Multivalent, Stabilized Mannose-6-Phosphates for the Targeted Delivery of Toll-Like Receptor Ligands and Peptide Antigens

Article abstract of DOI:10.1002/cbic.202000538

Mannose-6-phosphate (M6P) is recognized by the mannose-6-phosphate receptor and plays an important role in the transport of cargo to the endosomes, making it an attractive tool to improve endosomal trafficking of vaccines. We describe herein the assembly of peptide antigen conjugates carrying clusters of mannose-6-C-phosphonates (M6Po). The M6Po's are stable M6P mimics that are resistant to cleavage of the phosphate group by endogenous phosphatases. Two different strategies for the incorporation of the M6Po clusters in the conjugate have been developed: the first relies on a “post-assembly” click approach employing an M6Po bearing an alkyne functionality; the second hinges on an M6Po C-glycoside amino acid building block that can be used in solid-phase peptide synthesis. The generated conjugates were further equipped with a TLR7 ligand to stimulate dendritic cell (DC) maturation. While antigen presentation is hindered by the presence of the M6Po clusters, the incorporation of the M6Po clusters leads to increased activation of DCs, thus demonstrating their potential in improving vaccine adjuvanticity by intraendosomally active TLR ligands.

Full text of DOI:10.1002/cbic.202000538

Combining Chemistry and Biology
Accepted Article
Title: Multivalent, Stabilized Mannose-6-Phosphates for the Targeted
Delivery of Toll-Like Receptor Ligands and Peptide Antigens
Authors: Niels R. M. Reintjens, Elena Tondini, Christopher Vis, Toroa
McGlinn, Nico J. Meeuwenoord, Tim P. Hogervorst, Herman
S. Overkleeft, Dmitri V. Filippov, Gijsbert A. van der Marel,
Ferry Ossendorp, and Jeroen D. C. Codée
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.202000538
Link to VoR: https://doi.org/10.1002/cbic.202000538
01/2020

10.1002/cbic.202000538
ChemBioChem
FULL PAPER
Multivalent, Stabilized Mannose-6-Phosphates for the Targeted
Delivery of Toll-Like Receptor Ligands and Peptide Antigens
Niels R. M. Reintjens,[a] Elena Tondini,[b] Christopher Vis,[a] Toroa McGlinn,[a] Nico J. Meeuwenoord,[a]
Tim P. Hogervorst,[a] Herman S. Overkleeft,[a] Dmitri V. Filippov,[a] Gijsbert A. van der Marel,[a] Ferry
Ossendorp,[b] and Jeroen D. C. Codée*[a]
[a]
[b]
Dr. N. R. M. Reintjens, C. Vis, T. McGlinn, N. J. Meeuwenoord, T. P. Hogervorst, Prof. Dr. H. S. Overkleeft, Dr. D. V. Filippov, Prof. Dr. G. A. van der Marel,
Dr. J. D. C. Codée
Leiden Institute of Chemistry, Leiden University
Einsteinweg 55, 2333 CC Leiden (The Netherlands)
E-mail: jcodee@chem.leidenuniv.nl
E. Tondini, Prof. Dr. F. Ossendorp
Dept. of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden University (The Netherlands)
Albinusdreef 2, 2333 ZA Leiden
Supporting information for this article is given via a link at the end of the document.
Abstract: Mannose-6-phosphate (M6P) is recognized by the
mannose-6-phosphate receptor and plays an important role in the
transport of cargo to the endosomes, making it an attractive tool to
improve endosomal trafficking of vaccines. We here describe the
assembly of peptide antigen conjugates carrying clusters of mannose-
6-C-phosphonates (M6Po). The M6Po’s represent stable M6P mimics
that are resistant to cleavage of the phosphate group by endogenous
phosphatases. Two different strategies for the incorporation of the
M6Po clusters in the conjugate have been developed: the first relying
on a “post-assembly” click approach employing an M6Po bearing an
alkyne functionality; the second hinges on an M6Po C-glycoside
amino acid building block that can be used in solid-phase peptide
synthesis. The generated conjugates were further equipped with a
TLR7-ligand to stimulate dendritic cell (DC) maturation. While antigen
presentation is hindered by the presence of the M6Po clusters, the
incorporation of the M6Po clusters leads to increased activation of
DCs, demonstrating their potential in improving vaccine adjuvanticity
by intraendosomally active TLR-ligands.
