Mono- and bivalent ligands bearing mannose 6-phosphate (M6P) surrogates: Targeting the M6P/ insulin-like growth factor II receptor

Article abstract of DOI:10.1021/ol0479444

(Chemical Equation Presented) Mannose 6-phosphate mimics locked into the α-configuration and bearing hydrolase-resistant phosphate surrogates were synthesized and evaluated for binding affinity to the mannose 6-phosphate/insulin-like growth factor II rece

Full text of DOI:10.1021/ol0479444

ORGANIC
LETTERS
2004
Vol. 6, No. 26
4921-4924
Mono- and Bivalent Ligands Bearing
Mannose 6-Phosphate (M6P)
Surrogates: Targeting the M6P/
Insulin-Like Growth Factor II Receptor
David B. Berkowitz,*,† Gourhari Maiti,† Bradley D. Charette,†
Christine D. Dreis,‡ and Richard G. MacDonald*,‡
Department of Chemistry, UniVersity of Nebraska, Lincoln, Nebraska 68588-0304, and
Department of Biochemistry & Molecular Biology, UniVersity of Nebraska Medical
Center, Omaha, Nebraska 68198-5870
dbb@unlserVe.unl.edu; rgmacdon@unmc.edu
Received October 6, 2004
ABSTRACT
Mannose 6-phosphate mimics locked into the
r-configuration and bearing hydrolase-resistant phosphate surrogates were synthesized and
evaluated for binding affinity to the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R). Affinity increases as the phosphate
surrogate is varied in the order malonyl ether < malonate < phosphonate. An alkene cross-metathesis approach to sought-after bivalent
M6P-bearing ligands is also described. These compounds were designed to map onto biantennary sectors of high-mannose-type oligosaccharides
carried by glycoprotein M6P/IGF2R ligands.
Mammalian cells possess two receptors for mannose 6-phos-
phate (M6P)-decorated glycoproteins; namely, (i) the small
46 kDa cation-dependent mannose 6-phosphate receptor (CD-
MPR) and (ii) the large 300 kDa cation-independent,
mannose 6-phosphate/insulin-like growth factor II receptor
(CI-MPR or M6P/IGF2R).1 Both serve to mediate the
trafficking of hydrolytic Golgi enzymes. In addition to its
M6P-binding site, the CI-MPR also possesses a distinct
binding site for IGF-II. Its ability to internalize circulating
IGF-II has led to the suggestion that the CI-MPR may be a
tumor suppressor gene.2 IGF-II internalization appears to be
accelerated through the binding of bi- or multivalent high-
affinity ligands to the M6P sites of the receptor.3
including those of the liver,4 lung,5 GI tract,6 and breast.7 A
mouse model shows that CI-MPR deficiency leads to cardiac
hyperplasia.8
Whereas crystal structures are available for the free CD-
MPR, and its complexes with M6P and with pentamannosyl
phosphate (PMP),9 the much larger CI-MPR has proven to
be a greater challenge. The CI-MPR possesses 15 homolo-
(1) For a review, see: Ghosh, P.; Dahms, N.; Kornfeld, S. Nat. ReV.
Mol. Cell Biol. 2003, 4, 202-213.
(2) (a) O’Gorman, D. B.; Weiss, J.; Hettiaratchi, A.; Firth, S. M.; Scott,
C. D. Endocrinology 2002, 143, 4287-4294. (b) Hankins, G. R.; De Souza,
A. T.; Bentley, R. C.; Patel, M. R.; Marks, J. R.; Iglehart, J. D.; Jirtle, R.
L. Oncogene 1996, 12, 2003-2009.
(3) York, S. J.; Arneson, L. S.; Gregory, W. T.; Dahms, N. M.; Kornfeld,
S. J. Biol. Chem. 1999, 274, 1164-1171.
(4) Yamada, T.; De Souza, A. T.; Finkelstein, S.; Jirtle, R. L. Proc. Natl.
Acad. Sci. U.S.A. 1997, 94, 10351-10355.
(5) (a) Tsujiuchi, T.; Sasaki, Y.; Tsutsumi, M.; Konishi, Y. Mol.
Carcinog. 2002, 36, 32-37. (b) Kong, F.-M.; Anscher, M. S.; Washington,
M. K.; Killian, J. K.; Jirtle, R. L. Oncogene 2000, 19, 1572-1578.
(6) Pavelic, K.; Kolak, T.; Kapitanovic, S.; Radosevic, S.; Spaventi, S.;
Kruslin, B. J. Pathol. 2003, 201, 430-438.
Consistent with the important role this receptor plays in
cell growth and motility, altered structure, function, or
expression of the CI-MPR is seen in a number of cancers,
† University of Nebraska, Lincoln.
‡ University of Nebraska Medical Center.
10.1021/ol0479444 CCC: $27.50
© 2004 American Chemical Society
Published on Web 12/03/2004

