal cells. Their recognition
through a set of pattern-recognition receptors (PRRs), such as
Toll-like receptors (TLRs),18 provides appropriate “danger
signals” for the maturation of dendritic cells, which are
fundamental targets for many clinical situations.19−21 In this
respect, the resulting production of proinflammatory cytokines
influences innate and adaptive responses.22,23 Some of these
immune mediators are crucial for the differentiation of helper
T cells toward a Th-1 or Th-2 phenotype. Efforts have thus
been made to develop conjugates of tumor antigens and TLR
ligands.24−26 The Boons27 and Payne28 groups have for
example developed multicomponent glycosylated MUC-1
based vaccine candidates incorporating a T-cell helper peptide
and an immunostimulating lipopeptide TLR2 ligand. These
vaccines presented remarkably high immunogenicity establish-
ing the importance of TLR signaling for optimal immune
responses.
Herein, following a similar approach, we report the synthesis
and immunological evaluation of a structurally well-defined,
fully synthetic three-component construct 35 that combines all
the features required for a focused and effective activation of
DCs, T- and B-cells and to elicit relevant IgG immune
responses (Figure 1). This construct is composed of an original
glycotripeptoid core with α-O-linked GalNAc moieties (B
epitope). To strengthen its presentation to the immune system
and to overcome the acquired immunotolerance to TACAs
and their inherently T-cell independent nature, the glyco-
peptoid trimer was conjugated to the OVA 323−339 peptide
(TH epitope) and to a Toll-like receptor 7 (TLR7)
heterocyclic agonist (immunoadjuvant).29 The intracellular
TLR7s act as innate immune sensors of microbial nucleic
acids.30TLR7 (and TLR9) have also been proven to be
implicated in the development of some autoimmune diseases,
such as systemic lupus erythematosus (SLE),31 as a
consequence of the aberrant transportation of self-derived
nucleic acids. Cell surface TLR7s were also recently revealed
on bone marrow-derived macrophages.32 As TLR7-dependent
signaling is believed to promote autoimmune pathologies,
constructs incorporating a TLR7 agonist can also be useful
tools with respect to this type of disease. As immunoadjuvant,
the major benefit of TLR7 agonists is their capacity to
stimulate several cell types (APCs, CD4 and CD8 T cells, and
NK cells) resulting in a broad spectrum of activated immune
cells, cytokines, and chemokines at the tumor site.33
A
tricomponent construct analogue (36) in which the three
GalNac were replaced by three mannoses, was also synthesized
B
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a
Scheme 1. Synthesis of the C-Terminus Alkyne-Functionalized β-Glycotripeptoids 15 and 16
a
Reagents and conditions: (i) Fmoc-Cl, NaHCO3, 1,4-dioxane, rt, 4 h, 84% (3), 91% (4); (ii) TFA, CH2Cl2 (1:1, v/v), rt, 1 h, quantitative, then 1
or 2, HATU, DIEA, CH2Cl2, DMF, rt, overnight, 81% (5 from 1 and 3), 90% (6 from 2 and 4); (iii) TFA, CH2Cl2 (1:1, v/v), rt, 1 h, quantitative,
then 1 or 2, HATU, DIEA, CH2Cl2, DMF, rt, overnight, 75% (7 from 1 and 5), 87% (8 from 2 and 6); (iv) Et2NH, CH3CN (1:2, v/v), rt, 4 h,
quantitative; (v) NaOMe, MeOH, rt, 1 h, quantitative; (vi) 7 or 8, TFA, CH2Cl2 (1:1, v/v), rt, 1 h, quantitative then H2NCH2CCH, HATU, DIEA,
CH2Cl2, CH3CN, rt, overnight, 95% (13), 86% (14).
as a negative control with respect to the production of anti-Tn
IgG. A β-peptoid platform was chosen for bringing together all
the components of the constructs 35 and 36. β-Peptoids (N-
substituted β-alanine oligomers) are a class of homologous
peptoids,34,35 being defined as N-substituted glycine
oligomers.36,37 In other words, what distinguishes peptoids
from peptides is the fact that the side chains are located on the
nitrogen atoms of the backbone amides instead of being
attached to the main chain carbons atoms. This structural
change confers advantages over peptides, notably an enhanced
metabolic stability38,39 and a greater cell-permeability,40−42 two
important factors for their use as peptidomimetics or chemical
platforms. The presence of peptoid tertiary amide bonds,
prone to cis/trans isomerism,43−45 is also responsible for a
greater backbone flexibility. Different strategies aimed at
promoting either the cis- or trans-amide bond conformations
have been developed in the recent past,46−48 notably by our
group.49−53 These strategies might be harnessed in the future
to reduce flexibility of peptoid-based vaccine constructs. For
the initial design, we have selected a conformationally flexible
glycopeptoid moiety, assuming that this design will increase
the binding of the constructs to C-type lectin receptors
(CLRs) at the surface of DCs. In addition, the flexibility of B
cell epitopes plays a major role in their capacity to induce
antibody responses.54 It is also for that reason that we chose a
β-peptoid backbone rather than the more common α-peptoid
backbone.55
antigenicity of the construct were first investigated in vitro by
ELISA and T-cell stimulation assays, respectively. Its
immunogenicity, i.e., the ability to elicit anti-Tn IgG, was
then analyzed in vivo in mice. The synthetic and biological
results are reported in detail in this paper.
RESULTS AND DISCUSSION
■
Design of the Three-Component Constructs 35 and
36 Based on a TLR7 Agonist and Multivalent B/CD4+ T-
Cell Epitopes. We developed a convergent solution-phase
synthetic approach for the construction of the synthetic targets
35 and 36, based on two amide bond forming reactions and a
chemoselective copper azide−alkyne cycloaddtion (CuAAC)
reaction (Figure 1). The synthetic targets were composed of
four modules that were assembled sequentially: the TLR7
agonist 24 equipped with a carboxylic acid function, a 6-
aminocaproyl group used as a spacer, the glycopeptoid cores
15 and 16 ended by a propargyl amide function, and the OVA
323−339 peptide bearing a 6-azidohexanoyl arm. In this
modular approach, the TLR7 agonist 24 was first coupled to
the aminocaproic acid spacer, which in turn was coupled to the
N-terminus of the glycopeptoid cores. Finally, the resulting
constructs were conjugated to the OVA peptide by a CuAAC
reaction.
Two approaches were envisaged to access the conforma-
tionally flexible glycopeptoids56 cores 15 and 16: a monomer
approach consisting of the peptide coupling of orthogonally
protected glycosylated β-alanine monomers and the so-called
submonomer approach initially developed for the synthesis of
α-peptoid oligomers36 and adapted for β-peptoid synthesis.34
To date, this is the first time that a peptidomimetic structure
and a TLR7 adjuvant were used and attached together in an
anticancer vaccine prototype. The adjuvanticity and the T-
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a
Scheme 2. Biotinylation of Glycopeptoid Platforms 9 and 10
a
Reagents and conditions: (i) 9 or 10, HO2C(CH2)5NH-Fmoc, HATU, DIEA, CH2Cl2, DMF, rt, 14 h, 50% (9), 90% (10); (ii) 17 or 18, Et2NH,
CH3CN (1:2, v/v), rt, 4 h; (iii) biotin N-hydroxysuccinimide, Et3N, DMF, rt, 16 h, 81% (17), 37% (18); (iv) NaOMe, MeOH, rt, 1 h, quantitative.
a
Scheme 3. Biotinylation of TLR7 Agonist 24
a
Reagents and conditions: (i) biotin (1 equiv), IBCF 1.2 equiv, Bu3N 1.3 equiv., DMF, rt, then 0 °C, BocNH(CH2)4NH2 in DMF, 4h, rt; (ii)
TFA/DCM 1/1; (iii) HATU 2 equiv, DIEA 5 equiv, 24 (1 equiv) DMF.
The submonomer synthesis consists of creating each new
monomer in two steps, which are, in the case of β-peptoid
synthesis, acylation of the N-terminus of the growing chain
with acryloyl chloride followed by the aza-Michael addition of
a primary amine to the formed acrylamide system. It is known
that this two-step methodology is not as effective as the
submonomer synthesis of α-peptoids,57 but it could reasonably
be considered for the synthesis of the short glycosyl-β-peptoid
trimers 15 and 16. Both monomer and submonomer
methodologies entailed the synthesis of the peracetylated
building blocks 1 and 2 (Scheme 1, Supporting Informa-
tion).58,59 The solution-phase submonomer synthesis of β-
peptoids has been optimized in our group, notably when
volatile amines are used in the Michael addition steps.60,61
Application of our optimized protocol to form β-glycopeptoids
trimers was however unsuccessful. We subsequently turned to
the use of the “monomer method” for preparing trimers 15 and
16.
with Toll-like receptor 7.29 This leads to maturation of DCs
and production of cytokines which promote the development
and activation of T helper cells. Moreover, its pharmacological
potency is improved after conjugation to chemical entities that
facilitate endocytosis.62,63 Aminocaproic acid was used as a
spacer between the TLR7 agonist 24 and glycopeptoid
components. The choice of the peptide fragment OVA 323−
339 (ISQAVHAAHAEINEAGR) from ovalbumin protein was
guided by past work showing that it contains multiple T helper
epitopes involved in high affinity humoral responses.64−67 The
CuAAC was envisioned as a convenient strategy for chemo-
selective assembling of OVA 323−339 to the TLR7-
glycopeptoid conjugates.68,69 Indeed, this very popular ligation
method allows in a single step for the formation of an amide
bioisostere70−72 between unprotected fragments and is
compatible with a plethora of chemical functions. In this
respect, the OVA 323−339 moiety was purchased with an
azide-functionalized arm at the N-terminus and the glyco-
conjugates 15 and 16 were engineered with a C-terminal
alkyne function.
The adenine-like heterocyclic moiety 24 is a potent
immunoadjuvant eliciting its activity through the interaction
D
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a
Scheme 4. Synthesis of the Three-Component Carbohydrate-Based Constructs
a
Reagents and conditions: (i) aminocaproic acid, IBCF, N(Bu)3, DMF, H2O (3:1, v/v), 0 °C, 24 h, 55%; (ii) 9 or 10 or 15 or 16, HATU, DIEA,
CH2Cl2, DMF, rt, overnight, 53% (27 from 9), 53% (28 from 10), 48% (29 from 15), 50% (30 from 16); (iii) NaOMe, MeOH, rt, 1 h,
quantitative; (iv) N3-OVA 323−339, THPTA, aminoguanidine hydrochloride, HEPES buffer (100 mM, pH 7.5), H2O, HFIP, 1−3 h, 80% (35
from 33), 77% (36 from 34).
