A set of phosphatase-inert "molecular rulers" to probe for bivalent mannose 6-phosphate ligand-receptor interactions

Article abstract of DOI:10.1016/j.bmcl.2007.11.094

A set of bivalent mannose 6-phosphonate 'molecular rulers' has been synthesized to examine ligand binding to the M6P/IGF2R. The set is estimated to span a P-P distance range of 16-26 A (MMFF energy minimization on the hydrated phosphonates). Key synthetic

Full text of DOI:10.1016/j.bmcl.2007.11.094

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3085–3089
A set of phosphatase-inert ‘‘molecular rulers’’ to probe for
bivalent mannose 6-phosphate ligand–receptor interactions
Xiang Fei,a, Christopher M. Connelly,b, Richard G. MacDonaldb,*
and David B. Berkowitza,*
aDepartment of Chemistry, University of Nebraska, Lincoln, NE 68588, USA
bDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA
Received 16 October 2007; revised 19 November 2007; accepted 19 November 2007
Available online 28 November 2007
Abstract—A set of bivalent mannose 6-phosphonate ‘molecular rulers’ has been synthesized to examine ligand binding to the M6P/
˚
IGF2R. The set is estimated to span a P–P distance range of 16–26 A (MMFF energy minimization on the hydrated phosphonates).
Key synthetic transformations include sugar triflate displacement for phosphonate installation and Grubbs I cross-metathesis to
achieve bivalency. Relative binding affinities were tested by radioligand displacement assays versus PMP-BSA (pentamannosyl
phosphate-bovine serum albumin). These compounds exhibit slightly higher binding affinities for the receptor (IC50’s = 3.7–
5 lM) than the parent, monomeric mannose 6-phosphonate ligand and M6P itself (IC50 = 11.5 2.5 lM). These results suggest that
the use of an a-configured anomeric alkane tether is acceptable, as no significant thermodynamic penalty is apparently paid with this
design. On the other hand, the modest gains in binding affinity observed suggest that this ligand set has not yet found true bivalent
interaction with the M6P/IGF2R (i.e., simultaneous binding to two distinct M6P-binding pockets).
Ó 2007 Elsevier Ltd. All rights reserved.
The mannose 6-phosphate/insulin-like growth factor II
receptor (M6P/IGF2R) is a type I transmembrane glyco-
protein that cycles through the Golgi, endosomes, and
the plasma membrane to carry out its role in the trans-
port of lysosomal enzymes to their cellular destination.1
The receptor also functions in the binding, uptake, and
degradation of the mitogen, insulin-like growth factor
II (IGF-II) and facilitates activation of the growth inhib-
itor, transforming growth factor-b. The ability of the
M6P/IGF2R to inhibit cell proliferation, or stimulate
apoptosis, by these mechanisms has implicated the recep-
tor as a tumor suppressor. The IGF-II binding activity of
the M6P/IGF2R is mainly responsible for its growth sup-
pressor function. Many cancers become growth factor-
independent by high-level expression of IGF-II, which
not only binds to the M6P/IGF2R, but also to the
IGF1R. The high-affinity interaction of IGF-II with
the IGF1R leads to activation of IGF1R signaling path-
ways that promote cell division and survival.2
The extracellular portion of the M6P/IGF2R contains
15 homologous repeat domains of ꢀ147 amino acid res-
idues each. There are two M6P binding sites located in
domains 3 and 9, and there is one IGF-II binding site
in domain 11.3 Binding of high-affinity, M6P-based li-
gands and rapid internalization of extracellular ligands,
such as IGF-II, are aided by the M6P/IGF2R’s ability to
dimerize.4,5 York et al. demonstrated that b-glucuroni-
dase (hGUS), a homotetrameric lysosomal enzyme bear-
ing multiple M6P moieties, stabilized the receptor’s
dimeric structure by cross-bridging the M6P binding
sites on two adjacent subunits.5 These data support a di-
meric model for binding of bivalent M6P-based ligands
by the M6P/IGF2R (Fig. 1). Importantly, they also ob-
served that hGUS binding increased the rate of internal-
ization of the receptor and consequently stimulated the
degradation of any passenger ligands, including IGF-
II, by 3- to 4-fold. The long-term goal of the present
work is to exploit this unique property of bivalent
M6P ligands as a potential strategy for therapeutic inter-
vention in IGF-II-dependent cancer.
Keywords: Mannose 6-phosphate; Insulin-like growth factor II recep-
tor; Phosphonate; Bivalent ligand; Cross-metathesis; Triflate
displacement.
Multivalent interactions between receptors and their li-
gands,6 which are common in biology, involve a multi-
step mechanism in which most of the entropic cost is
paid by the initial binding event and subsequent con-
*
Corresponding authors. Tel./fax: +1 402 472 2738 (D.B.B.); e-mail:
dbb@unlserve.unl.edu
Note: These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.094