the endosomes, as was shown by the conjugation of M6P
analogues to acid α-glucosidase leading to improved delivery of
this enzyme in the treatment of the lysosomal myopathy Pompe
disease.[7] The MPR has also been exploited as a drug delivery
system for cancer therapy,[8,9] for example, doxorubicin was
delivered via mannose-6-phosphate-modified human serum
albumin as carrier and N-hexanoyl-D-erythro-sphingosine with
M6P-functionalized liposomes.[10,11] Our group has previously
reported that a cathepsin inhibitor that is covalently attached to an
M6P cluster could effectively be delivered into the endolysosomal
pathway.[12]
The activity of peptide-based anti-cancer vaccines may be
enhanced by the targeted delivery of these antigens to antigen
presenting cells. In this context, mannosylated antigens, destined
for the mannose receptor or DC-SIGN present on dendritic cells
(DCs), have been widely explored.[13–16] We, therefore, reasoned
that conjugate vaccines in which an M6P moiety is covalently
bound to an antigenic peptide might be targeted effectively to
immune cells expressing the MPR, leading to improved uptake
and more efficient delivery to the lysosomes where the cargo is
processed for MHC loading, ultimately resulting in enhanced
antigen presentation. Since dephosphorylation by endogenous
phosphatases is one of the potential drawbacks of the use of M6P,
several stable M6P analogues have previously been evaluated,
such as a malonyl ether, a malonate or an isosteric C-
phosphonate ester.[17,18] The C-phosphonate proved to be a
stable and effective replacement for the phosphate monoester.[19]
We here describe the incorporation of a cluster of mannose-6-
phosphonates (M6Po) in two types of peptide antigen-conjugates
(1-8, Figure 1), wherein two different M6Po building blocks, 9 and
10, respectively, are used to construct M6Po-clusters and
conjugate them to either the N- or the C-terminus of a synthetic
long peptide (SLP). In this study, two different ovalbumin derived
SLPs are used: DEVA5K (DEVSGLEQLESIINFEKLAAAAAK),
which harbours the MHC-I presented epitope SIINFEKL, and
HAAHA (ISQAVHAAHAEINEAGRK), which contains an MHC-II
epitope. Our goal is to enhance the immune response by
increasing endosomal trafficking of the peptide vaccine via the
MPR which led to the design of bis-conjugates (2, 4, 6, and 8) in
which the Toll-like receptor 7 ligand (TLR7L) 2-alkoxy-8-oxo-
Introduction
Carbohydrates play an important role in many biological
processes, such as cell-cell communication, pathogen recognition
and protein folding. Mannose-6-phosphate (M6P),
a
D-
mannopyranose bearing a phosphate group at the C-6 position,
serves as a signaling moiety on the termini of glycan branches
mounted on newly synthesized proteins in the trans-Golgi network
and is essential for the transportation of these proteins to the late
endosomes and lysosomes. The mannose-6-phosphate receptor
(MPR),
a P-type lectin, plays an important role in this
transportation through binding to M6P.[1,2] There are two members
of this lectin family: the cation-dependent mannose-6-phosphate
receptor (CD-MPR) and the cation-independent mannose-6-
phosphate receptor (CI-MPR). The latter has a high affinity for
ligands containing multiple M6Ps due to the presence of two M6P
binding domains, which can simultaneously bind two M6Ps.[3–5]
A
small fraction of MPRs can be found on the cell surface of which
only CI-MPR binds and internalizes M6P-bound substrates.[6]
Therefore, the CI-MPR is an efficient tool for targeted delivery to
1
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10.1002/cbic.202000538
ChemBioChem
FULL PAPER
M6Po
M6Po
H
N
SLP
NH2
R
SLP
Ac
N
N
R
H
H
O
O
6
6
DEVA5K
DEVA5K
1
2
5
6
O-M6P SLP = DEVA5K R = H
O-M6P SLP = DEVA5K R = TLR7L O-M6Po
C-M6P SLP = HAAHA R = H
3 O-M6P SLP = DEVA5K R = Ac
O-M6Po
O-M6Po
O-M6Po
C-M6Po
C-M6Po
DEVA5K
HAAHA
TLR7L
TLR7L
4
DEVA5K
HAAHA
HAAHA
O-M6P SLP = DEVA5K R = TLR7L
TLR7L
TLR7L
7 C-M6P SLP = HAAHA R = Ac
C-M6Po
HAAHA
O
8 C-M6P SLP = HAAHA R = TLR7L
C-M6P SLP = HAAHA R = TLR7L C-M6Po
O
O
HO
HO
HO
HO
P
P
N
HN
OH
OH
O
O
HO
HO
HO
HO
TLR7L =
N
H2N
O
O-M6Po =
C-M6Po =
O
N
O
HN
O
O
O
O
N
N
N
O
tBuO
tBuO
P
O
O
HO
HO
O
PMBO
P
O
OH
O
DEVA5K = DEVSGLEQLESIINFEKLAAAAAK
HAAHA = ISQAVHAAHAEINEAGRK
HO
HO
O
O
O
HN
OH
NHFmoc
9
10
Figure 1. Structures of the O-M6Po conjugates 1-4, C-M6Po conjugates 5-8, and building blocks 9 and 10.
adenine is added at either the N- or the C-terminus of the M6Po-
SLP.[20,21] We coupled an α-configured spacer,[12,18,22] carrying an
alkyne function to the O-M6Po building block 9 to allow for the
copper mediated 1,3-dipolar cycloaddition to azide-functions
incorporated in the SLP, while C-M6Po building block 10 was
used to generate the HAAHA-conjugates via solid-phase peptide
synthesis (SPPS). This building block is a C-analogue[23] of O-
M6Po, in which the anomeric oxygen is replaced with a CH2,
preventing hydrolysis under the acidic conditions used in SPPS.
