Synthesis of novel bivalent mimetic ligands for mannose-6-phosphate receptors

Article abstract of DOI:10.1016/j.bmcl.2013.02.068

Mannose-6-phosphate (M6P)-containing N-linked glycans are essential signaling molecules for sorting hydrolases in eukaryotic cells. Their receptors, especially the cation-independent M6P receptors (CI-MPRs), have emerged as promising protein targets for targeted drug delivery for the treatment of lysosomal storage disease and liver fibrosis. In this Letter, we describe the design and synthesis of novel bivalent mimetic ligands for CI-MPRs. We report that for the first time, a newly-discovered binding motif, GlcNAc-M6P, has been incorporated in mimetic ligands. M6P- and GlcNAc-M6P-containing building blocks, equipped with NH₂ and CO₂H handles, have been prepared and assembled with an ornithine linker through amide coupling reactions. Efficient global deprotection protocols have also been developed which have been showcased in the synthesis of our novel bivalent mimetic ligands.

Full text of DOI:10.1016/j.bmcl.2013.02.068

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2328–2331
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel bivalent mimetic ligands
for mannose-6-phosphate receptors
Yunpeng Liu a, Jared Marshall a, Qiong Li a, Nicola Edwards b, Gong Chen a,
⇑
a Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, United States
b Department of Chemistry, The Pennsylvania State University, Worthington Scranton, Dunmore, PA 18512, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Mannose-6-phosphate (M6P)-containing N-linked glycans are essential signaling molecules for sorting
hydrolases in eukaryotic cells. Their receptors, especially the cation-independent M6P receptors
(CI-MPRs), have emerged as promising protein targets for targeted drug delivery for the treatment of
lysosomal storage disease and liver fibrosis. In this Letter, we describe the design and synthesis of novel
bivalent mimetic ligands for CI-MPRs. We report that for the first time, a newly-discovered binding motif,
GlcNAc-M6P, has been incorporated in mimetic ligands. M6P- and GlcNAc-M6P-containing building
blocks, equipped with NH2 and CO2H handles, have been prepared and assembled with an ornithine lin-
ker through amide coupling reactions. Efficient global deprotection protocols have also been developed
which have been showcased in the synthesis of our novel bivalent mimetic ligands.
Received 11 January 2013
Revised 10 February 2013
Accepted 13 February 2013
Available online 24 February 2013
Keywords:
Mannose-6-phosphate
GlcNAc-M6P
CI-MPR
Ó 2013 Elsevier Ltd. All rights reserved.
N-glycan
Mimetic ligands
Mannose-6-phosphate (M6P)-containing N-glycans are a class
of oligosaccharide-based signaling molecules that plays an essen-
tial role in intracellular transport of hydrolases into the
endosomal/lysosomal compartments in eukaryotic cells (Fig. 1).1
Two major types of M6P receptors (MPRs), cation-dependent
(CD-MPRs) and cation-independent (CI-MPRs), are responsible for
recognizing M6P moieties of hydrolases, and facilitating their
transport to the lysosome.2 Additionally, CI-MPR, also known as
insulin-like growth factor 2 receptor (IGF2R), functions as a mem-
brane-associated receptor that is responsible for the internaliza-
tion of a number of exogenous proteins in the extracellular
matrix. Defects in the M6P and MPR mediated sorting system are
known to cause lysosomal storage diseases (LSD). Currently, en-
zyme replacement therapy (ERT), via the infusion of enzymes pro-
duced in vitro, is the only viable method for treating certain LSDs.3
However, this method suffers from low efficiency and high cost
due to the poor uptake of the infused enzymes that lack the critical
targeting elements. To improve ERT and develop other MPR-tar-
geted drug delivery strategies, it is crucial that we better under-
stand the M6P-coded sorting system.4
occurring M6P carrying N-glycans provides valuable insight in
the MPR system in their native format and these complex N-gly-
cans are well-suited for biomedical applications. There is, however,
a formidable cost associated with the total synthesis of these com-
pounds. An alternative strategy is to synthesize simpler model
scaffolds containing the key binding motifs from these N-glycans.
These scaffolds could exhibit comparable or even enhanced bind-
ing ability, when compared to the naturally occurring N-glycans.
Herein, we report our efforts to design and synthesize novel biva-
lent mimetic ligands for MPR-targeted biomedical applications. For
the first time, a newly discovered binding motif, GlcNAc-M6P, has
been incorporated in mimetic ligands.
The concept of developing simplified mimetic ligands based on
the M6P motif has been pioneered by the groups of Hindsgaul,
Bock, Berkowitz and MacDonald.7 Although some promising
results were obtained, truly unimolecular multivalent ligands that
can span the long distance between the two known M6P binding
domains 3 and 9 in CI-MPRs have not be well-established.
Recently, the Dahms laboratory made a key discovery by identify-
ing N-acetylglucosamine M6P (GlcNAc-M6P), a biosynthesis pre-
cursor of M6P, as a new binding element to CI-MPRs, at a new
binding domain 5 located between domains 3 and 9 (Fig. 1).8
Although this structural motif has been long found in many
N-glycoproteins, their binding interaction with CI-MPRs was not
studied for several decades. We speculate that this was due to its
relatively weaker binding to CI-MPRs and difficulty in obtaining
homogenous N-glycan carrying this motif. Different from the
previously-reported mimetic ligands, we sought to incorporate
the newly-discovered binding element, GlcNAc-M6P, in our ligand
The ability to elucidate this M6P/MPR mediated recognition/
transport system has been hampered by the heterogeneity of
M6P N-glycans and the complex multivalency of their binding
towards MPRs.5 Recently, we successfully developed the first
chemical synthesis of N-glycans carrying well-defined M6P motifs
for in vitro uptake studies.6 The total synthesis of the naturally
⇑
Corresponding author. Tel.: +1 814 867 2590.
E-mail address: guc11@psu.edu (G. Chen).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.068