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X. Fei et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3085–3089
Figure 1. M6P ligand binding to the M6P/IGF2R: The alternative
‘hook and ladder’ models: (A) One monomeric unit of the M6P/
IGF2R consisting of the 15 extracellular repeating domains, trans-
membrane domain, and a short cytoplasmic tail. The M6P/IGF2R is
depicted as forming a hook-like structure when a ligand bearing two
M6P groups binds to domains 3 and 9 (lighter shaded ovals). (B) Two
monomeric units of the M6P/IGF2R are connected through binding
by ligands that interact with either domain 3 or 9 of two individual
monomeric units to form a dimeric ladder-like structure.
tacts contribute a favorable enthalpy without further
sacrifice of rotational and translational entropy.7 The
resultant high binding affinity in these interactions is
due to a reduced rate of ligand–receptor dissociation.
This type of interaction occurs in carbohydrate binding
to lectins and is particularly important in the binding of
M6P-bearing oligosaccharides by P-type lectins such as
the M6P/IGF2R.
Figure 2. Hindsgaul’s model biantennary ligands.
mannose disaccharides capped with phosphate con-
nected by a core peptide of 3–5 amino acids. A tripep-
tide version of this compound bound the M6P/IGF2R
with high-affinity, which led to the hypothesis of a biva-
lent M6P-based mechanism.5,11 However, upon closer
inspection, it appears that the exceptional binding affin-
ity of this compound was attributable to an anthranoyl
group present on the lysine e-amino group within the
core peptide (Fig. 3). This modification increased the
affinity by ꢀ200-fold relative to the same compound
with an unmodified peptide,11 presumably through
interaction with a hydrophobic patch on the receptor
proximal to the M6P-binding site. Considering that
the high affinity of this compound did not arise from a
bivalent M6P-based binding mechanism, it is not sur-
prising that York et al. found that the compound failed
to stabilize the receptor’s dimeric structure or to stimu-
late its rapid internalization.5 In summary, there is cur-
rently no evidence in the literature of a small synthetic
compound capable of bivalent binding to the M6P/
IGF2R by a M6P-based mechanism.
Tong et al. demonstrated that there are two M6P-bind-
ing sites per monomeric unit of the M6P/IGF2R.8 Some
native glycoprotein ligands and model compounds (e.g.,
PMP-BSA) display up to 100- to 1000-fold lower disso-
ciation constants, that is, higher affinities, than ligands
bearing a single phosphorylated mannoside. Given that
two M6P-binding pockets are available per receptor in
the monomeric binding model and four per receptor in
the dimeric model, bi- or multivalency may account
for this effect. This could result from simultaneous con-
tact with two M6P groups on two distinct oligosaccha-
rides. Alternatively, the pioneering work of Varki and
Kornfeld suggested that such high-affinity bivalent bind-
ing might also be achieved with a single N-linked oligo-
saccharide phosphorylated on the two ultimate mannose
residues, at the first and third antennae (Fig. 2).9 This
high affinity could arise either from intramolecular con-
tact between a single receptor molecule and the two
phosphate groups on the ligand or by intermolecular
cooperation between two subunits within a dimeric
receptor structure, as depicted in Figure 1.
Thus, the goal of this work is to develop high-affinity
bivalent M6P-based ligands that accelerate disposal of
IGF-II as a passenger ligand directly in tumors, by
cross-bridging the M6P/IGF2R thereby enhancing its
ability to internalize IGF-II. In our previous work, we
discovered that the phosphonate is an excellent surro-
gate for phosphate to promote equivalent interaction
with the M6P-binding domains of the M6P/IGF2R.12,13
The phosphonate has the advantage of resistance to
hydrolysis with the potential for improved pharmacoki-
netics and efficacy in vivo. In light of the aforemen-
tioned studies, we sought to improve affinity by
building bivalency into such ligands.
Later work by the Hindsgaul group demonstrated that
the linkage between the ultimate Man and penultimate
Man on the phosphorylated branch is important, as an
a-1,2-glycosidic linkage results in a higher binding affin-
ity to the receptor than an a-1,3 linkage (Fig. 2).10 A ser-
ies of synthetic multivalent ligands for the M6P/IGF2R
was prepared by Bock and coworkers, using a glycopep-
tide design.11 The best of these compounds bore two