An additional advantage of this SPPS-compatible building block
is the possibility to prepare conjugates of peptides that are not
suitable for copper mediated 1,3-dipolar cycloaddition. It also
allows one to incorporate azide or alkyne click handles in
conjugate vaccine constructs that can be exploited for labelling or
visualization purposes.
triflated, to enable the subsequent substitution by a phosphonate
anion (See supporting information Scheme S1), we placed an
isopropylidene group over the 2,3-cis-diol system to prevent this
intramolecular side reaction.[24,25] Thus, tritylation of 11 and
subsequent installation of the isopropylidene gave 13, of which
the remaining alcohol was masked as a p-methoxybenzyl ether to
give the fully protected mannose 14. The alkyne in 14 was
protected with a TMS group using TMSCl and n-BuLi at -78°C.
Removal of the trityl in the thus obtained 15 with a catalytic
amount of p-toluenesulfonic acid in DCM/MeOH was
accompanied by partial removal of the isopropylidene ketal.
Reinstallation of the isopropylidene and subsequent deprotection
of the mixed ketal simultaneously formed on the primary alcohol
gave 16 in 98% over three steps. Alcohol 16 was treated with Tf2O
and pyridine at -40°C[26] and the obtained crude triflate was added
to a mixture of dimethyl methylphosphonate and n-BuLi in THF at
-70°C, yielding compound 17 in 72% over two steps. Removal of
the TMS protecting group gave 18, which was transformed into
key building block 9 by a two-step deprotection sequence. The
deprotection of the phosphonate using TMSBr was followed by
the removal of the p-methoxybenzyl and isopropylidene groups
by treatment with AcOH/H2O at 90°C to deliver key building block
We here report the synthesis and immunological evaluation of
these dual conjugated peptide vaccine constructs.
Results and Discussion
Synthesis of M6Po-conjugates
9 in 27% yield over 15 steps starting from D-mannose.
The first type of O-M6Po conjugates comprises the six-fold
addition of α-propargyl mannose-6-phosphonate (O-M6Po)
building block (9) to azide containing peptides. Synthesis of the
En route to mannose-6-phosphonate SPPS building block 10
(Scheme 1B), the trityl group of known compound 19[23] was
removed as described above to give alcohol 20 in 72% over three
steps. Conversion of 20 to the primary triflate, followed by
nucleophilic substitution with the anion of di-tert-butyl
methylphosphonate[27] gave phosphonate 21 in 72% over two
required building block 9 started from propargyl-D-mannose 11
(Scheme 1A). As we found that the use of para-methoxybenzyl
ethers for the protection of the secondary alcohols led to the
formation of a 3,6-ether bridge when the C-6-hydroxyl was
2
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10.1002/cbic.202000538
ChemBioChem
FULL PAPER
HO
HO
HO
HO
HO
HO
TrtO
R1O
R2O
TrtO
OH
O
OH
O
OR3
O
O
O
b
e
a
PMBO
O
OH
O
O
O
11
12 R1 = R2 = R3 = H
13 R1 = H, R2 = R3 = C(CH3)2
14 R1 = PMB, R2 = R3 = C(CH3)2
15
TMS
c
d
f
O
P
O
O
HO
HO
MeO
MeO
MeO
P
P
MeO
HO
OH
O
O
O
O
O
O
O
i
h
g
HO
HO
PMBO
PMBO
PMBO
O
O
O
O
O
O
O
A
9
18
17
16
TMS
TMS
O
O
P
tBuO