Synthesis of the Core Glycopeptoids 15 and 16.
Various strategies have been developed for synthesizing
glycopeptoids including postmodifications of functionalized
glycotripeptoids 7 and 8 in 75% and 87%, respectively. One
portion of each of them was further deprotected at the N-
́
terminus (Et2NH/CH3CN, 1:2, v/v) followed by Zemplen
side chains for the anchoring of carbohydrate moieties.73
A
deacetylation of the carbohydrate moieties for biological
“submonomer” approach has been adapted for the synthesis of
β-peptoid oligomers in which the side chains are incorporated
by aza-Michael addition to acrylamide intermediates.34 In the
present work, the strategy that has proved the most effective in
synthesizing the core glycopeptoids 15 and 16 is the peptide
coupling of the synthesized N-substituted β-alanine monomers
1 and 2 (Scheme 1). They were prepared in six steps from D-
GalNAc and D-mannose, respectively (Supporting Informa-
tion). First, monomers 1 and 2 were protected as Fmoc
carbamates by treatment with Fmoc-Cl and saturated
NaHCO3 in dioxane to provide 3 (84%) and 4 (91%),
respectively. With these orthogonally protected monomers in
hand, the construction of trimers 15 and 16 was then
accomplished using an iterative sequence, consisting of
forming the free acid partners and their subsequent coupling
with the amine 1 or 2. From monomers 3 and 4, the
corresponding free carboxylic acids were quantitatively
testing (compounds 11 and 12).
As the strategy for conjugation of the core glycopeptoid
trimers 7 and 8 with the OVA 323−339 peptide was based on
the non-native CuAAC reaction, it was therefore necessary to
equip the C-termini of trimers 7 and 8 with a terminal alkyne
arm. Accordingly, the two trimers were treated with TFA/
CH2Cl2 (1:1, v/v) for removal of the tBu esters, followed by a
peptide coupling with propargylamine by employing HATU-
DIEA conditions to give 13 (95%) and 14 (86%). 13 and 14
were further deprotected at the N-terminus (Et2NH/CH3CN,
1:2, v/v) to yield compounds 15 and 16 ready for conjugation
with the TLR7 agonist moiety 26.
At this stage of the synthetic work, the TLR7 agonist (24)
and glycopeptoids components (9, 10) were biotinylated for
enzyme-linked immunosorbent assay (ELISA) aimed at
analyzing the specificity of the antibodies induced by the
constructs (Schemes 2, 3).
t
produced by hydrolysis of the Bu esters (TFA/CH2Cl2, 1:1,
Conjugation to the TLR7 Agonist and OVA 323−339
Peptide. The TLR7 heterocyclic agonist 24, chosen in the
present work, was prepared from xanthine following
adaptations of published procedures (Supporting Informa-
tion).29 The TLR7 heterocyclic agonist 24 was first coupled to
aminocaproic acid, used as a spacer, employing IBCF and
v/v) and coupled with their amine partner (1 or 2) with
HATU/DIEA-optimized conditions (CH2Cl2/DMF (7:3, v/
v)). Glycopeptoids dimers 5 and 6 were isolated in 81% and
90%, respectively, after silica gel column chromatography.
Reiteration of the deprotection−coupling process furnished
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Figure 2. In vitro adjuvanticity and T-antigenicity of the glycoconjugates 31, 35, and 36. Mouse BMDCs (105) were cultured at 37 °C either with
increasing concentrations of 11, 24, 31, 35, or 36 for 48 h or with increasing concentrations of the same compounds for 3 days in the presence of
OT-II CD4+ T lymphocytes isolated from OT-II transgenic mice. IL-12p40 (A), IL-6 (B), and IFN-γ (C) were measured in cell supernatants by
ELISA.
Figure 3. In vivo T-immunogenicity of the three-component glycopeptoids 35 and 36. After administration of 2 × 105 OT-II CD4+ T cells,
C57BL/6 mice (n = 3 per group) were intradermally immunized with 24, 31, 35, 36, or OVA 323−339 (11.7 nmol per mouse). After 5 days, mice
were sacrificed and then the spleen and draining lymph node were collected. OT-II CD4+ T cells proliferation (A) was detected by flow cytometry
using FITC labeled antibodies anti-CD4, anti-Vα2, and anti-Vβ5. Among CD4+ population, the number of OT-II CD4+ T cells in lymph node that
produce IFN-γ (Β) was determined using FITC labeled antibody anti-IFN-γ. The splenocytes were cultured (5 × 105) in the presence or not of
OVA 323−339 (1 μg/mL) at 37 °C for 48 h, and IFN-γ (C) and IL-5 (D) levels were measured in the supernatants by ELISA.
N(Bu)3 in DMF/H2O (3:1, v/v) (Scheme 4). The formed
compound 26 was then conjugated to the glycotripeptoid
scaffolds 9, 10, 15, and 16 to yield glycoconjugates 27 (53%),
28 (53%), 29 (48%), and 30 (50%), respectively. All of them
were deacetylated, giving compounds 31, 32, 33, and 34 in
essentially quantitative yields and high purity.
The deacetylated conjugates 31 and 32 made of only two
components of the three composing the final constructs (35,
36) were intended to be subject to the immunological tests.
On the other hand, the deacetylated oligomers 33 and 34
bearing an alkynyl group were intended to be used for the
conjugation to the azide-functionalized OVA 323−339 peptide
employing the CuAAC reaction. The OVA peptide was indeed
purchased with a 6-azidohexanoyl arm at its N-terminus to
allow the CuAAC to proceed. An air-free procedure developed
by Finn et al.74 was applied to avoid damage of the peptide due
to copper-mediated production of reactive oxygen species. In
addition to the CuSO4/sodium ascorbate reagents, the
THPTA copper(I) stabilizing ligand, and aminoguanidine
hydrochloride, an additive known to intercept byproducts of
ascorbate oxidation was also used.
The CuAAC ligations were conducted under an argon
atmosphere, and all the solvents were carefully degassed. For
the first attempt, 34 and the N3-OVA 323−339 peptide were
engaged (1:1 ratio, ∼5 mM) in a HFIP/HEPES buffer mixture
at pH 7.5. The catalytic solution was composed of CuSO4 (2
equiv), sodium ascorbate (4 equiv), THPTA (6 equiv), and
aminoguanidine (5 equiv). Under these conditions, 34 and
OVA were not totally consumed after 1 h. Another catalytic
solution was added with the same amount of each compound
except for CuSO4 (4 equiv) and sodium ascorbate (8 equiv).
After 2 h of reaction, CuSO4 and sodium ascorbate were added
again. Finally, a complete conversion was observed within 3 h
and a C18 SPE cartridge purification afforded the pure O-
mannosylated three-component construct 36 in a good 77%
yield. In order to reduce the reaction time between 33 and N3-
F
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Figure 4. In vivo B-immunogenicity of the three-component glycopeptoids 35. C57BL/6 mice (n = 3 per group) were intradermally immunized on
days 0 and 21 with 11.7 nmol of 31, 35, or OVA 323−339. Sera were collected on day 0 and before (day 20) or after (day 28) the second injection
and were tested for IgG antibodies by ELISA using 31, OVA 323−339, or biotinylated Tn3G7K coated at 1 μg/mL.
Figure 5. Superior efficacy of the three-component glycopeptoids 35 and 36 in their ability to induce anti-TLR7 agonist IgG. (A−H) C57BL/6
mice (n = 3 per group) were intradermally immunized on days 1 and 20 and then intravenously on day 92 with 11.7 nmol of the compounds 24,
31, 35, 36 or the peptide OVA 323−339. Sera were collected after the second (A−D, day 27) and the third (E−H, day 111) injections and were
analyzed for IgG antibodies by ELISA using 21, 22, 25, or biotinylated Tn3G7K coated at 1 μg/mL.
OVA 323−339, we envisioned directly introducing a catalytic
solution with more significant amounts of CuSO4 (4 equiv),
sodium ascorbate (8 equiv), and aminoguanidine (10 equiv)
but with the same amount of THPTA. Hence, a complete
conversion was obtained within 1 h. After a C18 SPE cartridge
purification, the O-GalNAc three-component construct 35 was
provided in high purity and in good 80% yield.
Immunology. We first evaluated in vitro the capacity of the
constructs to retain the immunological properties of the
original components, through the analysis of the adjuvant
activity of the TLR7 ligand and the T cell antigenicity of the
OVA 323−339 peptide. The adjuvant property of the various
constructs was analyzed for their capacity to trigger the
production of cytokines by murine DC (Figure 2).
Compounds 31, 35, and 36, containing a TLR7 agonist,
induced strong and comparable IL-12 and IL-6 secretion
(Figure 2A and Figure 2B). In contrast, the GalNAc
glycopeptoid trimer 11 and TLR7 agonist 24 alone were
unable to induce the production of cytokines. Taken together,
these results confirm the functional role of the TLR7 agonist
moiety to stimulate mouse BMDCs and highlight the
importance of the conjugation of this TLR7 ligand to the
glycopeptoid moiety for the activation of the TLR7 endosomal
receptors. Recruitment of MGL receptors is likely to promote
DC activation.75 Further, it has been documented that the
immunostimulatory activity of a TLR7 ligand can be amplified
by conjugation.29 We next tested the IFN-γ production by
OVA-specific OT-II T cells in response to the constructs
(Figure 2C). Compounds 35 and 36, which contain the OVA
323−339 peptide but not 31, potently activated OT-II T cells
in the presence of DCs.
The in vivo immunogenicity of the constructs was then
evaluated. C57BL/6 mice received OT-II cells specific for the
OVA 323−339 peptide and were then immunized with
compounds 24, 31, 35, 36 or with the OVA 323−339 peptide.
Five days later, we analyzed the OT-II T cell responses in
spleen and lymph nodes (LN). In the spleen of mice
immunized with the compounds 35 and 36, we detected 30-
fold more OT-II T cells, as compared to the mice immunized
with the OVA 323−339 peptide (Figure 3A). These results
showed that the TLR7 agonist, whatever the GalNAc or Man
glycosylation, potently enhanced the activation of OT-II cells
by the OVA T cell epitope, most likely through DC activation.
