Synthesis and immunostimulatory activity of sugar-conjugated TLR7 ligands

Article abstract of DOI:10.1016/j.bmcl.2019.126840

Toll-like receptors (TLRs) are a type of pattern recognition receptors (PRRs), which are activated by recognizing pathogen-associated molecular patterns (PAMPs). The activation of TLRs initiates innate immune responses and subsequently leads to adaptive immune responses. TLR agonists are effective immuomodulators in vaccine adjuvants for infectious diseases and cancer immunotherapy. In exploring hydrophilic small molecules of TLR7 ligands using the cell-targeted property of a vaccine adjuvant, we conjugated 1V209, a small TLR7 ligand molecule, with various low or middle molecular weight sugar molecules that work as carriers. The sugar-conjugated 1V209 derivatives showed increased water solubility and higher immunostimulatory activity in both mouse and human cells compared to unmodified 1V209. The improved immunostimulatory potency of sugar-conjugates was attenuated by an inhibitor of endocytic process, cytochalasin D, suggesting that conjugation of sugar moieties may enhance the uptake of TLR7 ligand into the endosomal compartment. Collectively our results support that sugar-conjugated TLR7 ligands are applicable to novel drugs for cancer and vaccine therapy.

Full text of DOI:10.1016/j.bmcl.2019.126840

Journal Pre-proofs
Synthesis and Immunostimulatory Activity of Sugar-conjugated TLR7 ligands
Akihito Baba, Masahiro Wakao, Hiroyuki Shinchi, Michael Chan, Tomoko
Hayashi, Shiyin Yao, Howard B. Cottam, Dennis A. Carson, Yasuo Suda
PII:
S0960-894X(19)30812-1
DOI:
https://doi.org/10.1016/j.bmcl.2019.126840
BMCL 126840
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
18 September 2019
5 November 2019
15 November 2019
Please cite this article as: Baba, A., Wakao, M., Shinchi, H., Chan, M., Hayashi, T., Yao, S., Cottam, H.B., Carson,
D.A., Suda, Y., Synthesis and Immunostimulatory Activity of Sugar-conjugated TLR7 ligands, Bioorganic &
Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126840
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will
undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing
this version to give early visibility of the article. Please note that, during the production process, errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
©
2019 Published by Elsevier Ltd.

Synthesis and Immunostimulatory Activity of Sugar-conjugated TLR7 ligands
1
1
1
2
Akihito Baba , Masahiro Wakao *, Hiroyuki Shinchi , Michael Chan , Tomoko
2
2
2
2
1,3
Hayashi , Shiyin Yao , Howard B. Cottam , Dennis A. Carson , Yasuo Suda *
1Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate
School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto,
Kagoshima 890-0065, Japan
2Moores Cancer Center, University of California, San Diego, 9500 Gilman Drive,
La Jolla, CA 92093-0695, USA
3SUDx-Biotec Corporation, 1-41-1 Shiroyama, Kagoshima 890-0013, Japan
AUTHOR EMAIL ADDRESS
wakao@eng.kagoshima-u.ac.jp, ysuda@eng.kagoshima-u.ac.jp
AUTHOR PHONE&FAX
TEL: (+81)99-285-7843, FAX: (+81)99-285-7856 for M. Wakao
TEL & FAX: (+81)99-285-8369 for Y. Suda
1

Abstract
Toll-like receptors (TLRs) are a type of pattern recognition receptors (PRRs),
which are activated by recognizing pathogen-associated molecular patterns
(PAMPs). The activation of TLRs initiates innate immune responses and
subsequently leads to adaptive immune responses. TLR agonists are effective
immuomodulators in vaccine adjuvants for infectious diseases and cancer
immunotherapy. In exploring hydrophilic small molecules of TLR7 ligands using
the cell-targeted property of a vaccine adjuvant, we conjugated 1V209, a small
TLR7 ligand molecule, with various low or middle molecular weight sugar
molecules that work as carriers. The sugar-conjugated 1V209 derivatives showed
increased water solubility and higher immunostimulatory activity in both mouse
and human cells compared to unmodified 1V209. The improved
immunostimulatory potency of sugar-conjugates was attenuated by an inhibitor of
endocytic process, cytochalasin D, suggesting that conjugation of sugar moieties
may enhance the uptake of TLR7 ligand into the endosomal compartment.
Collectively our results support that sugar-conjugated TLR7 ligands are applicable
to novel drugs for cancer and vaccine therapy.
2