tBuO
tBuO
tBuO
P
TrtO
PMBO
HO
PMBO
O
O
O
O
O
O
O
O
j
k
l
PMBO
PMBO
O
O
O
O
O
19
20
21
OR
22
R = Me
m
23 R = H
O
O
tBuO
tBuO
tBuO
tBuO
P
P
n
O
O
O
O
PMBO
PMBO
O
O
o
O
O
O
O
HN
HN
OH
NHFmoc
OMe
NHFmoc
B
10
24
Scheme 1. Synthesis of alkyne building blocks 9 and 10. Reagents and conditions: a) i. Ac2O, pyridine; ii. propargyl alcohol, BF3ꞏOEt2, 50°C; iii. NaOMe, MeOH,
70% over three steps; b) TrtCl, Et3N, DMF, 60°C, 83%; c) p-toluenesulfonic acid, 2,2-dimethoxypropane, 87%; d) p-methoxybenzyl chloride, NaH, DMF, 95%; e)
TMSCl, n-BuLi, THF, -78°C, 97%; f) i. p-toluenesulfonic acid, DCM/MeOH; ii. p-toluenesulfonic acid, 2,2-dimethoxypropane; iii. 1 M HCl, EtOAc, 0°C, 98% over
three steps: g) i. Tf2O, pyridine, DCM, -40°C; ii. n-BuLi, dimethyl methylphosphonate, THF, -70°C to -50°C, 72% over two steps; h) TBAF, THF, quant.; i) i. TMSBr,
pyridine, MeCN; ii. AcOH/H2O, 90°C, 81% over two steps; j) i. p-toluenesulfonic acid, DCM/MeOH; ii. p-toluenesulfonic acid, 2,2-dimethoxypropane; iii. 1 M HCl,
EtOAc, 0°C, 75% over three steps; k) i. Tf2O, pyridine, DCM, -40°C; ii. n-BuLi, di-tert-butyl methylphosphonate, THF, -70°C to -50°C, 72% over two steps; l) i. methyl
acrylate, CuI, Grubbs 2nd gen. catalyst, DCE, 60°C; ii. NaBH4, RuCl3, MeOH, DCE, 45°C, 72% over two steps; m) LiOH, THF/H2O, quant; n) Fmoc-
L-Lys-OMe,
HCTU, DIPEA, DMF, 86%; o) LiOH, THF/H2O, 0°C, 80%.
steps on 3 mmol scale.[28] Cross metathesis with methyl acrylate,
followed by the reduction of the double bond with NaBH4 and
ruthenium trichloride then resulted in compound 22.[29,30]
Hydrolysis of the obtained methyl ester, was followed by
group at the C-terminal lysine of 25 was selectively deprotected
using a cocktail of TFA/TIS/DCM (2/2/96 v/v/v). The released
amine was subsequently coupled with the spacer 33 and the Boc-
protected TLR7-ligand building block 34.[21] After deprotection,
release from the resin and RP-HPLC purification peptides 27 and
30 were obtained in a 2% yield. Coupling of O-M6Po building
block 9 to peptides 26, 27, 29 and 30 was performed using a
condensation with Fmoc-L-Lys-OMe and subsequent treatment of
24 with LiOH at 0°C, which left the Fmoc group unaffected, then
delivered the key SPPS building block 10 in 80% yield.
Next, the assembly of the (O-M6Po)6-SIINFEKL conjugates was
undertaken. Immobilized peptides 25 and 28 were prepared
through standard SPPS HCTU/Fmoc chemistry using Tentagel S
Ram as solid support (Scheme 2A). The C-terminal lysine of 25
was protected with a MMT group since we aimed to make M6Po-
peptide conjugates as well as conjugates containing both an
M6Po-cluster and a TLR7-ligand. TFA/TIS/H2O (95/2.5/2.5 v/v/v)
treatment removed all protecting groups and cleaved the peptides
from the resin to give peptides 26 and 29 in 1% and 6% yield
respectively, after purification. Alternatively, the MMT protecting
cocktail
of
CuSO4,
sodium
ascorbate
and
tris(benzyltriazolylmethyl)amine in DMSO/H2O,[31] with the
addition of a 20 mM Tris/150 mM NaCl buffer. After RP-HPLC,
conjugates 1-4 were obtained in 5% (0.3 mg), 18% (0.9 mg), 18%
(1.0 mg) and 31% (3.3 mg) yield respectively.