Lymph node IFN-γ producing T cells were then analyzed by
intracellular cytokine staining using flow cytometry (Figure
3B). In mice immunized with the OVA 323−339 peptide in
the absence of adjuvant, no IFN-γ production was detected
despite the proliferation of OT-II cells. In contrast, in mice
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immunized with compounds 35 and 36, we observed a high
and comparable IFN-γ production by LN OT-II T cells,
demonstrating no major influence of the type of glycosylation.
In vitro stimulation by the OVA peptide of the splenocytes of
mice immunized with the compounds 35 and 36 induced the
production of high level of IFN-γ but not of IL-5 (Figure 3C
and Figure 3D), suggesting a Th1 phenotype of the activated T
cells, in agreement with the capacity of these compounds to
induce the production of IL-12 by DC (Figure 2A). Control
24 and 31 compounds, devoid of the OVA peptide, did not
induce any T cell proliferation or activation (Figure 3, all
panels).
We then assessed the antibody responses of mice immunized
twice at days 0 and 21 with 31, 35, or peptide OVA 323−339
(Figure 4). No antibody response was detected against the
OVA peptide or against the biotinylated Tn3G7K.76 However,
in the group immunized with compound 35, high antibody
responses were detected against compound 31, demonstrating
the B cell immunogenicity of the immunogen containing
glycans, the peptoid moiety, and the TLR7 agonist (Figure 4).
To further investigate the fine specificity of the antibodies
induced by the immunogen 35 and 36, we analyzed their
reactivity by ELISA against the compounds 21, 22, and 25. For
this purpose, the serum of mice immunized by the constructs
35 and 36 or by the controls 24 and 31 were collected and
analyzed at days 0 and 21 for their capacity to recognize the
glycan biotinylated Tn3G7K, the peptoid moiety 21 and 22 or
the TR7 agonist 25 (Figure 5A−D). Mice also received a third
injection of the compounds at day 92 (Figure 5E−H), and
their sera were analyzed at day 111.
capturing TLR7 activating molecules could represent an
alternative strategy to inhibit TLR7 stimulation. The capacity
of constructs to induce the production of antibodies against
TLR7 agonists was never analyzed and thus clearly deserves
further investigations.
CONCLUSION
■
We have designed, synthesized, and evaluated the three-
component construct 35 as a novel potential anticancer
vaccine candidate. In this respect, we have investigated the use
of a β-peptoid scaffold for the preparation of this three-
component construct. By sequential amide bond forming
reactions and a CuAAC ligation, a β-glycotripeptoid molecule
(B epitope), displaying clustered Tn antigen analogues to
mimic the epithelial tumor surface, a TLR7 immunoadjuvant
and an OVA 323−337 peptide (T-helper epitope) were
conjugated together. To the best of our knowledge, this
structure represents the first example of a self-adjuvanted
vaccine candidate built on a β-peptoid platform. The
immunological evaluation of our original multiepitopic
glycoconjugate 35 clearly demonstrated an effective activation
of innate and adaptive immune cells (DCs, T-helper, and B-
cells), leading to robust IgG antibody responses. We can
conclude from these results that compound 35 is highly
immunogenic and that the functionality of the three
components within the conjugate has been validated in vitro
and in vivo. We have also demonstrated that the incorporation
of a TLR7 agonist is crucial for the immunogenicity of the
construct, providing the expected cytokines to induce
maturation of DCs.
Both compounds 21 and 22 were recognized by the sera of
mice immunized by 35 and 36, showing that independent of
the glycosidic part, the peptoid backbone is slightly
immunogenic (Figure 5A,B,E,F). However, the recognition
of the peptoid remained low as compared to the very high level
of antibodies (up to 100 000) directed against the TLR7
agonist (Figure 5C and Figure 5G). Tn3G7K, expressing the
Tn antigen, was not recognized by the sera of mice immunized
by 35 and 36, in contrast to the 9A7 monoclonal antibody
used as a positive control, demonstrating that the antibody
responses induced by these compounds are in large part
directed against the TLR7 agonist (Figure 5D and Figure 5H)
Thus, in the present study, we have validated in vitro and in
vivo the functionality and efficiency of the three conjugated
components (TLR7 agonist, glycoclusters, and OVA 323−
339). The novel synthetic constructs 35 and 36 are highly
immunogenic but induce unexpected immunological re-
sponses. Indeed, instead of the production of IgG antibodies
against the Tn trimeric cluster as observed with other synthetic
constructs, these compounds elicited in mice low titers of anti-
glycopeptoid (mannose and GalNAc) IgG but very high levels
of IgG antibodies that recognize the TLR7 agonist. The lack of
Tn reactivity probably reflects the absence of natural Ser/Thr
amino acid backbone for the GalNAc within the constructs
(Figure 4B and Figure 4F). As the TLR7 (and TLR9) have
been implicated in the development of some autoimmune
pathologies, such as systemic lupus erythematosus,31,32 the
vaccination against TLR7 agonists could represent a potential
therapeutic approach for the treatment of lupus and other
autoimmune diseases. Several compounds that bind to
endosomal TLR7 and/or TLR9 are indeed being developed
with the goal to inhibit IFN production and activation of
autoimmune B cells.77Thus, circulating antibodies capable of
However, a surprising finding was that, instead of the
production of IgG antibodies against the target Tn antigen, 35
elicited very high levels of IgG antibodies against the TLR7
agonist. Altogether, these preliminary studies could suggest
that the organization of the Tn Ag analogs on the flexible β-
peptoid scaffold does not provide a convenient clustered
conformation for eliciting such antibodies responses. Alter-
natively, it could be suggested that the conformation of
compound 35 is favorable for the presentation of the TLR7
agonist to specific B cells. This could be exploited for the
induction of powerful antibody responses against circulating
DNA. The TLR7 and TLR9 have indeed been implicated in
the development of some autoimmune pathologies, such as
systemic lupus erythematosus.31,32 The vaccination against
TLR7 agonists could represent a potential therapeutic
approach for the treatment of lupus and other autoimmune
diseases.
Several improvements must be made to the tricomponent
vaccine candidate presented here to induce antibody responses
against the Tn antigen. The modularity of our construct and
the efficiency of the synthetic strategy have to be further
exploited to improve the anti-Tn response from a novel design
of glycopeptoids displaying clustered Tn Ags, possibly closer to
the modified mucins comprising other heterocyclic TLR7
immunoadjuvants.
EXPERIMENTAL SECTION
■
Chemistry. Materials and Methods. All anhydrous reactions
were performed under an argon atmosphere. CH2Cl2 was distilled
from CaH2 under argon before use. MeOH was distilled from MgI2
under argon before use. THF was distilled from sodium/
benzophenone under argon before use. DMF and CH3CN were
obtained from commercial sources and stored over 4 Å molecular
H
DOI: 10.1021/acs.jmedchem.8b00960
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Journal of Medicinal Chemistry
Article
sieves. EtOAc was distilled and stored over 4 Å molecular sieves.
MeOH, EtOAc, cyclohexane, and CH2Cl2 for column chromatog-
raphy were distilled before use. Et3N was distilled from KOH and
stored over 4 Å molecular sieves. All other solvents and chemicals
were obtained from commercial sources and used as supplied. The
N3-OVA 323−339 peptide (immunograde purity) equipped with a 6-
azidohexanoyl arm was purchased from Polypeptide Laboratories,
France. TLC was performed on Merck TLC aluminum sheets, silica
gel 60, F254. The extent of reactions was, when applicable, followed by
TLC and/or HPLC. Components were made visual with UV light
and/or vanilline in EtOH/H2SO4 and/or 30% sulfuric acid in MeOH.
Flash chromatography was performed with Merck silica gel 60, 40−63
μm. IR spectra were recorded on a Shimadzu FITR-8400S
spectrometer equipped with a Pike Technologies MIRacle ATR,
and wavenumbers (ν) are expressed in cm−1. NMR spectra were
recorded on a 400 MHz Bruker Avance 400 spetrometer. Chemical
shifts, reported in δ ppm, are referenced to the residual solvent peak.
The following multiplicity abbreviations are used: (s) singlet, (d)
doublet, (t) triplet, (quint) quintuplet, (dd) doublet of doublets, (dt)
doublet of triplets, (ddd) doublet of doublet of doublets, (m)
multiplet. All NMR spectral data for glycopeptoids are of rotameric
mixtures. HRMS were recorded on a Micromass Q-Tof Micro
(3000V) apparatus. RP-HPLC analyses were performed on a Dionex
instrument equipped with an Uptisphere strategy column (C18, 5 μm,
100 Å, 4.6 mm × 250 mm) and a UVD340U detector. All tested
compounds had purity of ≥95% as determined by analytical HPLC.
N-Fmoc Protected O-GalNAc Glycoside 3. Compound 1 (360
mg, 0.7 mmol) was suspended in 1,4-dioxane (1.4 mL) and saturated
NaHCO3 solution (3.7 mL) at 0 °C under an argon atmosphere.
Then, Fmoc-Cl (350 mg, 1.3 mmol) in 1,4-dioxane (1 mL) was added
and the mixture was stirred at room temperature for 4 h. The reaction
was dissolved in AcOEt (30 mL). The organic layer was washed with
H2O (3 × 10 mL), dried (MgSO4), and concentrated under reduced
pressure. The crude was purified by column chromatography
(AcOEt/cyclohexane, 95:5) to give compound 3 as a yellow oil
(422 mg, 84%). RP-HPLC tR, 20.5 min (91.7%) and 22.6 min (8.2%),
MeOH/H2O 80:20, 99.9% purity, 210 nm. IR (ATR): 3353, 3323,
2979, 2927, 1747, 1728, 1700, 1695, 1679, 1560, 1529, 1479, 1458,
1451, 1369, 1224, 1221, 1153, 1131, 1075, 1044, 953, 739, 737 cm−1.