KEYWORDS:
TLR7; ligand; agonist; sugar conjugate
3

Introduction
Toll-like receptors (TLRs), which are membrane type 1 proteins, are classified as
pattern recognition receptors (PRRs), and play significant roles in the innate and
1
-4
adaptive immune responses. In mammals, 13 kinds of TLRs (TLR1 to TLR13)
have been found to date. Each TLR recognizes specifically microbial components
called pathogen-associated molecular patterns (PAMPs); for example, lipoproteins
5
and lipopeptides interact with TLR1, TLR2, and TLR6, lipopolysaccharide with
6
7
TLR4, or nucleic acids with TLR3, TLR7, TLR8, and TLR9 . Although most
PAMPs are macromolecules, small molecule TLR ligands have been identified
among natural or synthetic compounds.8-11 These compounds have attracted much
attention as new candidates for cancer immunotherapy and as vaccine adjuvants
because they modulate host immune responses.
Several TLR7 ligands based on scaffolds of imidazoquinoline,12,13 purine,14-19 or
2
0
guanine analogs have been studied intensively, and some of them have already
been used in clinical treatment. Imiquimod (R837) is approved by the FDA for
treatment of external genital warts, superficial basal cell carcinoma, and actinic
keratosis.21,22 However, its clinical use is limited to only topical administration due
to undesired side effects such as cytokine release syndrome following systemic
administration.23-25 To improve bioavailability and pharmacokinetic properties of
4

small TLR7 ligand molecules, conjugates to a variety of accessory molecules such
as proteins, lipids, PEG, or polysaccharides have been performed and showed
higher immunostimulatory activity in vitro.18,19,26,27 In vivo analysis of these
conjugates in mice indicated that immune response varied depending on accessory
molecules. For example, the lipid-PEG conjugate induced Th-2 immune responses
1
9
in mice, and the dextran conjugate induced a strong Th-1 biased immune
2
7
response. It was suggested that these differences were derived from the uptake
mechanism and the location of TLR7 conjugates, and different pathway for the
activation of TLR7 signals. Thus, the accessory molecules may contribute to
potency and characteristics of immune response in vivo. In this study, we focused
on sugar molecules, which are involved in cell-selective transport in living
2
8
system, and prepared several sugar-conjugated TLR7 ligands and evaluated their
immunostimulatory activities.
Result and discussion
Synthesis of sugar-conjugated TLR7 ligands
We prepared 13 types of sugar molecules containing amino groups, which are
commercially available or readily synthesized by amination from commercially
available sugar molecules (Figure 1). Monosaccharide (glucosamine,
5

galactosamine, glucose, galactose, and mannose) and disaccharide (maltose and
lactose) components were chosen since they are widely distributed as biomolecules.
Cyclodextrines (CDs) were chosen since their derivatives are often utilized as
additives for food and drug.
NH2
NH2
N
N
N
N
N
N
OH
Sugar amines
OH
O
N
O
O
N
O
OH
R
O
O
1
V209
Sugar Conjugates 1-13
Sugar amines
OH
HO OH
O
OH
O
HO
OH
HO
HO
OH
O
O
H
HO
HO
HO
OH
O
OH
OH
H
HO
H
N
N
HO
N
HO
NH
HO
NH
OH
1
2
3
4
5
OH
O
H
N
HO
HO
OH
OH
O
OH
O
OH
O
H
HO
O
O
OH
N
O
OH
HO
OH
HO
HO
O
O
O
O
6
OH
OH
NH
OH
n
n
HO
HO
OH
O
OH
H
N
O
8 (n=5), 9 (n=6), 10 (n=7)
11 (n=5), 12 (n=6), 13 (n=7)
O
HO
HO
HO
7
Figure 1. Sugar-conjugated TLR7 ligands.
The
carboxylic
acid
functionalized
TLR7
ligand,
1V209
(2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine), is easily coupled to the
amino containing molecules with the coupling reagent, HATU
6