The HAAHA peptide contains two histidines, which can
coordinate to copper and thereby inhibit the reduction of Cu(II) to
Cu(I).[32] Therefore, conjugates 5-8 were generated by an online
SPPS synthesis (Scheme 2B) using C-M6Po building block 10,
which is equipped with acid-labile protecting groups that can be
3
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10.1002/cbic.202000538
ChemBioChem
FULL PAPER
b,c
or
N3
K
N3
K
N3
K
R
K
R
K
R
K
e, f, b, c
a
H
H
DEVA5K
MMT
DEVA5K
Fmoc
N
K
K
K
N
K
R
K
R
K
R
N3
N3
N3
R'
Fmoc Tentagel S Ram
25
26 R = N3, R' = H
27 R = N3, R' = TLR7L
d, c
R = R1, R' = H
R = R1, R' = TLR7L
1
d, c
2
h, b,c
or
f, b, c
N3
K
N3
N3
K
g
R
K
R
K
R
K
H
H2N
DEVA5K
K
K
K
K
N
R'
DEVA5K
K
R
K
R
K
R
N3
N3
N3
28
29 R = N3, R' = H
R = N , R' = TLR7L
d, c
30
3
3
R = R1, R' = H
d, c
R = R1, R' = TLR7L
A
4
h, b,c
or
j, f, b, c
R2
R2
R2
R3
R3
K
R3
K
i
H
H
HAAHA
MMT
HAAHA
Fmoc
N
K
R2
K
K
R2
K
K
R2
K
N
K
R3
K
K
R3
K
R3
R'
5
6
R' = H
R' = TLR7L
Fmoc Tentagel S Ram
31
h, b,c
or
f, b, c
R2
R2
K
R2
R3
K
R3
R3
K
k
H
N
H2N
R'
HAAHA
HAAHA
K
K
K
K
K
K
K
K
K
R2
R2
R2
R3
R3
R3
32
7
8
R' = H
R' = TLR7L
B
NH2
O
P
O
P
O
P
NH2
N
HO
HO
tBuO
tBuO
HO
HO
Boc
N
H
N
N
N
O
O
OH
O
O
O
OH
O
N
N
O
N
HO
HO
PMBO
HO
HO
O
O
O
34
R1
R2
R3
O
O
HO
N
HN
O
O
O
N
N
O
O
O
O
NHFmoc
HO
O
DEVA5K = DEVSGLEQLESIINFEKLAAAAAK
HAAHA = ISQAVHAAHAEINEAGRK
33
TLR7L
Scheme 2. Synthesis of A) O-M6Po conjugates 1-4 and B) C-M6Po conjugates 5-8. Reagents and conditions: a) i. 20% piperidine, DMF; ii. Fmoc SPPS cycle for
K(N3)-K(N3)-K(N3)-K(N3)-K(N3)-K(N3)-DEVSGLEQLESIINFEKLAAAAAK; iii. 20% piperidine, DMF; iv. Ac2O, DIPEA, DMF; b) TFA/TIS/H2O (95/2.5/2.5 v/v/v), 3h; c)
RP-HPLC; d) 9, 20 mM Tris/150 mM NaCl buffer, CuSO4/NaAsc/TBTA, H2O/DMSO; e) TFA/TIS/DCM (2/2/96 v/v/v); f) i. {2-[2-(Fmoc-amino)ethoxy]ethoxy}acetic
acid, HCTU, DIPEA, DMF; ii. 20% piperidine, DMF; iii. 4-((2-butoxy-6-((tert-butoxycarbonyl)amino)-8-oxo-7,8-dihydro-9H-purin-9-yl)methyl)benzoid acid, HCTU,
DIPEA, DMF; g) i. 20% piperidine, DMF; ii. Fmoc SPPS cycle for DEVSGLEQLESIINFEKLAAAAAK-K(N3)-K(N3)-K(N3)-K(N3)-K(N3)-K(N3); iii. 20% piperidine, DMF;
h) Ac2O, DIPEA, DMF; i) i. 20% piperidine, DMF; ii. Fmoc SPPS cycle for ISQAVHAAHAEINEAGRK; iii. 20% piperidine, DMF; iv. 10, HCTU, DIPEA, DMF; v. 5x
repeat of iii. and iv.; vi. 20% piperidine, DMF; vii. Ac2O, DIPEA, DMF; j) AcOH/TFE/DCM (1/2/7 v/v/v); k) i. 20% piperidine, DMF; ii. 10, HCTU, DIPEA, DMF; iii. 5x
repeat of i. and ii.; iv. 20% piperidine, DMF v. Fmoc SPPS cycle for ISQAVHAAHAEINEAGRK; iii. 20% piperidine, DMF; Yield peptides and conjugates: 26) 4.6 mg,
1%; 27) 4.0 mg, 2%; 29) 17.4 mg, 6%; 30) 8.2 mg, 2%; 1) 0.3 mg, 5%; 2) 1.0 mg, 18%; 3) 0.9 mg, 18%; 4) 3.3 mg, 31%; 5) 13.3 mg, 10%; 6) 11.0 mg, 8%; 7) 3.1
mg, 2%; 8) 17.0 mg, 11%.