1H NMR (400 MHz, CDCl3) δ 1.42 (m, 9H, t-Bu), 1.67−1.81 (m,
2H, CH2CH2CH2), 1.93−2.19 (m, 13H, 3 × OAc, NHAc, 0.5 ×
removed by coevaporation with CH2Cl2 under reduced pressure to
give the acid as a yellow foam (328 mg, quantitative), used in the next
step without further purification. A stirred solution of the previous
acid (328 mg, 0.5 mmol) in anhydrous CH2Cl2 (3.0 mL) and DMF
(1.8 mL) was treated with DIEA (0.2 mL, 1.2 mmol) for 10 min at
room temperature under an argon atmosphere. HATU (358 mg, 0.94
mmol) was added, and the reaction mixture was stirred for an
additional 10 min. After that, the monomer amine 1 (251 mg, 0.5
mmol) in anhydrous CH2Cl2 (1.0 mL) was added, and the mixture
was stirred overnight at the same temperature. After evaporation of
the solvent, the crude material was dissolved in CH2Cl2 (30 mL),
washed with 5% citric acid solution (2 × 30 mL), saturated NaHCO3
solution (2 × 30 mL), H2O (2 × 30 mL), and brine (2 × 30 mL).
The organic layer was dried (MgSO4) and concentrated under
reduced pressure and the crude was purified by column chromatog-
raphy (CH2Cl2/MeOH, 97:3) to provide compound 5 (480 mg,
81%) as a white foam. RP-HPLC tR, 16.7 min (77.7%) and 18.1 min
(19.9%), MeOH/H2O 80:20, 97.6% purity, 210 nm. IR (ATR): 3307,
2953, 1745, 1715, 1676, 1648, 1555, 1522, 1479, 1426, 1370, 1223,
1
1153, 1132, 1047, 1044, 944, 769, 745 cm−1. H NMR (400 MHz,
CDCl3) δ 1.42 (m, 9H, t-Bu), 1.49−1.84 (m, 4H, 2 × CH2CH2CH2),
1.93−2.14 (m, 24H, 6 × OAc, 2 × NHAc), 2.26−2.71 (m, 4H, 2 ×
CH2CO), 3.06−3.76 (m, 12H, 4 × CH2-N, 2 × CH2-O-GalNAc),
3.98−4.23 (m, 7H, 2 × H5′, 4 × H6′, CH Fmoc), 4.38−4.63 (m, 4H, 2
× H2′, CH2 Fmoc), 4.84 (m, 2H, 2 × H1′), 5.13 (m, 2H, 2 × H3′),
5.35 (m, 2H, 2 × H4′), 6.34−7.10 (m, 2H, 2 × NHAc), 7.30 (t, 2H, J
= 7.4 Hz, 2 × CH aromatic), 7.38 (t, 2H, J = 7.4 Hz, 2 × CH
aromatic), 7.54 (m, 2H, 2 × CH aromatic), 7.75 (d, 2H, J = 7.4 Hz, 2
× CH aromatic). HRMS (ESI): calculated for C59H81N4O23 [M +
H]+, 1213.5292; found, 1213.5251.
β-Dipeptoid O-Mannosylated Scaffold 6. Compound 4 was
conjugated to monomer amine 2 following the procedure described
for 5 to yield compound 6 (690 mg, 90%) as a white foam. RP-HPLC
tR, 22.3 min, MeOH/H2O 80:20, 96.9% purity, 214 nm. IR (ATR):
2947, 1746, 1698, 1643, 1479, 1450, 1440, 1422, 1368, 1220, 1136,
1084, 1047, 980, 743 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.43 (m,
9H, t-Bu), 1.57−1.83 (m, 4H, 2 × CH2CH2CH2), 1.95−2.14 (m,
24H, 8 × OAc), 2.30−2.60 (m, 4H, 2 × CH2CO), 3.10−3.71 (m,
12H, 4 × CH2-N, 2 × CH2-O-Man), 3.96 (m, 2H, 2 × H5′), 4.1 (m,
2H, 2 × H6′), 4.27 (m, 3H, 2 × H6′, CH Fmoc), 4.52 (m, 2H, CH2
Fmoc), 4.77 (m, 2H, 2 × H1′), 5.25 (m, 6H, 2 × H2′, 2 × H3′, 2 ×
H4′), 7.3 (m, 2H, 2 × CH aromatic), 7.38 (t, 2H, J = 7.5 Hz, 2 × CH
aromatic), 7.57 (d, 2H, J = 7.5 Hz, 2 × CH aromatic), 7.75 (d, 2H, J
= 7.5 Hz, 2 × CH aromatic). HRMS (ESI): calculated for
C59H79N2O25 [M + H]+, 1215.4972; found, 1215.4957.
β-Tripeptoid O-GalNAc Scaffold 7. Compound 5 was
conjugated to monomer amine 1 following the procedure described
for 5 to yield compound 7 (380 mg, 75%) as a white foam. RP-HPLC
tR, 14.3 min (70.2%) and 15.4 min (27.5%), MeOH/H2O 80:20,
97.7% purity, 210 nm. IR (ATR): 3342, 2954, 1747, 1703, 1680,
1634, 1626, 1555, 1531, 1480, 1452, 1435, 1371, 1223, 1155, 1131,
1047, 1044, 949, 734 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.42 (m,
9H, t-Bu), 1.72−2.13 (m, 42H, 9 × OAc, 3 × NHAc, 3 ×
CH2CH2CH2), 2.18−2.65 (m, 6H, 3 × CH2CO), 3.06−3.69 (m,
18H, 6 × CH2-N, 3 × CH2-O-GalNAc), 4.04−4.27 (m, 10H, 3 × H5′,
6 × H6′, CH Fmoc), 4.47−4.62 (m, 5H, 3 × H2′, CH2 Fmoc), 4.88
(m, 3H, 3 × H1′), 5.13 (m, 3H, 3 × H3′), 5.36 (m, 3H, 3 × H4′),
6.23−7.09 (m, 3H, 3 × NHAc) 7.29 (t, 2H, J = 7.2 Hz, 2 × CH
aromatic), 7.38 (t, 2H, J = 7.2 Hz, 2 × CH aromatic), 7.53 (m, 2H, 2
× CH aromatic), 7.74 (d, 2H, J = 7.2 Hz, 2 × CH aromatic). HRMS
(ESI): calculated for C79H112N6O33 [M + H]2+, 836.3630; found,
836.3607.
β-Tripeptoid O-Mannosylated Scaffold 8. Compound 6 was
conjugated to monomer amine 2 following the procedure described
for 5 to yield compound 8 (738 mg, 87%) as a white foam. RP-HPLC
tR, 21.4 min, MeOH/H2O 80:20, 96.6% purity, 214 nm. IR (ATR):
2937, 1749, 1699, 1645, 1640, 1480, 1458, 1441, 1423, 1370, 1223,
1136, 1084, 1047, 970 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.4 (m,
9H, t-Bu), 1.60−1.80 (m, 6H, 3 × CH2CH2CH2), 1.91−2.15 (m,
36H, 12 × OAc), 2.40−2.62 (m, 6H, 3 × CH2CO), 3.10−3.70 (m,
t
t
CH2CO2 Bu), 2.45 (m, 1H, 0.5 × CH2CO2 Bu), 3.06−3.66 (m, 6H, 2
× CH2-N, CH2-O-GalNAc), 4.03−4.22 (m, 4H, H5′, 2 × H6′, CH
Fmoc), 4.50 (m, 2H, CH2 Fmoc), 4.60 (m, 1H, H2′), 4.80 (m, 1H,
H1′), 5.13 (dd, 1H, J = 3.0, 11.7 Hz, H3′), 5.37 (d, 1H, J = 3.0 Hz,
H4′), 6.19−6.79 (m, 1H, NHAc), 7.32 (t, 2H, J = 7.6 Hz, 2 × CH
aromatic), 7.39 (t, 2H, J = 7.6 Hz, 2 × CH aromatic), 7.55 (m, 2H, 2
× CH aromatic), 7.75 (d, 2H, J = 7.6 Hz, 2 × CH aromatic). HRMS
(ESI): calculated for C39H51N2O13 [M + H]+, 755.3391; found,
755.3402.
N-Fmoc Protected O-Mannosylated Glycoside 4. A procedure
similar to that for compound 3 was used to obtain compound 4 as a
yellow oil (535 mg, 91%) from 2. RP-HPLC tR, 26.1 min, MeOH/
H2O 80:20, 100% purity, 214 nm. IR (ATR): 2964, 2939, 2920, 1747,
1701, 1479, 1450, 1423, 1368, 1220, 1151, 1136, 1084, 1047, 980,
1
760, 742 cm−1. H NMR (400 MHz, CDCl3) δ 1.43 (m, 9H, t-Bu),
1.50−1.80 (m, 2H, CH2CH2CH2), 1.98 (s, 3H, OAc), 2.01 (s, 3H,
OAc), 2.08 (s, 3H, OAc), 2.14 (s, 3H, OAc), 2.23 (t, 1H, J = 6.4 Hz,
t
t
0.5 × CH2CO2 Bu), 2.43 (t, 1H, J = 6.4 Hz, 0.5 × CH2CO2 Bu),
3.11−3.69 (m, 6H, 2 × CH2-N, CH2-O-Man), 3.95 (m, 1H, H5′),
4.07 (dd, 1H, J = 2.1, 12.1 Hz, 0.5 × H6′), 4.25 (m, 2H, CH Fmoc, 0.5
× H6′), 4.52 (m, 2H, CH2 Fmoc), 4.75 (m, 1H, H1′), 5.25 (m, 3H,
H2′, H3′, H4′), 7.30 (t, 2H, J = 7.4 Hz, 2 × CH aromatic), 7.38 (t, 2H,
J = 7.4 Hz, 2 × CH aromatic), 7.56 (m, 2H, 2 × CH aromatic), 7.75
(d, 2H, J = 7.4 Hz, 2 × CH aromatic). HRMS (ESI): calculated for
C39H50NO14 [M + H]+, 756.3231; found, 756.3228.