Y. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2328–2331
2329
TrtO
OBn
a
BnO
HO
TrtO
BnO
OBn
O
OBn
O
b
O
BnO
BnO
BnO
BnO
O
O
CO2All
1
O
CO2H
2
3
5
(BnO)2OPO
BnO
(BnO)2OPO
OBn
OBn
O
c
d
O
BnO
BnO
BnO
O
CO2All
O
CO2H
4
AcO
AcO
O
3
O
AcO
AcO
AcO
AcO
AcHN
AcO
AcHN
O
AcO
α
O
AcO
AcHN
O
P
O
P
OBn
O
OBn
O
OBn
P
O
O
6
OBn
NiPr2
OBn
f
O
O
BnO
BnO
BnO
BnO
e
O
CO2H
O
CO2All
7
8
Scheme 1. Synthesis of building blocks 5 and 8 equipped with CO2H handle.
Reagents and conditions: (a) RuCl3, NaIO4, CH3CN/CH2Cl2/H2O (2:2:3), rt; (b) (i) allyl
bromide, K2CO3, DMF, rt, 72% over two steps, (ii) FeCl3Á6H2O, CH2Cl2, 0 °C, 75%; (c)
(i) (BnO)2PN(iPr2), 1H-tetrazole, 4 Å MS, CH2Cl2, rt, (ii) t-BuO2H, rt, 91% over two
steps; (d) Pd(PPh3)4, morpholine, CH3CN, 0 °C; (e) (i) 6, 1H-tetrazole, 4 Å MS, CH2Cl2,
rt, (ii) t-BuO2H, À40 °C, 90% over two steps; (f) Pd(PPh3)4, morpholine, CH3CN, 0 °C.
position of 3 was effected by dibenzyl N,N-diisopropyl phospho-
ramidite (BnO)2PN(iPr2) and the subsequent oxidation by tBuO2H
gave intermediate 4 in 91% yield. The allyl group of 4 was removed
efficiently using Pd(Ph3P)4/morpholine to provide the M6P-OH
building block 5. Installation of the GlcNAc phosphoester on the
6-OH position of 3 was effected using the GlcNAc-derived phospho-
ramidite 6 and the subsequent oxidation with tBuO2H. Reagent 6
was originally developed by Boons and co-workers for the synthesis
Figure 1. Representative structures of naturally occurring M6P-containing N-
glycans and mimetic ligands for CI-MPRs.
design. We expect that unimolecular multivalent ligands carrying
suitable M6P and/or GlcNAc-M6P elements are uniquely suited
to achieve the elusive high-affinity binding toward the monomeric
unit of CI-MPR. To pursue suitable multivalent ligands, we need to
prepare and test a series of multivalent ligands that M6P and Glc-
NAc-M6P moieties are presented on linker molecules with varying
lengths and spatial arrangements. Due to the challenging physical
and chemical properties of these phosphorylated sugar com-
pounds, their synthesis routes need to be carefully orchestrated
to accommodate the labile phosphoester moieties, so as to avoid
any difficult purification steps and to ensure that these synthetic
routes are amenable to library synthesis.9
of GlcNAc-P-serine motifs containing a similar
a phosphodiester
linkage.11 Removal of the allyl group of 7 using Pd(Ph3P)4/morpho-
line gave the GlcNAc-M6P-OH building block 8 in good yield.
We then prepared M6P and GlcNAc-M6P building blocks 13 and
14, equipped with NH2 handles (Scheme 3). Stereoselective
glycosylation of the mannose trichloroacetimidate 96a with Boc-
protected 3-aminopropanol 10, under the promotion of TMSOTf,
yielded compound 11. The TIPS group of compound 11 was then
removed by the treatment of TBAF to give compound 12. OBn-pro-
TIPSO
In contrast to the late-stage phosphorylation strategy used in
our previous total synthesis routes, we adopted a more modular
synthetic strategy for these mimetic ligands. Our strategy is out-
lined as follows. First, as shown in Schemes 1 and 2, protected
M6P and GlcNAc-M6P building blocks bearing NH2 or CO2H han-
dles, were prepared as standard cassette-type building blocks.
Second, these building blocks were then coupled with the linker
molecules through amide bonds to provide the desired ligands.
For the first-generation bivalent ligands, amino acid ornithine
TIPSO
OAc
O
OAc
O
BnO
HO
NHBoc
BnO
BnO
BnO
b
10
O
NHBoc
O
CCl3
NH
a
9
11
(BnO)2OPO
OAc
O
c
BnO
BnO
O
NH2
13
HO
OAc
O
was used as a linker to tether two building blocks on its
a-NH2
BnO
AcO
BnO
and CO2H ends; the remaining d-NH2 group on side chain can be
potentially used for conjugation with biophysical tags.
O
AcO
AcO
AcHN
O
NHBoc
12
O
As shown in Scheme 1, building blocks 5 and 8 bearing a CO2H
handle were first prepared. OBn-protected phosphoester linkages
are labile under nucleophilic conditions but reasonably stable
under acidic conditions. Accordingly, the allyl ester group, was cho-
sen as the protecting group for the intermediates 4 and 7. The allyl
O
P
OBn
O
OAc
O
BnO
BnO
6
d
O
NH2
14
group of the a
-O-allyl mannoside 110 was first cleaved using RuCl3/
Scheme 2. Synthesis of building blocks 13 and 14 equipped with NH2 handle.
Reagents and conditions: (a) 10, TMSOTf, 4 Å MS, CH2Cl2, À20 °C, 70%; (b) TBAF,
THF, rt, 78%; (c) (i) (BnO)2PN(iPr2), 1H-tetrazole, 4 Å MS, CH2Cl2, rt, (ii) t-BuO2H, rt,
89% over two steps, (iii) TFA, CH2Cl2, rt; (d) (i) 6, 1H-tetrazole, 4 Å MS, CH2Cl2, rt, (ii)
t-BuO2H, À40 °C, 90% over two steps, (iii) TFA, CH2Cl2, rt.
NaIO4 to furnish mannosyl glycolic acid 2. The CO2H group of 2 was
then protected with allyl bromide to give an allyl ester intermediate,
whose trityl group was subsequently removed by FeCl3Á6H2O to give
3. Installation of the OBn-protected phosphoester on the 6-OH