removed at the end of the SPPS concomitantly with all other acid-
labile peptide protecting groups and release of the peptide from
removal cycles with building block 10 gave immobilized peptide
31. Peptide 32, bearing the C-M6Po cluster at the C-terminal end,
was generated by assembling the hexa-C-M6Po peptide through
manual couplings of building block 10, followed by automated
SPPS to assemble the rest of the peptide. Immobilized and
protected peptides 31 and 32 were deprotected and
the resin. Tentagel
S
Ram resin was elongated with
ISQAVHAAHAEINEAGRK using automated SPPS, of which the
lysine(MMT) at the C-terminus will be used for elongation at a
later stage of the synthesis. Six consecutive coupling and Fmoc
4
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10.1002/cbic.202000538
ChemBioChem
FULL PAPER
simultaneously cleaved from the resin with the TFA/TIS cocktail Conclusion
to provide conjugates 5 (13.3 mg) and 7 (3.1 mg) after purification
by RP-HPLC in 10% and 8% yield respectively, demonstrating the
suitability of 10 for SPPS. To obtain conjugate 6, bearing the
TLR7-ligand, the MMT-group in 31 was selectively removed with
a cocktail of AcOH/TFE/DCM (1/2/7 v/v/v). The obtained free
amine was elongated with spacer 33 and Boc-protected TLR7-
ligand building block 34 to give conjugate 6 (11.0 mg) in 2% yield,
after removal of all the protecting groups, cleavage from the resin
and RP-HPLC purification. The same sequence of events was
applied to the N-terminal amine in immobilized peptide 32 to
afford conjugate 8 (17.0 mg) in 8% yield.
In conclusion, we have described the development of two
mannose-6-C-phosphonate (M6Po) building blocks which allow
for stabilized M6P-analogues to be incorporated in peptide
sequences to target the M6P receptor. To prevent
dephosphorylation by endogenous phosphatases, the O-
phosphate in the naturally occurring mannose-6-phosphate was
replaced by a C-phosphonate moiety. The first building block
carries an O-propargyl group at the anomeric center of the
mannose-6-C-phosphonate, which allows for the incorporation of
the M6Po in peptide sequences through an azide-alkyne click
reaction. The second building block, a C-mannoside, was
designed and synthesized for application in solid-phase peptide
synthesis. The acid-stable anomeric linkage and protecting
groups used enabled the streamlined in-line incorporation of the
building block during SPPS. With the building blocks, various
peptide conjugates were assembled, containing either an MHC-I
or an MHC-II epitope, an M6Po-cluster presenting six mannose-
6-phosphonates, and a TLR7-ligand. Although immunological
evaluation has shown that conjugation of the M6Po-clusters
inhibits antigen presentation, the ability of the TLR7-ligand
conjugates to induce DC maturation was significantly improved.
While the M6Po clusters effectively trafficked the conjugates to
the endosome, where the conjugates interacted with TLR7
receptor, the processing of the conjugates was impeded by the
M6Po-clusters. Future conjugates will be designed featuring
cleavable linkers that allow for the release of the clusters during
endolysosomal processing, effectively liberating the incorporated
peptide antigens for presentation.
Immunological evaluation
We assessed the capacity of the conjugates 1-8 to induce
maturation of dendritic cells (DCs) and stimulate antigen
presentation (Figure 2). In these assays reference compounds
35-40, lacking the mannose-6-phosphonate clusters, were used
as a control (See supporting information Scheme S2). The
activation of DCs can be measured by the detection of the
production of interleukin-12 (IL-12). First, the O-M6Po conjugates
1-4 were evaluated (Figure 2A). To this end, murine bone
marrow/derived DCs were stimulated for 24 hours with the
compounds and the amount of secreted IL-12 was measured in
the supernatant. As expected, conjugation with solely a cluster of
M6Po (as in conjugates 1 and 3) does not induce DC maturation.
In contrast, the TLR7-ligand SLP conjugates (36 and 37) induce
IL12 production due to stimulation of the TLR7 receptor.
Interestingly, the conjugates carrying the M6Po-cluster and the
TLR7-ligand (2 and 4) induced a stronger activation of the DCs
than their counterparts solely bearing a TLR7-ligand (i.e., SLP-
conjugates 36 and 37). The position of the M6Po-clusters and
TLR7 ligand in these conjugates did not seem to influence the
activity of the conjugates. Similar effects were observed for the
conjugates 6 and 8 with the C-M6Po-clusters, indicating that the
C-mannosyl-6-C-phosphonate is an adequate mimic of its O-
mannosyl counterpart and that the enhanced stimulatory effect is
independent of the peptide sequence (Figure 2B, see also
supporting Figure S1). Overall this indicates that conjugation of
the M6Po-cluster to a peptide antigen adjuvanted with a TLR7-
ligand enhances DC maturation by improving uptake of the
conjugate and/or trafficking of the conjugates to the endosomally
located TLR7 receptor.
Processing of the two peptides was investigated by assessing
presentation of the SIINFEKL epitope on MHC-I by the DCs to
CD8+ T cells and presentation of the helper epitope (HAAHA) to
CD4+ T cells. For the former assay, a hybridoma (B3Z) CD8+ T
cell line was used, while the latter employed an OTIIZ hybridoma
CD4+ T cell line. As can be seen in Figures 2C and 2D, attachment
of the TLR7-ligand to the SLPs led to the enhanced presentation
of the antigens (See for example 35 vs. 36/37 and 38 vs. 40),
however the inclusion of the M6Po-clusters hampered
presentation through both the MHC class I and MHC class II
pathways (See for example 36 vs. 4, 37 vs. 2 and 40 vs. 6). This
indicates that the conjugation of M6Po-clusters affects
intracellular trafficking or processing of the peptides.