β-Dipeptoid O-GalNAc Scaffold 5. Compound 3 (355 mg, 0.5
mmol) was dissolved in TFA/CH2Cl2 (1:1, 6.8 mL) at 0 °C and the
reaction was stirred for 1 h at room temperature under an argon
atmosphere and then concentrated to dryness. The excess of TFA was
I
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Journal of Medicinal Chemistry
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18H, 6 × CH2-N, 3 × CH2-O-Man), 3.94 (m, 3H, 3 × H5′), 4.06 (m,
3H, 3 × H6′), 4.25 (m, 4H, 3 × H6′, CH Fmoc), 4.47 (m, 2H, CH2
Fmoc), 4.75 (m, 3H, 3 × H1′), 5.25 (m, 9H, 3 × H2′, 3 × H3′, 3 ×
H4′), 7.29 (m, 2H, 2 × CH aromatic), 7.36 (t, 2H, J = 7.5 Hz, 2 × CH
aromatic), 7.56 (d, 2H, J = 7.5 Hz, 2 × CH aromatic), 7.73 (d, 2H, J
= 7.5 Hz, 2 × CH aromatic). HRMS (ESI): calculated for
C79H109N3O36 [M + H]2+, 837.8395; found, 837.8367.
β-Tripeptoid O-GalNAc Scaffold 9. A stirred solution of
compound 7 (50 mg, 0.03 mmol) in CH3CN (0.8 mL) was treated
with Et2NH (0.4 mL) at 0 °C for 30 min and at room temperature for
4 h. After concentration to dryness, the excess of Et2NH was removed
by coevaporation with CH2Cl2 and the crude was purified by column
chromatography (CH2Cl2/MeOH, 95:5 to 85:15) to obtain
compound 9 (46 mg, quantitative) as a yellow oil. IR (ATR):
3322, 2942, 1745, 1732, 1654, 1636, 1542, 1536, 1432, 1426, 1371,
1222, 1225, 1156, 1130, 1048, 1036 cm−1. 1H NMR (400 MHz,
CDCl3) δ 1.39−1.42 (m, 9H, m, t-Bu), 1.70−2.13 (m, 43H, 9 × OAc,
3 × NHAc, 3 × CH2CH2CH2, NH peptoid), 2.18−2.83 (m, 6H, 3 ×
CH2CO), 2.90−4.01 (m, 18H, 6 × CH2-N, 3 × CH2-O-GalNAc),
4.03−4.36 (m, 9H, 3 × H5′, 6 × H6′), 4.58 (m, 3H, 3 × H2′), 4.82−
5.00 (m, 3H, 3 × H1′), 5.13 (m, 3H, 3 × H3′), 5.36 (m, 3H, 3 × H4′),
6.54−7.45 (m, 3H, 3 × NHAc). HRMS (ESI): calculated for
C64H102N6O31 [M + H]2+, 725.3295; found, 725.3262.
CH Fmoc), 4.56 (m, 5H, 3 × H2′, CH2 Fmoc), 4.86 (m, 3H, 3 × H1′),
5.14 (m, 3H, 3 × H3′), 5.35 (m, 3H, 3 × H4′), 6.54−7.18 (m, 4H, 3 ×
NHAc, NH-CH2CCH), 7.27 (m, 2H, 2 × CH aromatic), 7.37 (m,
2H, 2 × CH aromatic), 7.53 (m, 2H, 2 × CH aromatic), 7.73 (m, 2H,
2 × CH aromatic). HRMS (ESI): calculated for C78H107N7O32 [M +
H]2+, 826.8475; found, 826.8450.
β-Tripeptoid O-Mannosylated Scaffold 14. A procedure
similar to that for compound 5 was used. The reaction was carried
out with compound 8 (1.0 equiv), conjugated to propargyl amine (3.0
equiv) to yield compound 14 (278 mg, 86%) as a white foam. RP-
HPLC tR, 12.8 min, MeOH/H2O 80:20, 98.0% purity, 214 nm. IR
(ATR): 2941, 2360, 1744, 1693, 1672, 1635, 1477, 1456, 1445, 1426,
1
1369, 1219, 1135, 1083, 1047, 979, 747 cm−1. H NMR (400 MHz,
CDCl3) δ 1.56−1.82 (m, 6H, 3 × CH2CH2CH2), 1.94−2.12 (m,
36H, 12 × OAc), 2.20 (m, 1H, CCH), 2.45−2.7 (m, 6H, 3 ×
CH2CO), 3.15−3.7 (m, 18H, 6 × CH2-N, 3 × CH2-O-Man), 3.94
(m, 5H, CH2-NH, 3 × H5′), 4.07 (m, 3H, 3 × H6′), 4.25 (m, 4H, 3 ×
H6′, CH Fmoc), 4.47 (m, 2H, CH2 Fmoc), 4.75 (m, 3H, 3 × H1′),
5.22 (m, 9H, 3 × H2′, 3 × H3′, 3 × H4′), 7.22 (m, 1H, NH-CH2C
CH), 7.29 (m, 2H, 2 × CH aromatic), 7.36 (t, 2H, J = 7.1 Hz, 2 ×
CH aromatic), 7.56 (d, 2H, J = 7.1 Hz, 2 × CH aromatic), 7.73 (d,
2H, J = 7.1 Hz, 2 × CH aromatic). HRMS (ESI): calculated for
C78H104N4O35 [M + H]2+, 828.3241; found, 828.3207.
β-Tripeptoid O-Mannosylated Scaffold 10. A procedure
similar to that for compound 9 was used. The product 10 (33 mg,
quantitative) was obtained as a yellow oil from 8. IR (ATR): 2949,
2916, 1745, 1741, 1641, 1452, 1440, 1431, 1371, 1224, 1137, 1082,
1045 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.42 (m, 9H, t-Bu), 1.78−
2.14 (m, 42H, 12 × OAc, 3 × CH2CH2CH2), 2.24 (m, 1H, NH
peptoid), 2.47−2.79 (m, 4H, 2 × CH2CO), 2.93−3.86 (m, 20H,
0.5 × CH2CO, 6 × CH2-N, 3 × CH2-O-Man), 3.96 (m, 3H, 3 ×
H5′), 4.07 (m, 3H, 3 × H6′), 4.27 (dd, 3H, J = 5.2, 12.0 Hz, 3 × H6′),
4.77−4.84 (m, 3H, 3 × H1′), 5.17−5.34 (m, 9H, 3 × H2′, 3 × H3′, 3 ×
H4′). HRMS (ESI): calculated for C64H99N3O34 [M + H]2+,
726.8055; found, 726.8067.
β-Tripeptoid O-GalNAc Scaffold 15. A procedure similar to that
for compound 9 was used. The product 15 (53 mg, quantitative) was
obtained as a yellow oil from 13. IR (ATR): 3262, 2914, 2378, 1746,
1673, 1654, 1648, 1637, 1577, 1539, 1528, 1451, 1430, 1372, 1220,
1223, 1167, 1170, 1133, 1142, 1048, 1037 cm−1. 1H NMR (400 MHz,
CDCl3) δ 1.73−2.92 (m, 45H, 9 × OAc, 3 × NHAc, CCH, 3 ×
CH2CO, 3 × CH2CH2CH2, NH peptoid), 3.10−4.05 (20H, m,
NH−CH2-CCH, 6 × CH2-N, 3 × CH2-O-GalNAc), 4.07−4.47 (m,
9H, 3 × H5′, 6 × H6′), 4.59 (m, 3H, 3 × H2′), 4.92 (m, 3H, 3 × H1′),
5.12 (m, 3H, 3 × H3′), 5.36 (m, 3H, 3 × H4′), 6.67−8.00 (m, 4H, 3 ×
NHAc, NH-CH2CCH). HRMS (ESI): calculated for C63H97N7O30
[M + H]2+, 715.8135; found, 715.8030.
β-Tripeptoid O-GalNAc Scaffold 11. A stirred solution of
compound 9 (7.10 mg, 0.0049 mmol) in dry MeOH (1.3 mL) was
treated with a solution of NaOMe (130 μL) in dry MeOH for 1 h at
room temperature under an argon atmosphere. The reaction mixture
was neutralized with Dowex 50W-X8 (H+) resin and then filtered to
give the compound 11 (5.2 mg, quantitative) as a white solid. IR
(ATR): 3514, 3160, 2926, 2853, 1710, 1653, 1634, 1623, 1554, 1467,
1342, 1233, 1154, 1123, 1058, 1041 cm−1. 1H NMR (400 MHz,
CD3OD) δ 1.42 (m, 9H, t-Bu), 1.80−2.09 (m, 15H, 3 ×
CH2CH2CH2, 3 × NHAc), 2.50−3.00 (m, 6H, 3 × CH2CO),
3.20−3.90 (m, 33H, 3 × CH2CH2CO, 3 × CH2N, 3 × OCH2CH2,
3 × H3′, 3 × H4′, 3 × H5′, 6 × H6′), 4.30 (m, 3H, 3H2′), 4.70 (m, 3H,
3H1′). HRMS (ESI): calculated for C46H83N6O22 [M + H]+,
536.2819; found, 536.2825.
β-Tripeptoid O-Mannosylated Scaffold 16. A procedure
similar to that for compound 9 was used. Product 16 (51 mg,
quantitative) was obtained as a yellow oil from 14. IR (ATR): 2954,
2360, 1744, 1674, 1668, 1550, 1452, 1432, 1370, 1223, 1135, 1084,
1047 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.81 (m, 6H, 3 ×
CH2CH2CH2), 1.94−2.11 (m, 36H, 12 × OAc), 2.20 (m, 2H, C
CH, NH peptoid), 2.49−3.02 (m, 6H, 3 × CH2CO), 3.08−3.77
(m, 18H, 6 × CH2-N, 3 × CH2-O-Man), 3.95 (m, 5H, NH−CH2-
CCH, 3 × H5′), 4.07 (m, 3H, 6 × H6′), 4.25 (m, 3H, 6 × H6′), 4.80
(m, 3H, 3 × H1′), 5.22 (m, 9H, 3 × H2′, 3 × H3′, 3 × H4′), 7.75 (m,
1H, NH-CH2CCH). HRMS (ESI): calculated for C63H94N4O33 [M
+ H]2+, 717.2900; found, 717.2973.
TLR7 Agonist−Spacer Conjugate 26. A stirred solution of
agonist TLR7 24 (70 mg, 0.2 mmol) in dry DMF (4 mL) was treated
with tributylamine (70 μL, 0.3 mmol) for 10 min at room
temperature under an argon atmosphere, and then isobutyl
chloroformate (38 μL, 0.3 mmol) was added. After 10 min again,
aminocaproic acid (128 mg, 0.98 mmol) in DMF/H2O (1:1, 4 mL)
was added at 0 °C and the mixture was stirred for an additional 28 h.