(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxide
hexafluorophosphate). Coupling of commercially available hexosamines
(glucosamine: GlcN, galactosamine: GalN) and 3A-aminocyclodextrins
(
3A-NH --CD, 3A-NH --CD, and 3A-NH --CD) with 1V209 afforded
2
2
2
sugar-conjugates 1, 2, 11, 12, and 13, respectively. The additional sugar amine
derivatives were prepared according to two routes (Figure 2, Route A or B) and
then conjugated to 1V209. In Route A, sugars were treated with 7 M methanolic
ammonia in autoclave reactor according to the method reported by Zhang et al.29
with modifications, and the resulting sugar amines were reacted with 1V209 to
produce conjugates. In Route B, the transformation via azide groups was applied;30
sugar molecules were peracetylated, brominated at the one position using HBr in
AcOH, and azidated using NaN . Reduction of the azide group, subsequent
3
coupling with 1V209 using HATU, and then the successive deacetylation afforded
the sugar conjugates. The conjugates 3, 5, and 6 were synthesized by Route A,
3
1
whereas 4 and 7 were synthesized by Route B to avoid Amadori byproducts and
other side products produced in Route A. The monosaccharide and disaccharide
conjugates were used without isolation of anomers for the immunomodulating
experiments. On the other hand, cyclodextrin conjugates were prepared via
3
2
6
A-azido-cyclodextrins using a method previously reported (Route C), and were
7

purified by reversed phase (ODS) column chromatography with MeOH-H O
2
eluents. As expected, the water solubility of the obtained sugar conjugates was
higher than that of 1V209 (see supporting information, Table 1S).
NH2
Route A
N
N
a
b
OH
HO
O
OH
H N
2
O
OH
N
O
N
O
H
N
O
OH
O
NH2
Route B
N
N
N
c-e
f, b, g
NH2
OH
HO
O
OH
N3
O
OAc
O
N
O
H
N
O
OH
Route C
HO
O
N
N
OH
O
OH
O
N
O
N
O
H
OH
O
OH
h, i, f, b
N
OH
O
O
O
OH
OH
n
O
OH
OH
O
O
OH
OH
n
Figure 2. Synthesis of glycosyl amine (Route A or B) and 6A-amino-CD (Route
C) derivatives. a) 7 M NH in MeOH, 60 ˚C, b) HATU in DMF, c) Ac O, Pyr., d)
3
2
HBr in AcOH, e) NaN in DMF, 60 ˚C, f) H , Pd/C in MeOH, g) K CO in MeOH,
3
2
2
3
h) TsCl, Pyr. in DMF, i) NaN in DMF.
3
Immunostimulatory activity of sugar-conjugated TLR7 ligands
8

Immunostimulatory activity of the sugar-conjugated TLR7 ligands was evaluated
in two types of murine cells: macrophage cell line (RAW264.7) and primary bone
marrow-derived dendritic cells (mBMDC). The levels of tumor necrosis factor-
(TNF-) and interleukin-6 (IL-6) from those cells into the culture supernatants
were evaluated by ELISA. The results are shown in Figure 3 (A, B, C, D, E, and F)
and summary of half maximal effective concentrations (EC ) and the
5
0
concentration of maximum induction (E ) is shown in Table 1. Sugar-conjugated
max
TLR7 ligands showed higher or equal cytokine release potency compared to
unconjugated TLR7 ligand, 1V209. The potency of IL-6 production in mBMDC
with sugar conjugates varied with size and structure of sugar moiety, and tended to
be higher as the size of sugar molecule increased. Cyclodextrin conjugates linked
at 3A position showed 10-fold higher potency than 1V209 in mBMDC.
The conjugates showing favorable potencies in murine cells (sugar-conjugates 1, 6,
and 11) were also tested using human peripheral blood mononuclear cells (PBMC)
to assess whether the conjugates with different sugar size have activity in human
cells. It was found that the sugar-conjugates showed higher potencies compared to
1
V209 (conjugates 1 and 6: 2-fold, conjugate 11: 3-fold, Figure 3G).
Immune cells contain receptors or transporters that can facilitate uptake of sugar
molecules. On antigen presenting cells (APC), various types of lectins are
9