Keywords: C-glycoside • mannose-6-phosphate receptor •
peptide conjugate • solid-phase peptide synthesis • TLR-ligand
[1]
[2]
[3]
[4]
[5]
[6]
[7]
N. Dahms, Biochim. Biophys. Acta - Gen. Subj. 2002, 1572, 317–
340.
P. Ghosh, N. M. Dahms, S. Kornfeld, Nat. Rev. Mol. Cell Biol. 2003,
4, 202–213.
J. J. Distler, J. F. Guo, G. W. Jourdian, O. P. Srivastava, O.
Hindsgaul, J. Biol. Chem. 1991, 266, 21687–92.
P. Y. Tong, W. Gregory, S. Kornfeld, J. Biol. Chem. 1989, 264,
7962–9.
S. J. York, L. S. Arneson, W. T. Gregory, N. M. Dahms, S. Kornfeld,
J. Biol. Chem. 1999, 274, 1164–71.
K. Prydz, A. W. Brändli, M. Bomsel, K. Simons, J. Biol. Chem. 1990,
265, 12629–35.
K. El Cheikh, I. Basile, A. Da Silva, C. Bernon, P. Cérutti, F.
Salgues, M. Perez, M. Maynadier, M. Gary-Bobo, C. Caillaud, et al.,
Angew. Chemie Int. Ed. 2016, 55, 14774–14777.
E. Dalle Vedove, G. Costabile, O. M. Merkel, Adv. Healthc. Mater.
2018, 7, 1701398.
[8]
[9]
C. A. Parra-López, R. Lindner, I. Vidavsky, M. Gross, E. R. Unanue,
J. Immunol. 1997, 158, 2670–9.
[10]
R. Greupink, H. I. Bakker, W. Bouma, C. Reker-Smit, D. K. F.
Meijer, L. Beljaars, K. Poelstra, J. Pharmacol. Exp. Ther. 2006, 317,
514–521.
[11]
C. Minnelli, L. Cianfruglia, E. Laudadio, R. Galeazzi, M. Pisani, E.
Crucianelli, D. Bizzaro, T. Armeni, G. Mobbili, J. Drug Target. 2018,
26, 242–251.
5
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Figure 2. M6Po-conjugation enhances DC activation of theTLR7-ligand conjugates but inhibits antigen presentation. A) Murine bone marrow-derived dendritic cells
were stimulated in triplicates for 24 hours with the indicated conjugates of the DEVA5K peptide. The induction of DC maturation was analyzed by measuring IL-12
production with sandwich ELISA specific for the IL-12p40 subunit. The bars represent the standard deviation of the mean of the triplicates. B) The induction of IL-
12p40 of the DEVA5K and the HAAHA conjugates was compared by stimulating bone-marrow derived dendritic cells for 24 hours followed by sandwich
ELISA. C and D) Antigen uptake and presentation to T cells was measured by incubating dendritic cells in duplicates with the indicated compounds for 3 hours,
followed by incubation with the reporter hybridoma T cell lines. The SIINFEKL-specific hybridoma T cell line B3Z served as readout for the DEVA5K peptide, while
the OTIIZ hybridoma cell line was used to detect presentation of the HAAHA epitope. B3Z or OTIIZ activation was determined by colorimetric reaction of the lacZ
reporter enzyme. Results are representative of three independently performed experiments. The statistical differences between the compounds at the same
concentrations were calculated by 2-way ANOVA followed by multiple comparison and Tukey corrections. * p<0.05, ** p< 0.01, *** p<0.001, **** p<0.0001.and
Tukey corrections. * p<0.05, ** p< 0.01, *** p<0.001, **** p<0.0001.
6
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FULL PAPER
[12]
S. Hoogendoorn, G. H. M. van Puijvelde, J. Kuiper, G. A. van der
Filippov, Angew. Chemie Int. Ed. 2018, 57, 1659–1662.
Marel, H. S. Overkleeft, Angew. Chemie Int. Ed. 2014, 53, 10975–
10978.
[32]
J. Zhou, K. Xu, P. Zhou, O. Zheng, Z. Lin, L. Guo, B. Qiu, G. Chen,
Biosens. Bioelectron. 2014, 51, 386–390.
[13]
C. R. Becer, M. I. Gibson, J. Geng, R. Ilyas, R. Wallis, D. A.
Mitchell, D. M. Haddleton, J. Am. Chem. Soc. 2010, 132, 15130–
15132.