The reaction mixture was concentrated, dissolved in H2O (7 mL),
and acidified with 1.0 M HCl to pH 1.0. The mixture was cooled
down to 0 °C and then filtered to give compound 26 (51 mg, 55%) as
a white solid. IR (ATR): 3419, 3103, 3039, 2935, 2858, 1705, 1636,
1616, 1553, 1506, 1452, 1419, 1375, 1348, 1327, 1125, 1092, 949,
β-Dipeptoid O-Mannosylated Scaffold 12. A procedure similar
to that for compound 11 was used. The product 12 was obtained (8.0
mg, quantitative) as a white foam from 10. IR (ATR): 3517, 3168,
2926, 2857, 1715, 1656, 1630, 1625, 1555, 1475, 1345, 1233, 1154,
1
1123, 1060, 1047 cm−1. H NMR (400 MHz, CD3OD) δ 1.42 (m,
9H, t-Bu), 1.80−2.09 (m, 6H, 3 × CH2CH2CH2), 2.50−3.00 (m, 6H,
3 × CH2CO), 3.20−3.90 (m, 36H, 3 × CH2CH2CO, 3 × CH2N,
3 × OCH2CH2, 3 × H2′, 3 × H3′, 3 × H4′, 3 × H5′, 6 × H6′), 4.70 (m,
3H, 3 × H1′). HRMS (ESI): calculated for C40H74N3O22 [M + H]+,
948.4764; found, 948.4776.
β-Tripeptoid O-GalNAc Scaffold 13. A procedure similar to that
for compound 5 was used. The reaction was carried out with
compound 7 (1.0 equiv), conjugated to propargyl amine (3.0 equiv)
to yield compound 13 (138 mg, 95%) as a white foam. RP-HPLC tR,
10.0 min (75.8%) and 10.5 min (24.1%), MeOH/H2O 80:20, 99.9%
purity, 210 nm. IR (ATR): 3289, 2948, 2371, 1745, 1675, 1671, 1648,
1637, 1570, 1541, 1478, 1456, 1424, 1371, 1224, 1166, 1131, 1073,
1047, 948, 923, 742 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.70−2.21
(m, 43H, 9 × OAc, 3 × NHAc, CCH, 3 × CH2CH2CH2), 2.24−
2.68 (m, 6H, 3 × CH2CO), 3.11−3.72 (m, 18H, 6 × CH2-N, 3 ×
CH2-O-GalNAc), 3.96−4.26 (m, 12H, CH2-NH, 3 × H5′, 6 × H6′,
1
779, 717 cm−1. H NMR (400 MHz, DMSO-d6) δ 1.26 (m, 2H,
CH2(CH2)2CO2H), 1.50 (m, 4H, NHCH2CH2, CH2CH2CO2H),
2.19 (t, 2H, J = 7.2 Hz, CH2CO2H), 3.21 (m, 2H, NHCH2), 3.26 (s,
3H, CH3-O), 3.56 (t, 2H, J = 4.5 Hz, CH2-O), 4.24 (t, 2H, J = 4.5 Hz,
CH2-O), 4.89 (s, 2H, CH2-C6H4CO), 6.53 (s, 2H, NH2), 7.34 (d,
2H, J = 8.0 Hz, 2 × CH aromatic), 7.76 (d, 2H, J = 8.0 Hz, 2 × CH
aromatic), 8.39 (t, 1H, J = 5.0 Hz, NHCO), 10.0 (s, 1H, NH
cycle), 12.0 (s, 1H, CO2H). HRMS (ESI): calculated for C22H29N6O6
[M + H]+, 473.2149; found, 473.2146.
TLR7 Agonist−Spacer−O-GalNAc β-Tripeptoid Conjugate
27. A stirred solution of the acid 26 (10.4 mg, 0.022 mmol) in
J
DOI: 10.1021/acs.jmedchem.8b00960
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
anhydrous CH2Cl2 (0.5 mL) and DMF (0.45 mL) was treated with
DIEA (10 μL, 0.051 mmol) for 10 min at room temperature under an
argon atmosphere. HATU (16.7 mg, 0.044 mmol) was added, and the
reaction mixture was stirred for an additional 10 min. After that, the
monomer amine 9 (31.9 mg, 0.022 mmol) in anhydrous CH2Cl2
(0.55 mL) and the mixture were stirred for 40 h at the same
temperature. After evaporation of the solvent, the crude material was
dissolved in CH2Cl2 (15 mL), washed with 5% citric acid solution (2
× 15 mL), saturated NaHCO3 solution (2 × 15 mL), H2O (2 × 15
mL), and brine (2 × 15 mL). The organic layer was dried (MgSO4)
and concentrated under reduced pressure and the crude was purified
by column chromatography (AcOEt/MeOH, 90:10 to 80:20) to
provide compound 27 (22 mg, 53%) as a white foam. IR (ATR):
3354, 3330, 2960, 2923, 2853, 1746, 1733, 1656, 1649, 1640, 1633,
1622, 1615, 1570, 1541, 1460, 1436, 1428, 1371, 1259, 1233, 1227,
1158, 1153, 1129, 1081, 1075, 1035, 800 cm−1. 1H NMR (400 MHz,
CDCl3) δ 1.43 (s, 9H, t-Bu), 1.54−1.85 (m, 12H, 3 × CH2CH2CH2,
CH2CH2CO spacer, CH2CH2CH2CO spacer, CH2CH2NH
spacer), 1.90−2.16 (m, 36H, 9 × OAc, 3 × NHAc), 2.20−2.70 (m,
2957, 2924, 1746, 1647, 1644, 1618, 1575, 1540, 1458, 1440, 1419,
1
1370, 1342, 1225, 1134, 1090, 1047, 932, 899 cm−1. H NMR (400
MHz, CDCl3) δ 1.58−1.88 (m, 12H, 3 × CH2CH2CH2, CH2CH2C
O spacer, CH2CH2CH2CO spacer, CH2CH2NH spacer), 1.95−
2.15 (m, 39H, 12 × OAc, CCH, CH2CO spacer), 2.19−2.71 (m,
6H, 3 × CH2CO peptoid), 3.27−3.76 (m, 25H, 6 × CH2N
peptoid, 3 × CH2-O-Man, CH2NH spacer, CH2-O agonist, CH3-O),
3.9−4.01 (m, 5H, NH−CH2-CCH, 3 × H5′), 4.1 (m, 3H, 3 × H6′),
4.28 (dd, 3H, J = 4.8, 12.2 Hz, 3 × H6′), 4.41 (m, 2H, CH2-O
agonist), 4.81 (m, 3H, 3 × H1′), 5.00 (m, 2H, CH2N agonist), 5.17−
5.32 (m, 9H, 3 × H2′, 3 × H3′, 3 × H4′), 5.82 (m, 2H, NH2), 7.39−
7.88 (m, 6H, 4 × CH aromatic, NH spacer, NH-CH2CH), 10.1 (m,
1H, NH cycle). HRMS (ESI): calculated for C85H120N10O38 [M +
H]2+, 944.3882; found, 944.3833.
TLR7 Agonist−Spacer−O-GalNAc β-Tripeptoid Conjugate
31. A procedure similar to that for compound 11 was used. The
product 31 was obtained (12.6 mg, quantitative) as a white foam from
27. RP-HPLC tR, 7.4 min, MeOH/H2O 60:40, 95.9% purity, 214 nm.
IR (ATR): 3501, 3181, 2928, 2853, 1720, 1642, 1625, 1615, 1555,
1465, 1374, 1340, 1153, 1123, 1060, 1039, 971 cm−1. 1H NMR (400
MHz, CD3OD) δ 1.42−1.45 (m, 9H, t-Bu), 1.55−1.71 (m, 6H, m,
CH2CH2CO spacer, CH2CH2CH2CO spacer, CH2CH2NH
spacer), 1.75−1.93 (m, 6H, 3 × CH2CH2CH2 peptoid), 1.99−2.03
t
8H, 3 × CH2CO2 Bu, CH2CO spacer), 3.30−3.74 (m, 25H, 6 ×
CH2N peptoid, 3 × CH2-O-GalNAc, CH2NH spacer, CH2-O, CH3-
O), 4.05−4.27 (m, 9H, 3 × H5′, 6 × H6′), 4.43 (m, 2H, CH2-O),
4.53−4.64 (m, 3H, 3 × H2′), 4.85−5.08 (m, 5H, 3 × H1′, CH2N
agonist), 5.15 (m, 3H, 3 × H3′), 5.37 (m, 3H, 3 × H4′), 5.77 (m, 2H,
NH2), 6.76−7.73 (m, 8H, 4 × CH aromatic, NH spacer, 3 × NHAc),
10.16 (m, 1H, NH cycle). HRMS (ESI): calculated for
C86H128N12O36 [M + H]2+, 952.4277; found, 952.4245.
t
(m, 9H, 3 × NHAc), 2.38−2.88 (m, 8H, 3 × CH2CO2 Bu, CH2CO
spacer), 3.33−3.90 (m, 40H, 6 × CH2N peptoid, 3 × CH2-O-
GalNAc, 3 × H3′, 3 × H4′, 3 × H5′, 6 × H6′, CH2NH spacer, CH2-O,
CH3-O), 4.28 (m, 3H, 3 × H2′), 4.45 (m, 2H, CH2-O), 4.75−4.83 (m,
3H, 3 × H1′), 5.01 (s, 2H, CH2N agonist), 7.47 (d, 2H, J = 8.2 Hz, 2
× CH aromatic), 7.78 (d, 2H, J = 8.2 Hz, 2 × CH aromatic). HRMS
(ESI): calculated for C68H110N12O27 [M + H]2+, 763.3802; found,
763.3774.
TLR7 Agonist−Spacer−O-Mannosylated β-Tripeptoid Con-
jugate 28. A procedure similar to that for compound 27 was used.
Compound 26 was conjugated to the glycopeptoid 10 to yield
compound 28 (23 mg, 53%) as a white foam. IR (ATR): 3352, 3329,
2962, 2925, 2858, 1745, 1733, 1657, 1645, 1640, 1633, 1628, 1617,
1571, 1541, 1460, 1438, 1428, 1378, 1260, 1233, 1222, 1159, 1153,
TLR7 Agonist−Spacer−O-Mannosylated β-Tripeptoid Con-
jugate 32. A procedure similar to that for compound 11 was used.