expressed as scavenger receptors such as C-type lectins.33,34 Thus, we hypothesized
that enhancement of the immunostimulatory potencies of sugar-conjugated TLR7
ligands may be attributed to receptor-mediated cellular uptake facilitated by the
receptors and/or transporters on the cell surface. To test this hypothesis, we treated
RAW264.7 cells with the sugar-conjugated TLR7 ligands (3, 6, and 11) in the
presence or absence of cytochalasin D, an inhibitor of actin polymerization
preventing the endocytic process (Table 2, Figure 3H, I). In the presence of
cytochalasin D, the sugar conjugates showed decreased potencies. Especially, the
potency of the conjugate 11 with cyclodextrin was significantly decreased by
cytochalasin D. On the other hand, the unconjugated TLR7 ligand, 1V209, were
not inhibited by cytochalasin D, suggesting that 1V209 was incorporated by
passive diffusion. These results indicate that enhanced immunostimulatory
potencies of sugar conjugates are due to enhanced endocytic uptake via sugar
transporter and/or lectins on the cell surface.
10

Figure 3. In vitro cytokine induction by sugar-conjugated TLR7 ligands in murine
4
and human cells. (A−D) RAW264.7 cells (1 × 10 cells/well) were plated and
incubated with serially diluted sugar-conjugated TLR7 ligands (1−13) or
unconjugated TLR7 ligand (1V209) for 18 h. The levels of TNF- were measured
5
by ELISA. (E, F) The compounds were also tested in mBMDC (1 × 10 cell/well).
IL-6 was measured by ELISA. The potencies of representative compounds (1, 3, 6,
8
, and 11) are presented. Vehicle (0.25% DMSO)-stimulation was not observed,
5
respectively. (G) Human PBMC were plated at 2 × 10 cells/well and incubated
with 10 μM glycan-conjugated TLR7 ligands (1, 6, and 11) or 1V209 for 18 h.
Control cells were treated with 0.25% DMSO. (H, I) RAW264.7 cells (1.5 × 104
11

cell/well) were plated and pre-incubated with or without 1 μM of cytochalasin D,
and incubated with serially diluted sugar-conjugated TLR7 ligands (3, 6, and 11)
or unconjugated TLR7 ligand (1V209) for 6 h. TNF-α and IL-6 released in the
culture supernatants were determined by ELISA. All data shown are means ± SD
of triplicate and are representative of three independent experiments. **A value of
p < 0.01 by one-way ANOVA with Dunnett's multiple comparisons test (G).
Table 1. Immunostimulatory activity of sugar conjugates.
TNF-a
EC50
IL-6b
Compound
Emax
(%)d
100
103
83
EC50
(nM)c
434
Emax
(%)d
100
139
139
(
nM)c
252
234
153
1
V209
1
2
239
210
12

3
4
5
6
7
8
9
230
316
252
136
80
87
198
258
303
99
118
118
116
159
142
127
127
130
104
113
109
115
121
157
177
126
135
97
110
71
128
287
132
57
153
108
27
1
0
1
2
3
1
1
1
96
40
106
113
40
107
56
a
b
c
d
RAW264.7 cells were used for production of TNF-.
mBMDC cells were used for production of IL-6.
EC was calculated using Prism software.
5
0
Emax values normalized to 1V209.
Table 2. Immunostimulatory activity of sugar conjugates in the presence or
absence of cytochalasin D.
13

a
b
TNF- (EC /M)
5
0
Compound cytochalasin
D (-)
cytochalasin
D (+)
1
V209
2.15
0.490
0.277
0.186
2.11
3
6
0.909
0.585
1
1
1.06
a
RAW264.7 cells were used for production of TNF-.
EC was calculated using Prism software.
b
5
0
To evaluate the binding interaction of sugar conjugates to TLR7 ligand,
computational modeling was further performed (Figure 4). Modeling of the
3
5
monosaccharide conjugate 3 with monkey TLR7 (PDB: 5GMH ) suggested that
monosaccharide moiety of conjugate 3 is positioned outside of TLR7 dimer and no
interaction between the sugar moiety and TLR7 dimer was observed. The
disaccharide of 6 is also found to be positioned outside of the 1V209-TLR dimer
complex (see supporting information, Figure 1S). The results suggest that sugar
moiety is not responsible for dimerization of TLR7, and sugar moieties are
important for water solubility, cellular uptake, and trafficking of ligands. Although
14