gous extracytoplasmic repeats, each of which resembles the
single such domain of the CD-MPR. Of these, there are two
known high-affinity M6P binding sites at domains 3 and 9.10
The receptor is likely a dimer,3,11 implying that ligands could
have accessibility to several M6P binding pockets. Just this
year, the first X-ray structures of an M6P-binding CI-MPR
fragment, domain 1-3 constructs, with and without bound
M6P, were reported.12
Native glycoprotein ligands for the M6P site(s) on the CI-
MPR bear mannose-rich, N-linked oligosaccharides that are
derived from the characteristic triantennary undecasaccharide
illustrated in Figure 1. In glycopeptide ligands that bind well
Figure 2. Bivalent M6P-bearing ligand (2a) due to Bock and its
lower-affinity congener (2b).
used bioorganic tool of this sort. It does show the sought-
after 3 orders of magnitude in RBA relative to M6P, and
for this reason, it has been postulated that this is the first
molecular “ruler” that spans two M6P sites on the receptor.3
However, a closer look at the Bock work15 reveals that 2b
binds 220-fold less well than 2a, suggesting that the
anthranoyl group in 2a contributes significantly to binding.
Thus, it is not so surprising that 2a fails to stabilize receptor
dimers and to accelerate IGF-II internalization by the CI-
MPR, characteristics of the high-affinity multivalent ligand
hGUS.3
Therefore, we set out to explore new approaches to M6P-
bearing model ligands. Our goals were to develop a strategy
that would provide for simple mono- and bivalent16 ligands
with variable tether lengths. We would eliminate phosphate
esters and amide bonds from our model ligands to build in
phosphatase and protease resistance. A cross-metathesis
(CM) approach employing allyl (or related) glycosides of
M6P-mimics appeared to be viable.17 As can be seen
Figure 1. Structure of high-mannose-type oligosaccharides as
found in N-linked glycoproteins. Green ) N-acetylglucosamine;
blue ) mannose (labeling follows Kornfeld ref 13); red ) possible
phosphorylation sites).
(7) Oates, A. J.; Schumaker, L. M.; Jenkins, S. B.; Pearce, A. A.;
DaCosta, S. A.; Arun, B.; Ellis, M. J. C. Breast Cancer Res. Treat. 1998,
47, 269-281.
(8) (a) Ludwig, T.; Eggenschwiler, J.; Fisher, P.; D’Ercole, A. J.;
Davenport, M. L.; Efstratiadis, A. DeV. Biol. 1996, 177, 517-535. (b) Wang,
Z.-Q.; Fung, M. R.; Barlow, D. P.; Wagner, E. F. Nature 1994, 372, 464-
467. (c) Lau, M. M. H.; Stewart, C. E. H.; Liu, Z.; Bhatt, H.; Rotwein, P.;
Stewart, C. L. Genes DeV. 1994, 8, 2953-2963.
(9) Olson, L. J.; Zhang, J.; Dahms, N. M.; Kim, J.-J. P. J. Biol. Chem.
2002, 277, 10156-10161 and refs cited therein.
(10) Hancock, M. K.; Yammani, R. D.; Dahms, N. M. J. Biol. Chem.
2002, 277, 47205-47212.
(11) (a) Byrd, J. C.; MacDonald, R. G. J. Biol. Chem. 2000, 275, 18638-
18646. (b) Byrd, J. C.; Park, J. H. Y.; Schaffer, B. S.; Garmroudi, F.;
MacDonald, R. G. J. Biol. Chem. 2000, 275, 18647-18656.
(12) (a) Olson, L. J.; Dahms, N. M.; Kim, J.-J. P. J. Biol. Chem. 2004,
279, 34000-34009. (b) Olson, L. J.; Yammani, R. D.; Dahms, N. M.; Kim,
J.-J. P. EMBO J. 2004, 23, 2019-2028.
(13) (a) Varki, A.; Kornfeld, S. J. Biol. Chem. 1980, 255, 10847-10858.
(b) Varki, A.; Kornfeld, S. J. Biol. Chem. 1983, 258, 2808-2818. (c) Tong,
P. Y.; Gregory, W.; Kornfeld, S. J. Biol. Chem. 1989, 264, 7962-7969.
(14) Distler, J. J.; Guo, J.; Jourdian, G. W.; Srivastava, O. P.; Hindsgaul,
O. J. Biol. Chem. 1991, 266, 21687-21692.
(15) Franzyk, H.; Christensen, M. K.; Joergensen, R. M.; Meldal, M.;
Cordes, H.; Mouritsen, S.; Bock, K. Bioorg. Med. Chem. 1997, 5, 21-40.
(16) For the only other study, of which we are aware, that targets simple
tethered bivalent M6P ligands, see: Lehmann, J.; Schweizer, F.; Weitzel,
U. P. Carbohydr. Res. 1995, 270, 181-189.
(17) (a) Chatterjee, A. K.; Morgan, J. P.; Scholl, M.; Grubbs, R. H. J.
Am. Chem. Soc. 2000, 122, 3783-3784. (b) Dominique, R.; Das, S. K.;
Roy, R. Chem. Commun. 1998, 2437-2438.
to the MPRs, the nonamannopyranose run has been phos-
phorylated and trimmed. In pioneering work, Kornfeld stud-
ied the various phosphorylation patterns one typically sees.13
It appears that bi- to multivalent M6P-bearing ligands bind
more tightly to the CI-MPR than do monovalent ones. Less
clear is how many M6P residues are needed and how they
are optimally spaced. Kornfeld’s early studies involved
isolation of native mixtures of mannose-rich oligosaccharides
from cellular glycoproteins. These studies revealed that most
Asn-linked oligosaccharides carry one or two phosphorylated
mannoses, with the latter binding more tightly than the
former. Later, elegant work by Hindsgaul revealed that one
could gain nearly an order of magnitude in binding to the
CI-MPR with pentasaccharides bearing two M6P residues,
as opposed to one.14 However, neither of these studies could
reproduce the high relative binding affinities (RBAs g 103
vs M6P) seen with native ligands such as hGUS (human
â-glucuronidase) or the synthetic glycoprotein, PMP-BSA
(pentamannose phosphate-bovine serum albumin).
This led Bock and co-workers to make a series of
glycopeptide ligands.15 Compound 2a is the most commonly
4922
Org. Lett., Vol. 6, No. 26, 2004