2330
Y. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2328–2331
(BnO)2OPO
OAc
O
AcO
O
BnO
AcO
BnO
AcO
AcHN
CO2H
AcO
O
O
O
AcO
(BnO)2OPO
BnO
OAc
O
AcO
AcHN
BocHN
O
P
OBn
HN
O
NHCbz
O
BnO
15
16
O
OAc
O
P
OBn
O
O
BnO
BnO
H2N
15
a
13
O
OAc
NHCbz
NH2
O
BnO
BnO
O
a
HN
O
(BnO)2OPO
OAc
O
2-O3PO
HO
OH
O
O
NH2
BnO
BnO
NHCbz
HO
H2N
14
21
O
O
HN
HN
O
HN
HN
O
AcO
HO
O
O
AcO
HO
c
AcO
AcHN
HO
AcHN
5
O
O
P
b
O
O
O
P
OBn
O
O-
NHCbz
O
NH2
O
O
O
BnO
BnO
OAc
OH
O
HO
HO
O
O
BnO
BnO
HO
HO
17
18
O
(BnO)2OPO
OBn
2-O3PO
OH
c
5
O
O
b
16
HN
HN
O
HN
O
2-O3PO
(BnO)2OPO
OAc
OH
O
O
BnO
BnO
HO
HO
HN
O
O
O
O
O
NHCbz
NH2
O
HN
HN
O
HN
HN
O
BnO
BnO
(BnO)2OPO
HO
HO
22
O
23
O
8
2-O3PO
OH
OBn
e
d
O
O
21
NHCbz
NH2
O
O
HO
AcO
O
O
HO
HO
BnO
BnO
HO
AcO
HO
AcHN
AcO
AcHN
O
O
O
OH
O
OBn
OBn
O
P
O
P
19
20
O
P
O-
O
P
O
O-
O
OBn
O
O
O
O
AcHN
HO
AcHN
AcO
OH
O
OAc
O
HO
HO
BnO
BnO
HO
HO
AcO
AcO
O
O
O
O
Scheme 3. Synthesis of bivalent ligands 18 and 20. Reagents and conditions: (a) (i)
15, HOBt, EDCI, DIPEA, CH2Cl2, rt, 86%, (ii) TFA, CH2Cl2, rt; (b) 5, HOBt, EDCI, DIPEA,
CH2Cl2, rt, 63%; (c) (i) H2, Pd(OH)2/C, MeOH, rt, (ii) NH2NH2ÁH2O, MeOH, rt, 80% over
two steps; (d) 8, HOBt, EDCI, DIPEA, CH2Cl2, rt, 68%; (e) (i) H2, Pd(OH)2/C, MeOH, rt,
(ii) NH2NH2ÁH2O, MeOH, rt, 78% over two steps.
HN
HN
O
HN
HN
O
8
f
e
O
25
O
NH2
NHCbz
O
O
HO
HO
BnO
HO
24
O
O
O
OH
O
OBn
OBn
O
P
O-
O
P
tected phosphoester group was installed on compound 12 using
the standard (BnO)2PN(iPr2)/tBuO2H reagents in 89% yield. The
Boc group was removed with TFA/CH2Cl2 at room temperature to
give the desired M6P-NH2 building block 13 as a TFA salt. The Glc-
NAc-M6P-NH2 building block 14 was prepared from 12 and 6 in
analogous fashion to compound 13.
O
O
AcHN
HO
AcHN
AcO
HO
HO
AcO
AcO
O
O
Scheme 4. Synthesis of bivalent ligands 23 and 25. Reagents and conditions: (a) (i)
15, HOBt, EDCI, DIPEA, CH2Cl2, rt, 84%, (ii) TFA, CH2Cl2, rt; (b) 5, HOBt, EDCI, DIPEA,
CH2Cl2, rt, 65%; (c) (i) H2, Pd(OH)2/C, MeOH, (ii) NH2NH2.H2O, MeOH, rt, 85% over
two steps; (e) 8, HOBt, EDCI, DIPEA, CH2Cl2, rt, 58%; (f) (i) H2, Pd(OH)2/C, MeOH, rt,
(ii) NH2NH2.H2O, MeOH, rt, 75% over two steps.
With the building blocks 5, 8, 13 and 14 in hand, we next
assembled them with the Boc-Nd-Cbz-
L-ornithine linker 15 to pro-
vide the bivalent ligands 18, 20, 23 and 25 (Schemes 3 and 4).
EDCI-mediated amide coupling of M6P-NH2 13 with ornithine 15,
and the subsequent Boc deprotection with TFA gave the M6P-
Orn-NH2 16 in good yield. EDCI-mediated amide coupling of 16
with M6P-OH 5 then gave compound 17. The protection of all
the polar functional groups in compound 17 allowed us to easily