[14]
[15]
E. A. B. Kantchev, C.-C. Chang, D.-K. Chang, Biopolymers 2006,
84, 232–240.
R.-J. E. Li, T. P. Hogervorst, S. Achilli, S. C. Bruijns, T. Arnoldus, C.
Vivès, C. C. Wong, M. Thépaut, N. J. Meeuwenoord, H. van den
Elst, et al., Front. Chem. 2019, 7, 650.
[16]
[17]
T. P. Hogervorst, R. J. J. E. Li, L. Marino, S. C. M. Bruijns, N. J.
Meeuwenoord, D. V. Filippov, H. S. Overkleeft, G. A. Van Der
Marel, S. J. Van Vliet, Y. Van Kooyk, et al., ACS Chem. Biol. 2020,
15, 728–739.
V. Barragan-Montero, A. Awwad, S. Combemale, P. de Santa
Barbara, B. Jover, J.-P. Molès, J.-L. Montero, ChemMedChem
2011, 6, 1771–1774.
[18]
[19]
D. B. Berkowitz, G. Maiti, B. D. Charette, C. D. Dreis, R. G.
MacDonald, Org. Lett. 2004, 6, 4921–4924.
S. Banik, K. Pedram, S. Wisnovsky, N. Riley, C. Bertozzi, 2019.
Preprint available at chemRxiv https://doi.org/10.26434/
chemrxiv.7927061.v2.
[20]
[21]
J. J. Weterings, S. Khan, G. J. van der Heden, J. W. Drijfhout, C. J.
M. Melief, H. S. Overkleeft, S. H. van der Burg, F. Ossendorp, G. A.
van der Marel, D. V. Filippov, Bioorg. Med. Chem. Lett. 2006, 16,
3258–3261.
G. P. P. Gential, T. P. Hogervorst, E. Tondini, M. J. van de Graaff,
H. S. Overkleeft, J. D. C. Codée, G. A. van der Marel, F.
Ossendorp, D. V. Filippov, Bioorg. Med. Chem. Lett. 2019, 29,
1340–1344.
[22]
[23]
X. Fei, C. M. Connelly, R. G. MacDonald, D. B. Berkowitz, Bioorg.
Med. Chem. Lett. 2008, 18, 3085–3089.
N. Reintjens, T. Koemans, N. Zilverschoon, R. Castelli, R.
Cordfunke, J.-W. Drijfhout, N. Meeuwenoord, H. Overkleeft, D.
Filippov, G. Van der Marel, et al., European J. Org. Chem. 2020,
DOI 10.1002/ejoc.202000587.
[24]
[25]
L. M. Mikkelsen, S. L. Krintel, J. Jiménez-Barbero, T. Skrydstrup, J.
Org. Chem. 2002, 67, 6297–6308.
L. Liu, C.-Q. Wang, D. Liu, W.-G. He, J.-Y. Xu, A.-J. Lin, H.-Q. Yao,
G. Tanabe, O. Muraoka, W.-J. Xie, et al., Org. Lett. 2014, 16, 5004–
5007.
[26]
A fast work-up was necessary due to the instability of the formed
triflate as decomposition was observed when concentrating in vacuo
at 40°C instead of 30°C..
[27]
[28]
[29]
[30]
[31]
It was found to be important to co-evaporate the phosphonate
reagents with toluene before treatment with n-BuLi.
The yield dropped to 49% when increasing the scale to 30 mmol
due to the instability of the primary triflate.
P. K. Sharma, S. Kumar, P. Kumar, P. Nielsen, Tetrahedron Lett.
2007, 48, 8704–8708.
K. Voigtritter, S. Ghorai, B. H. Lipshutz, J. Org. Chem. 2011, 76,
4697–4702.
Q. Liu, H. A. V. Kistemaker, S. Bhogaraju, I. Dikic, H. S. Overkleeft,
G. A. van der Marel, H. Ovaa, G. J. van der Heden van Noort, D. V.
7
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ChemBioChem
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Entry for the Table of Contents
O
tBuO
tBuO
P
O
P
M6Po
HO
HO
O
O
PMBO
OH
O
Click chemistry
O
Enhanced
immune
HO
HO
response
H
N
O
Solid-phase
peptide chemistry
Peptide
O
TLR7L
O
Ac
N
H
NH
O
6
HO
NHFmoc
We report the synthesis of two mannose-6-C-phosphonate (M6Po) building blocks that can be incorporated in a conjugate via either a
“post-assembly” click or an online solid-phase peptide synthesis approach to improve endosomal trafficking of vaccines. The generated
M6Po-conjugates, which were also equipped with a TLR7-ligand, were able to enhance the immune response by activation and
maturation of dendritic cells.
8
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