the detailed mechanism is still unclear, further analysis will clarify how sugar
structures enhance the agonistic activity.
Figure 4. Computational docking studies of 1V209 and conjugate 3 to TLR7
dimer. Molecular docking of 1V209 (purple) and conjugate 3 (green) in the
resiquimod (R848) binding pocket of monkey TLR7 dimer complex (PDB:
5
GMH). The van der Waals surface area of binding pocket is shown in gray.
Overlay of binding geometry between 1V209 (purple) and conjugate 3 (green) is
shown in the magnified view.
Conclusion
15

We synthesized various sugar-conjugated TLR7 ligands and evaluated their
immune stimulatory potency in mice and human cells. The conjugation of a TLR7
ligand with the amino group in sugar molecules was accomplished by simple
amide condensation reaction using HATU. Synthesized sugar conjugates were
water soluble and showed higher immunostimulatory potency compared to the
unconjugated 1V209. Among them, the cyclodextrin conjugate 11 showed 10-fold
higher potency in mBMDC and 3-fold higher activity in hPBMC relative to 1V209.
We demonstrated that the higher potencies of sugar conjugates are due to
incorporation into cells via the endocytic process. Molecular modeling of TLR7
with monosaccharide conjugates suggests that the sugar moiety is not involved in
the interaction of TLR7 dimerization. Although the detailed analysis of the sugar
structures relative to cellular trafficking property is needed, this study may lead to
novel applications in cancer immunotherapy or infectious diseases aimed at
selective activation of immune cells using sugar properties.
Acknowledgements
The present studies were financially supported in part by grants from Strategic
Young Researcher Overseas Visits Program for Accelerating Brain Circulation
(G2504) from Japan Society for the Promotion of Science (YS), the Project of the
16

NARO Bio-oriented Technology Research Advancement Institution （ R&D
matching funds on the field for Knowledge Integration and innovation) (YS),
Takahashi Industrial and Economic Research Fundation (MW), and Sankei
Science Scholarship Foundation (MW).
References
1
2
3
4
5
6
. Akira, S.; Takeda, K. Toll-like receptor signaling. Nat. Rev. Immunol. 2004, 4,
99−511.
4
. Pandey, S.; Agrawal, D. K. Immunobiology of Toll-like receptors: Emerging
trend. Immunol. Cell Biol. 2006, 84, 333–3.
. Akira, S.; Uematsu, S.; Takeuchi, O. Pathogen Recognition and Innate
Immunity. Cell 2006, 124, 783–801.
. Kawai, T.; Akira, S. The role of pattern-recognition receptors in innate
immunity: update on Toll-like receptors. Nat. Immunol. 2010, 11, 373–384.
. Oliveira-Nascimento, L.; Massari, P.; Wetzler, L. M. The role of TLR2
ininfection and immunity. Front Immunol. 2012, 3, 79.
. Miller, S. I.; Ernst, R. K.; Bader, M. W. LPS, TLR4 and infectious disease
diversity. Nat. Rev. Microbiol. 2005, 3, 36–46.
17

7
8
9
. Kawai, T.; Akira, S. Antiviral signaling through pattern recognition receptors. J.
Biochem. 2007, 141, 137–145.
. Kaczanowska, S.; Joseph, A. M.; Davila, E. TLR agonists: our best frenemy in
cancer immunotherapy. J. Leukocyte Biol. 2013, 93, 847–863.
. Mancini, R. J.; Stutts, L.; Ryu, K. A.; Tom, J. K.; Esser-Kahn, A. P. Directing
the immune system with chemical compounds. ACS Chem. Biol. 2014, 9,
1
075–1085.
1
1
1
0.Dowling, J. K.; Mansell, A. Toll-like receptors: the swiss army knife of
immunity and vaccine development. Clin. Transl. Immunol. 2016, 5, e85.
1.Temizoz, B.; Kuroda, E.; Ishii, K. J. Vaccine adjuvants as potential cancer
immunotherapeutics. Int. Immunol. 2016, 28, 329–338.
2.Hemmi, H.; Kaisho, T.; Takeuchi, O.; Sato, S.; Sanjo, H.; Hoshino, K.;
Horiuchi, T.; Tomizawa, H.; Takeda, K.; Akira, S. Small anti-viral compounds
activate immune cells via the TLR7 MyD88–dependent signaling pathway. Nat.
Immunol. 2002, 3, 196–200.
1
3.Diebold, S. S.; Kaisho, T.; Hemmi, H.; Akira, S.; Reis e Sousa, C. Innate
antiviral responses by means of TLR7-mediated recognition of single-stranded
RNA. Science 2004, 303, 1529–1531.
18