The product 32 was obtained (3.9 mg, quantitative) as a white foam
from 28. IR (ATR): 3508, 3175, 2928, 2859, 1710, 1645, 1630, 1620,
1560, 1465, 1373, 1345, 1155, 1120, 1061, 1040, 971 cm−1. 1H NMR
(400 MHz, CD3OD) δ 1.42−1.45 (m, 9H, t-Bu), 1.57−1.71 (m, 6H,
CH2CH2CO spacer, CH2CH2CH2CO spacer, CH2CH2NH
spacer), 1.78−1.95 (m, 6H, 3 × CH2CH2CH2 peptoid), 2.32−2.84
1
1130, 1081, 1076, 1034, 800 cm−1. H NMR (400 MHz, CDCl3) δ
1.42 (s, 1H, t-Bu), 1.58−1.90 (m, 12H, 3 × CH2CH2CH2,
CH2CH2CO spacer, CH2CH2CH2CO spacer, CH2CH2NH
spacer), 1.93−2.15 (m, 36H, 12 × OAc), 2.25−2.72 (m, 8H, 3 ×
t
CH2CO2 Bu, CH2CO spacer), 3.34−3.76 (m, 25H, 6 × CH2N
peptoid, 3 × CH2-O-Man, CH2N spacer, CH2-O, CH3-O), 3.97 (m,
3H, 3 × H5′), 4.09 (m, 3H, 3 × H6′), 4.28 (dd, 3H, J = 4.7, 12.1 Hz, 3
× H6′), 4.41 (m, 2H, CH2-O), 4.81 (m, 3H, 3 × H1′), 5.00 (m, 2H,
CH2N agonist), 5.16−5.31 (m, 9H, 3 × H2′, 3 × H3′, 3 × H4′), 5.73
(m, 2H, NH2), 7.36−7.44 (m, 3H, 2 × CH aromatic, NH spacer),
7.80 (d, 2H, J = 7.9 Hz, 2 × CH aromatic), 10.07 (m, 1H, NH cycle).
HRMS (ESI): calculated for C86H125N9O39 [M + H]2+, 953.9037;
found, 953.9047.
t
(m, 8H, 3 × CH2CO2 Bu, CH2CO spacer), 3.39−3.98 (m, 43H, 6
× CH2N peptoid, 3 × CH2-O-Man, 3 × H2′, 3 × H3′, 3 × H4′, 3 × H5′,
6 × H6′, CH2NH spacer, CH2-O, CH3-O), 4.45 (m, 2H, CH2-O), 4.72
(m, 3H, 3 × H1′), 5.07 (s, 2H, CH2N agonist), 7.50 (d, 2H, J = 8.1
Hz, 2 × CH aromatic), 7.80 (d, 2H, J = 7.7 Hz, 2 × CH aromatic).
HRMS (ESI): calculated for C62H101N9O27 [M + H]2+, 701.8403;
found, 701.8415.
TLR7 Agonist−Spacer−O-GalNAc β-Tripeptoid Conjugate
33. A procedure similar to that for compound 11 was used. The
product 33 was obtained (12.1 mg, quantitative) as a white foam from
29. RP-HPLC tR, 11.0 min, 10−60% MeOH/H2O, 85.6% purity, 205
nm. IR (ATR): 3342, 3264, 2950, 2925, 2853, 2364, 1718, 1710,
1648, 1637, 1618, 1560, 1545, 1460, 1450, 1439, 1419, 1377, 1342,
1154, 1121, 1039, 787 cm−1. 1H NMR (400 MHz, CD3OD) δ 1,58−
1,71 (m, 6H, CH2CH2CO spacer, CH2CH2CH2CO spacer,
CH2CH2NH spacer), 1.78−1.93 (m, 6H, 3 × CH2CH2CH2), 1.99−
2,05 (m, 9H, 3 × NHAc), 2.39−2.85 (m, 9H, CCH, 3× CH2CO
peptoid, CH2CO spacer), 3.36−3.95 (m, 42H, NH−CH2-CCH,
6 × CH2N peptoid, 3 × CH2-O-GalNAc, 3 × H3′, 3 × H4′, 3 × H5′, 3
× H5′, CH2NH spacer, CH2-O agonist, O−CH3), 4.28 (m, 3H, 3 ×
H2′), 4.40 (m, 2H, CH2-O agonist), 4.76−4.84 (m, 3H, 3 × H1′), 5.04
(s, 2H, CH2N agonist), 7.47 (d, 2H, J = 8.2 Hz, 2 × CH aromatic),
7.78 (d, 2H, J = 8.2 Hz, 2 × CH aromatic). HRMS (ESI): calculated
for C67H105N13O26 [M + H]2+, 753.8642; found, 753.8641.
TLR7 Agonist−Spacer−O-GalNAc β-Tripeptoid Conjugate
29. A procedure similar to that for compound 27 was used.
Compound 26 was conjugated to the glycopeptoid 15 to yield
compound 29 (20 mg, 48%) as a white foam. RP-HPLC tR, 9.0 min,
MeOH/H2O 70:30, 98.4% purity, 254 nm. IR (ATR): 3306, 3230,
2960, 2923, 2853, 2354, 1745, 1731, 1648, 1639, 1615, 1571, 1546,
1466, 1426, 1415, 1371, 1343, 1229, 1162, 1130, 1129, 1075, 1045,
1
781 cm−1. H NMR (400 MHz, CDCl3) δ 1.39−1.88 (m, 12H, 3 ×
CH2CH2CH2, CH2CH2CO spacer, CH2CH2CH2CO spacer,
CH2CH2NH spacer), 1.94−2.16 (m, 39H, 9 × OAc, 3 × NHAc, C
CH, CH2CO spacer), 2.3−2.68 (m, 6H, 3 × CH2CO peptoid),
3.30−3.74 (m, 25H, 6 × CH2N peptoid, 3 × CH2-O-GalNAc,
CH2NH spacer, CH2-O agonist, CH3-O), 3.94−4.19 (m, 11H, NH−
CH2-CCH, 3 × H5′, 6 × H6′), 4.43 (m, 2H, CH2-O agonist), 4.58
(m, 3H, 3 × H2′), 4.83−5.04 (m, 5H, 3 × H1′, CH2N agonist), 5.09−
5.22 (m, 3H, 3 × H3′), 5.38 (m, 3H, 3 × H4′), 5.76 (m, 2H, NH2),
6.86−7.72 (m, 9H, 4 × CH aromatic, NH spacer, 3 × NHAc, NH-
CH2CH), 10.16 (m, 1H, NH cycle). HRMS (ESI): calculated for
C85H123N13O35 [M + H]2+, 942.9122; found, 942.9157.
TLR7 Agonist−Spacer−O-Mannosylated β-Tripeptoid Con-
jugate 30. A procedure similar to that for compound 27 was used.
Compound 26 was conjugated to the glycopeptoid 16 to yield
compound 30 (22 mg, 50%) as a white foam. RP-HPLC tR, 11.7 min,
MeOH/H2O 70:30, 92.6% purity, 210 nm. IR (ATR): 3355, 3333,
TLR7 Agonist−Spacer−O-Mannosylated β-Tripeptoid Con-
jugate 34. A procedure similar to that for compound 11 was used.
The product 34 was obtained (10.8 mg, quantitative) as a white foam
from 30. RP-HPLC tR, 10.6 min, 10−60% MeOH/H2O, 89.8% purity,
210 nm. IR (ATR): 3532, 3120, 2924, 2855, 2359, 1727, 1707, 1663,
1617, 1564, 1465, 1419, 1343, 1200, 1132, 1062, 1030, 1028, 972
cm−1
.
1H NMR (400 MHz, CD3OD) δ 1.57−1.71 (m, 6H,
K
DOI: 10.1021/acs.jmedchem.8b00960
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CH2CH2CO spacer, CH2CH2CH2CO spacer, CH2CH2NH
spacer), 1.77−1.92 (m, 6H, 3 × CH2CH2CH2), 2.35−2.84 (m, 9H,
CCH, 3× CH2CO peptoid, CH2CO spacer), 3.36−3.97 (m,
45H, NH−CH2-CCH, 6 × CH2N peptoid, 3 × CH2-O-Man, 3 ×
H2′, 3 × H3′, 3 × H4′, 3 × H5′, 3 × H5′, CH2NH spacer, CH2-O
agonist, O−CH3), 4.41 (m, 2H, CH2-O agonist), 4.74 (3H, m, 3 ×
H1′), 5.04 (m, 2H, CH2N agonist), 7.46 (d, 2H, J = 8.1 Hz, 2 × CH
aromatic), 7.78 (d, 2H, J = 7.7 Hz, 2 × CH aromatic). HRMS (ESI):
calculated for C61H96N10O26 [M + H]2+, 692.3243; found, 692.3209.
Three-Component O-GalNAc Construct 35. All solvents were
degassed by several vacuum (15 mbar)/argon cycles. A stirred
solution of compound 33 (3.9 mg, 0.0026 mmol) and OVA 323−339
peptide (5.0 mg, 0.0026 mmol) was dissolved in HFIP (150 μL),
H2O (50 μL), and HEPES buffer (100 μL, 100 mM, pH 7.5) at room
temperature under an argon atmosphere. This solution was
neutralized to pH 7.5 with 0.5 N NaOH. Then, a catalytic solution
(350 μL) composed of CuSO4·5H2O (2.6 mg, 0.01 mmol), THPTA
(6.8 mg, 0.016 mmol), aminoguanidine hydrochloride (2.9 mg, 0.026
mmol), and sodium ascorbate (4.2 mg, 0.02 mmol) was added. The
reaction mixture was stirred for 1 h, quenched with 0.5% aqueous
TFA, and purified on SPE cartridge (H2O/CH3CN, 100:0 to 0:100)
to afford the three-component O-GalNAc 35 (7.1 mg, 80%) as a
white solid after lyophilization. RP-HPLC tR, 7.1 min, CH3CN/H2O
(0.1% TFA) 30:70, 96.5% purity, 214 nm. HRMS (ESI): calculated
for C147H235N42O52 [M + H]3+, 1140.2340; found, 1140.5516; for
C147H236N42O52 [M + H]4+, 855.4273; found, 855.6634. MALDI-
TOF: calculated for [M + H]+ 3418.6874; found,3418.6375.