purify it using normal silica gel chromatography. The final global
deprotection of 17 was first effected by the removal of the Bn
and Cbz groups using Pd(OH)2/C-catalyzed hydrogenolysis condi-
tions. The remaining OAc group of 17 was then removed with
NH2NH2ÁH2O in MeOH at room temperature to give the fully
deprotected bivalent M6P-Orn-M6P ligand 18. A filtration through
a sephadex G-25 column (eluted with H2O) provided 18 in high
yield and purity. The bivalent ligand M6P-Orn-GlcNAc-M6P 20
was prepared in analogous fashion to compound 18.
the free phosphate group was stable to nucleophilic conditions. This
order is particularly important for the successful deprotection of the
intermediate 19, which carries a more labile GlcNAc-M6P moiety.
Following the same synthetic routes for 18 and 20, another pair of
bivalent ligands 23 and 25, carrying a GlcNAc-M6P moiety on the
upper arm, were prepared in good yield from the building block 5,
8 and 14 (Scheme 4).
In summary, we designed and successfully synthesized novel
bivalent mimetic ligands for cation-independent mannose-6-phos-
phate receptors (CI-MPRs). For the first time, a newly discovered
MPR-binding element GlcNAc-M6P has been incorporated in mi-
metic ligands. A convergent and efficient synthesis strategy based
on modular assembly of cassette-type M6P- and GlcNAc-M6P-con-
taining building blocks via amide coupling was developed. Synthe-
sis of other bi- and higher valent mimetic ligands with different
linker molecules and in vitro binding analysis with CI-MPRs are
under current investigation and will be reported in due course.
It should be noted that the order of the debenzylation and
deacetylation steps of the deprotection sequence of 17 is important
because the OBn-protected phosphoester is not stable under nucle-
ophilic deacetylation conditions.11 Upon removal of the Bn group,

Y. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2328–2331
2331
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This work is supported by The Pennsylvania State University,
Glycomimetics Inc, and The Center for LignoCellulose Structure
and Formation, an Energy Frontier Research Center funded by the
US Department of Energy, Office of Science under Award Number
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Supplementary data
Supplementary data (synthetic procedures and structural char-
acterization data for all new compounds) associated with this arti-
cle can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2013.02.068.
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