1
1
1
1
4.Kurimoto, A.; Ogino, T.; Ichii, S.; Isobe, Y.; Tobe, M.; Ogita, H.; Takaku, H.;
Sajiki, H.; Hirota, K.; Kawakami, H. Synthesis and evaluation of 2-substituted
8
-hydroxyadenines as potent interferon inducers with improved oral
bioavailabilities. Bioorg. Med. Chem. 2004, 12, 1091–1099.
5.Jin, G.; Wu, C. C. N.; Tawatao, R. I.; Chan, M.; Carson, D. A.; Cottam, H. B.
Synthesis and immunostimulatory activity of 8-substituted amino
9
-benzyladenines as potent Toll-like receptor 7 agonists. Bioorg. Med. Chem.
Lett. 2006, 16, 4559–4563.
6.Kurimoto, A.; Hashimoto, K.; Nakamura, T.; Norimura, K.; Ogita, H.; Takaku,
H.; Bonnert, R.; McInally, T.; Wada, H.; Isobe, Y. Synthesis and Biological
Evaluation of 8-Oxoadenine Derivatives as Toll-like Receptor 7 Agonists
Introducing the Antedrug Concept. J. Med. Chem. 2010, 53, 2964–2972.
7.Biffen, M.; Matsui, H.; Edwards, S.; Leishman, A. J.; Eiho, K.; Holness, E.;
Satterthwaite, G.; Doyle, I.; Wada, H.; Fraser, N. J.; Hawkins, S. L.; Aoki, M.;
Tomizawa, H.; Benjamin, A. D.; Takaku, H.; McInally, T.; Murray, C. M.
Biological characterization of a novel class of toll-like receptor 7 agonists
designed to have reduced systemic activity. Brit. J. Pharmacol. 2012, 166,
5
73–586.
19

1
1
8.Wu, C. C.; Hayashi, T.; Takabayashi, K.; Sabet, M.; Smee, D. F.; Guiney, D.
D.; Cottam, H. B.; Carson, D. A. Immunotherapeutic activity of a conjugate of
a Toll-like receptor 7 ligand. Proc. Natl. Acad. Sci. USA 2007, 104, 3990–3995.
9.Chan, M.; Hayashi, T.; Kuy, C. S.; Gray, C. S.; Wu, C. C.; Corr, M.; Wrasidlo,
W.; Cottam, H. B.; Carson, D. A. Synthesis and immunological
characterization of Toll-like receptor 7 agonistic conjugates. Bioconjugate
Chem. 2009, 20, 1194–1200.
2
2
2
0.Lee, J.; Chuang, T.-H.; Redecke, V.; She, L.; Pitha, P. M.; Carson, D. A.; Raz,
E.; Cottam, H. B. Molecular basis for the immunostimulatory activity of
guanine nucleoside analogs: activation of Toll-like receptor 7. Proc. Natl. Acad.
Sci. USA 2003, 100, 6646–6651.
1.Vacchelli, E.; Galluzzi, L.; Eggermont, A.; Fridman, W. H.; Galon, J.;
Sautes-Fridman, C.; Tartour, E.; Zitvogel, L.; Kroemer, G. Trial watch:
FDA-approved Toll-like receptor agonists for cancer therapy.
Oncoimmunology 2012, 1, 894–907.
2.Dockrell, D. H.; and Kinghorn, G. R. Imiquimod and resiquimod as novel
immunomodulators. J. Antimicrob. Chemother. 2001, 48, 751–755.
20