X. Fei et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3085–3089
3087
Figure 3. Bock’s tripeptide bis-M6P-bearing ligands.
We have recently demonstrated proof of principle for a
Ru-mediated cross-metathesis (CM) route to joining
two M6P surrogates, of both the malonate and phos-
phonate varieties with a hydrocarbon tether.12 Herein,
we describe the exploitation of this methodology to
synthesize a series of bis-M6P-phosphonates, with incre-
mentally increasing tether lengths as a sort of ‘molecular
ruler’ set to probe for such a bivalent interaction with
the receptor. To systematically increase tether length
in two carbon increments, the initial mannosidation
reaction was performed with a series of terminally
unsaturated alcohols bearing 4–7 carbons [from 3-bu-
ten-1-ol through 6-hepten-1-ol (Scheme 1)].
likely results from the availability of 2 moles of M6P
per mole of ligand providing a 2-fold increase in the
effective competitor concentration, as opposed to any ef-
fect of tether length. Thus, we conclude that these syn-
thetic compounds are binding the M6P/IGF2R in a
monovalent manner. Moreover, these results imply that
the two M6-phosphonate moieties are binding essen-
tially independently, and with no apparent thermody-
namic or conformational penalty paid for the linker
(possible issues include: position of attachment, hydro-
phobicity, trajectory, etc.).
Dahms and coworkers performed very preliminary
modeling studies of the whole receptor, based on the
crystal structure of domains 1–3 using topographical
information based on the amino acid sequence of each
domain.16 Using this approach, they estimated the intra-
molecular distance of closest approach between the do-
Initial studies pointed to the need for a modified glyco-
sylation protocol. The previous work employed HCl
gas-mediated glycosylation for allyl alcohol itself, but
this approach gave low yields, in the present work, when
applied to longer chain alcohols. Instead, it was found
˚
main 3 and domain 9 M6P-binding sites to be ꢀ45 A. In
that TMSOTf-mediated, Vorbruggen-type glycosyla-
¨
contrast, they estimated the interphosphate distance be-
tween the M6P caps of a bis-phosphorylated oligosac-
tion, using an a-mannosyl acetate glycosyl donor was
quite an efficient reaction. Alkene cross-metathesis14
and triflate displacement15 then followed as the key
steps, as before, in constructing these compounds. Fol-
lowing a final alkene hydrogenation/global debenzyla-
tion step, the free tethered sugar phosphonates were
obtained. Pleasingly, even with the longest hydrocarbon
tether lengths studied here, no solubility issues were
encountered in preparing stock solutions up to
200 mM in a HEPES-saline buffer, pH 7.4.
˚
charide to be ꢀ30 A. For comparison, we conducted
modeling studies of our bis-phosphonate ligands to
determine if they could span these distances. Using
Spartan 04, we built the model structures of the four
compounds and added a cluster of six waters around
each phosphonate. In clustering these waters, water–
˚
proton/phosphonate oxygen distances were set at 2.5 A
(hydrogen bonding distance). From energy minimiza-
tion by molecular mechanics methods (Spartan 04)
using the Merck Molecular Force Field (MMFF),17
one obtains an estimate of the distance between the
two phosphorus atoms in each synthetic bis-phospho-
nate (Table 1). The longest ligand could have a maxi-
Relative binding affinities to M6P/IGF2R were deter-
mined by displacement assay using radiolabeled PMP-
BSA as the tracer in the presence of increasing concen-
trations of each of the synthetic ligands (Table 1). All
the compounds in this new series showed IC50 values
in the micromolar range with ꢀ2-fold increase in RBA
compared to M6P alone. This small increase in RBA
˚
mum span of ꢀ26 A (Fig. 4). Based on these
estimates, our compounds may still be too short to bind
in a bivalent manner, regardless of which receptor bind-
ing model is correct.