Three-Component O-Mannosylated Construct 36. All solvent
were degassed by several vacuum (15 mbar)/argon cycles. A stirred
solution of compound 34 (3.6 mg, 0.0026 mmol) and OVA 323−339
peptide (5.0 mg, 0.0026 mmol) was dissolved in HFIP (150 μL),
H2O (50 μL) and HEPES buffer (100 μL, 100 mM, pH 7.5) at room
temperature under an argon atmosphere. This solution was
neutralized to pH 7.5 with 0.5 N NaOH. Then, a catalytic solution
(350 μL) composed of CuSO4·5H2O (1.3 mg, 0.013 mmol), THPTA
(6.8 mg, 0.016 mmol), aminoguanidine hydrochloride (1.4 mg, 0.013
mmol), and sodium ascorbate (2.1 mg, 0.01 mmol) was added. Under
these conditions, 34 and OVA were not totally consumed after 1 h.
Another catalytic solution was added with the same amount of each
compound except for CuSO4·5H2O (2.6 mg, 0.026 mmol) and
sodium ascorbate (4.2 mg, 0.02 mmol). After 2 h of reaction, the
same amounts of CuSO4·5H2O and sodium ascorbate were added
again. Finally, a complete conversion was observed within 3 h and the
reaction mixture was quenched with 0.5% aqueous TFA and purified
on SPE cartridge (H2O/CH3CN, 100:0 to 0:100) to afford the three-
component O-mannosylated 36 (6.6 mg, 80%) as a white solid after
lyophilization. RP-HPLC tR, 7.2 min, CH3CN/H2O (0.1% TFA)
30:70, 95.8% purity, 214 nm. HRMS (ESI): calculated for
C141H226N39O52 [M + H]3+, 1099.2074; found, 1099.5420; for
C141H227N39O52 [M + H]4+, 824.6574; found, 824.8961. MALDI-
TOF: calculated for [M + H]+, 3295.6077; found, 3295,6024.
Immunology: Experimental Part. Stimulation of Mouse
Bone Marrow-Derived DCs (BMDCs) by the Compounds
(Figure 2A and Figure 2B). Bone marrow-derived DCs were
generated from bone marrow precursors from C57BL/6 mice in
RPMI medium (Gibco) containing 50 mM 2-ME (Gibco), 10% fetal
calf serum (Thermo Hyclone), antibiotics, and 1% of a GM-CSF-
containing supernatant. Cells were recovered on day 6 with PBS
EDTA and usually contained 60−70% of CD11c+CD11b+ cells. They
were then incubated at 37 °C with increasing concentrations of the
various compounds (11, 24, 31, 35, and 36) for 48 h. IL-12p40 and
IL-6 were measured in cell supernatants by ELISA.
the cultures for 3 days. IFN-γ was then measured in cell supernatants
by ELISA.
In Vivo T-Immunogenicity (Figure 3). C57BL/6 mice from
Charles River Laboratories (Les Oncins, France) were used at 6−10
weeks of age. OT-II cells were harvested from OT-II Rag2 T cell
receptor-transgenic knockout mice, bred at the animal facilities of the
Pasteur Institute. OT-2 cells are specific for the OVA323−339 T-cell
epitope presented by I-Ab molecules. After administration of 2 × 105
OT-II CD4+ T cells, C57BL/6 mice (n = 3 per group) were
intradermally immunized with 24, 31, 35, 36, or OVA 323−339
compounds (11.7 nmol per mouse).
OT-II CD4+ T Cells Proliferation (Figure 3A). Five days after
immunization, C57BL/6 mice were sacrificed and the spleen was
collected. The in vivo proliferation of OT-II CD4+ T cells was
analyzed by flow cytometry using anti-CD4 (e-bioscience 25-0042-
82), anti-Vα2 (e-bioscience 47-5812-82) and anti-Vβ5 (e-bioscience
46-5796-82). Cells were acquired on a Fortessa flow cytometer and
analyzed using FlowJo software.
Measurement of T Cell Response by Intracellular Staining
(Figure 3B). For the detection of IFNγ-producing CD8 T cells by
intracellular staining (ICS), mice were sacrificed 5 days after
immunization and isolated lymph node cells were set up in 96-well
plates at a concentration of 5 × 105 cells per well in RPMI medium
(Gibco) containing 50 mM 2-ME (Gibco), 10% fetal calf serum
(Thermo Hyclone), and antibiotics and incubated with 50 μg/mL of
the OVA 323−339 peptide in the presence of BD GolgiPlug (BD
Biosciences) for 2 h at 37 °C. Cells were fixed, permeabilized, and
stained using the BD Cytofix/Cytoperm kit (BD Biosciences).
Intracellular staining (ICS) was performed according to the
manufacturer’s instructions, with anti-CD4-FITC, anti-CD3-APC-
eFluor780, and anti-IFNγ from eBioscience. Cells were acquired on a
Fortessa flow cytometer and analyzed using FlowJo software.
Measurement of T Cell Response by ELISA (Figure 3C and
Figure 3D). Mice were sacrificed 5 days after immunization, and the
splenocytes were cultured in RPMI medium (Gibco) containing 50
mM 2-ME (Gibco), 10% fetal calf serum (Thermo Hyclone), and
antibiotics (5 × 105 cells per well), in the presence or not of OVA
323−339 (1 μg/mL) at 37 °C for 48 h. IFN-γ and IL-5 levels were
measured in the supernatants by ELISA using antibodies against IFN-
γ and IL-5 (BD Biosciences).
Immunization of Mice (Figures 4 and 5). Six-week-old female
C57BL/6 mice were purchased from Charles River Laboratories.
Mice were kept in the Pasteur Institute animal house and supplied
with water and food ad libitum. All procedures involving mice were in
accordance with the French ethical committee CETEA (Comite
́
́
d’Ethique en Experimentation Animale) (Project No. 2013-126).
Mice were intramuscularly (IM) or intradermally (ID) immunized
with 11.7 nmol of the various compounds on days 0 and 21. Sera were
collected at days 1, 21, and 28. In the experiment depicted on Figure
5, mice also received a third injection of the compounds at day 92 and
their sera were collected at day 111
Analysis of Antibody Response by ELISA (Figures 4 and 5).
Sera from immunized mice were tested for the presence of antibodies
by ELISA, using MaxiSorp plates (Nunc) coated with the various
compounds. For the detection of anti-Tn antibodies, the biotinylated
Tn3-G6K(Biot)G peptide (1 μg/mL) was incubated for 1 h at 37 °C
on streptavidin-coated microtiter plates (Pierce). Then, serial
dilutions of sera were performed and bound Abs were revealed
using anti-mouse-IgG-HRP peroxidase conjugate (Sigma) and o-
phenyldiamine/H2O2 substrates. Plates were read photometrically at
492 nm in an ELISA autoreader (Dynatech, Marnes la Coquette,
France). The negative control consisted of naive mouse sera diluted
100-fold. ELISA Ab titers were determined by linear regression
analysis, plotting dilution vs absorbance at 492 nm. The titers were
calculated to be the log10 highest dilution that gave twice the
absorbance of normal mouse sera diluted 1/100. Titers were given as
the arithmetic mean SD of the log10 titers.
IFN-γ Production by OVA-Specific OT-II T Cells in Response
to the Constructs (Figure 2C). C57BL/6 bone marrow-derived DC
were incubated in RPMI medium (Gibco) containing 50 mM 2-ME
(Gibco), 10% fetal calf serum (Thermo Hyclone), and antibiotics at
37 °C with increasing concentrations of the compound 31, 35, or 36.
CD4+ T lymphocytes isolated from OT-II Rag2 T cell receptor-
transgenic knockout mice, bred at the animal facilities of the Institut
Pasteur, and specific for the I-Ab-OVA323−339 epitopes were added to
L
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.8b00960.
■
S
Synthesis of compounds 1 and 2 and TLR7 agonist 24;
HPLC chromatograms for compounds 3−8, 13, 14, 29,
30, 31, 33−36; HRMS results for compounds 3, 4, 6, 9,
10, 12, 13, 15−17, 19−22, 24, 26−36; HSQC spectra
for compounds 3, 4, 6, 9, 11, 13, 15, 16, 27, 29, 30, 31,
33, 34, and MALDI-TOF spectra for 35 and 36 (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
*C.L.: e-mail, claude.leclerc@pasteur.fr.
*C.T.: e-mail, claude.taillefumier@uca.fr.
ORCID
Claude Taillefumier: 0000-0003-3126-495X
Author Contributions
T.S., C.L. and C.T. contributed equally. The manuscript was
written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge Dr.Vincent Aucagne for helpful discussions
and advice regarding conjugation of the OVA 323−339
peptide. Also, we kindly thank Prof. Bernadette Bouchon for
the MALDI-TOF analyses. This work was supported by
funding from the French Ministry of Higher Education and
Research (MESR to T.S.) for the chemical syntheses and by
́
the Ligue Nationale Contre le Cancer (Equipe Labellisee
2017) to C.L. for the immunological analyses.
ABBREVIATIONS USED
■
Boc, tert-butoxycarbonyl; CLR, C-type lectin receptor;
CuAAC, copper azide−alkyne cycloaddition; DIEA, N,N-
diisopropylethylamine; DC, dendritic cell; DMF, N,N-
dimethylformamide; Fmoc, 9-fluorenylmethoxycarbonyl; Gal-
NAc, N-acetylgalactosamine; HATU, azabenzotriazole-
tetramethyluranium hexafluorophosphate; HFIP, hexafluoroi-
sopropanol; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid; IBCF, isobutyl chloroformate; MGL,
macrophage galactose-type C-type lectin; Man, mannose;
OVA, ovalbumin; NHS, N-hydroxysuccinimide; PRR, pat-
tern-recognition receptor; SLE, lupus erythematosus; TACA,
tumor-associated carbohydrate antigen; TFA, trifluoroacetic
acid; TLR, Toll-like receptor; TF, Thomsen−Friedenreich
(12) Geraci, C.; Consoli, G. M. L.; Galante, E.; Bousquet, E.;
Pappalardo, M.; Spadaro, A. Calix[4]arene decorated with four Tn
antigen glycomimetic units and P3CS immunoadjuvant: synthesis,
characterization, and anticancer immunological evaluation. Bioconju-
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DOI: 10.1021/acs.jmedchem.8b00960
J. Med. Chem. XXXX, XXX, XXX−XXX