2
2
3.Savage, P.; Horton, V.; Moore, J.; Owens, M.; Witt, P.; Gore, M. E. A phase I
clinical trial of imiquimod, an oral interferon inducer, administered daily. Brit.
J. Cancer 1996, 74, 1482–1486.
4.Goldstein, D.; Hertzog, P.; Tomkinson, E.; Couldwell, D.; McCarville, S.;
Parrish, S.; Cunningham, P.; Newell, M.; Owens, M.; Cooper, D. A.
Administration of imiquimod, an interferon inducer, in asymptomatic human
immunodeficiency virus-infected persons to determine safety and biologic
response modification. J. Infect. Dis. 1998, 178, 858–861.
2
5.Gunzer, M.; Riemann, H.; Basoglu, Y.; Hillmer, A.; Weishaupt, C.; Balkow, S.;
Benninghoff, B.; Ernst, B.; Steinert, M.; Scholzen, T.; Sunderkötter C.; Grabbe.
S. Systemic administration of a TLR7 ligand leads to transient immune
incompetence due to peripheral-blood leukocyte depletion. Blood 2005, 106,
2
424–2432.
2
6.Chan, M.; Hayashi, T.; Mathewson, R. D.; Yao, S.; Gray, C.; Tawatao, R. I.;
Kalenian, K.; Zhang, Y.; Hayashi, Y.; Lao, F. S.; Cottam, H. B.; Carson, D. A.
Synthesis and characterization of PEGylated toll like receptor 7 ligands.
Bioconjugate Chem. 2011, 22, 445–454.
2
7.Shinchi, H.; Crain, B.; Yao, S.; Chan, M.; Zhang, S. S.; Ahmadiiveli, A.; Suda,
Y.; Hayashi, T.; Cottam, H. B.; Carson, D. A. Enhancement of the
21

Immunostimulatory Activity of a TLR7 Ligand by Conjugation to
Polysaccharides. Bioconjugate Chem. 2015, 26, 1713−1723.
2
2
3
8.Monsigny, M.; Roche, A.-C.; Midoux, P.; Mayer, R. Glycoconjugates as
carriers for specific delivery of therapeutic drugs and genes. Adv. Drug Deliv.
Rev. 1994, 14, 1–24.
9.Shen, C.; Zhao, Q.; Zheng, H.; Zhang, P. Synthesis of novel Schiff bases from
the reaction of 3-O-methyl-4,6-O-benzylidene-β-D-glucopyranosylamine with
substituted aldehydes. J. Chem. Res. 2009, 317–318.
0.Pető, C.; Batta, G.; Györgydeák, Z.; Sztaricskai, F. Synthesis of the anomers of
hepta-O-acetylcellobiosyl, -lactosyl, -maltosyl, and -melibiosyl Azide. Liebigs
Ann. Chem. 1991, 505–507.
3
3
1.Isbell, H. S.; Frush, H. L. Mutarotation, hydrolysis, and rearrangement
reactions of glycosylamines. J. Org. Chem. 1958, 23, 1309–1319.
2.Tang, W.; Ng, S. Facile synthesis of mono-6-amino-6-deoxy-α-, β-,
γ-cyclodextrin hydrochlorides for molecular recognition, chiral separation and
drug delivery. Nat. Protoc. 2008, 3, 691–697.
3
3.Osorio, F.; Reis e Sousa, C. Myeloid C-type lectin receptors in pathogen
recognition and host defense. Immunity 2011, 34, 651–664.
22

3
3
4.Yan, H.; Kamiya, T.; Suabjakyong, P.; Tsuji, N. M. Targeting C-type lectin
receptors for cancer immunity. Front. Immunol. 2015, 6, 408.
5.Zhang, Z.; Ohto, U.; Shibata, T.; Krayukhina, E.; Taoka, M.; Yamauchi, Y.;
Tanji, H.; Isobe, T.; Uchiyama, S.; Miyake, K.; Shimizu, T. Structural analysis
reveals that Toll-like receptor 7 is a dual receptor for guanosine and
single-stranded RNA. Immunity 2016, 45, 737–748.
Declaration of interests
☒
The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in
this paper.
☐
The authors declare the following financial interests/personal relationships
which may be considered as potential competing interests:
23

24

25

Graphical Abstract
NH2
OH
O
OH
O
N
N
OH
HO
OH
N
O
O
O
N
O
NH
OH
n
O
TLR7 ligand moiety
Sugar conjugates with TLR7 ligand
26