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X. Fei et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3085–3089
Scheme 1. Reagents and conditions: (a) NaH, BnBr, DMF, 0 °C to rt (90%); (b) AcOH, Ac2O, H2SO4, 0 °C (82%); (c) 2 TMSOTf, 3-buten-1-ol, rt
(86%), 3 TMSOTf, 4-penten-1-ol, rt (70%), 4 TMSOTf, 5-hexen-1-ol, rt (63%), 5 TMSOTf, 6-hepten-1-ol, rt (62%); (d) Grubbs I catalyst, DCM,
40 °C: 6 (81%), 7 (78%), 8 (69%), 9 (70%); (e) sodium methoxide, methanol, rt (quantitative); (f) 2,6-di-tert-butyl-4-methylpyridine, Tf2O, DCM,
À40 °C; (g) n-BuLi, dibenzyl methyl-phosphonate, THF, À78 °C (two step yields): 14 (67%), 15 (59%), 16 (33%), 17 (18%); (h) H2 (balloon pressure),
Pd/C, rt; (i) NH4HCO3 (50 mM) aqueous solution, rt (two step yields): 18 (66%), 19 (71%), 20 (68%), 21 (62%).
Table 1. Relative M6P/IGF2R binding affinities
Ligand
IC50 (n)a (lM)
RBAb
Mr
Lengthc
M6P
G6P
11.5 2.51 (4)
>10 (4)
1.0
NA
340
282
666
694
722
750
NA
NA
˚
18 (6C)
19 (8C)
20 (10C)
21 (12C)
4.76 2.50 (4)
5.03 1.34 (4)
4.44 1.40 (4)
3.70 0.56 (4)
2.63 0.74
2.39 0.83
2.65 0.52
3.02 0.41
16.2–19.5 A
˚
19.2–20.9 A
˚
19.6–22.7 A
˚
24.6–26.0 A
a IC50’s for competitive displacement of radiolabeled PMP-BSA from
the receptor (n = no. of trials, see SI for details); G6P = glucose 6-
phosphate (units in mM).
b RBA = relative binding affinity, normalized to free M6P.
c Length = P–P distance, as estimated by molecular mechanics mini-
mization (MMFF). For each compound, minimizations were run
from five different, chain-extended starting conformers. The P–P
distances given represent the ranges seen for the set of low energy,
chain-extended conformers found.
Figure 4. One of several low energy chain-extended conformers found
by an MMFF molecular mechanics minimization on the hydrated 12-
carbon-spaced ligand. See Table 1 for the P,P-distance range found for
the set of such conformers.

X. Fei et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3085–3089
3089
In addition to the importance of tether length, the struc-
Supplementary data
ture of the tether is critical to development of a high-
affinity bivalent ligand. The adventitious binding
properties of the Bock compound suggest that addi-
tional binding energy may be achieved by adjusting
hydrophobicity, charge, and/or p-surface of the tether.11
Moreover, the success enjoyed by the groups of Bock
and Hindsgaul, respectively, in attaining 2–3 orders of
magnitude improved receptor binding over M6P, sug-
gests that peptide- or carbohydrate-based linkers may
be advantageous. Both such tethers present H-bond do-
nor/acceptor functionality across the M6P–M6P span.
They also confer more rigidity than a simply sp3-hybrid-
ized alkane tether. Thus, each peptide bond really repre-
sents a degree of pseudo-unsaturation (planarity) with
an expected bias toward a transoid amide geometry.
Experimental procedures, spectral data, copies of NMR
spectra, and details of the radioligand displacement as-
says. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.bmcl.2007.11.094.
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In conclusion, this study introduces the design and suc-
cessful synthesis of the first array of bis-M6-phospho-
nate-presenting ‘molecular rulers’ to measure distances
between M6P-binding pockets at MPRs, and to distin-
guish between intramolecular and intermolecular modes
of bivalent binding. Although the highest M6P/IGF2R
binding affinity seen in the ligand set is in the micromo-
lar range [IC50 ꢀ 4 lM], no solubility problems or tether
penalty issues were encountered. Moreover, the replace-
ment of the M6P ester with a hydrolytically stable phos-
phonate surrogate persists as an effective design, across
the entire set, and reinforces the notion that phospha-
tase resistance can be incorporated into such small mol-
ecule probes. Completion of these studies will require
that we find a high-affinity ligand that stabilizes the di-
meric structure of the receptor and thereby promotes ra-
pid internalization of IGF-II in a cellular model.
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Acknowledgments
The authors are indebted to the American Heart Associ-
ation (GIA-060014Z to D.B.B.), the NIH (CA-91885 to
R.G.M.), and to the University of Nebraska Biomedical
Research Retreat 2006 Collaboration Seed Grant Pro-
gram (jointly to R.G.M. and D.B.B.) for funding.
C.M.C. was supported by the Nebraska Center for Cel-
lular Signaling (NIH Grant P20 RR18759), UNMC
Graduate Studies assistantship, the Dr. Fred W. Upson
Grant-in-Aid award through UNMC, and the UNMC
Faculty Women’s Club Scholarship. We acknowledge
the NSF (CHE-0091975, MRI-0079750) and NIH
(SIG-1-510-RR-06307) for NMR instrumentation, and
the NIH (RR016544) for facilities renovation.
16. Olson, L. J.; Yammani, R. D.; Dahms, N. M.; Kim, J.-J.
P. EMBO J. 2004, 23, 2019.
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