Scheme 1. Design and Retrosynthetic Analysis of a Bivalent
Scheme 3. Bivalent M6P-Type Ligand via a Triflate
Displacement/Alkene Cross-Metathesis Sequence
M6P-Presenting Liganda
a Y ) Phosphate surrogate functionality or leaving group
precursor.
(Scheme 1 and Figure 1), this approach has the further
advantage of providing direct access to bivalent ligands that,
in terms of spacing, map nicely onto trimannose regions of
the high-mannose oligosaccharide. Such model com-
pounds could thus serve as independent probes for specific
biantennary bisphosphorylation binding motifs.
Synthetically, the success of such an approach would
require either carrying a relatively polar phosphate surrogate
functionality into the CM reaction or its installation post-
CM. Given that we had in mind to exploit our sugar triflate
chemistry, as this had proven to be a versatile method for
introducing functionality at the 6-position of hexopyranoses,18
the latter approach raised the intriguing notion of synthesizing
and handling bis-sugar triflates.
Three phosphate surrogates were chosen, all diacids, (i) a
malonyl ether, (ii) a C2′-linked malonate, and (iii) a
phosphonate.19 To streamline the synthesis, a divergent
strategy was employed whereby methyl 2,3,4-tri-O-benzyl-
R-D-mannopyranoside (4) served as the precursor to all target
compounds. On one hand, Rh-mediated carbene insertion
chemistry produced 6. On the other hand, conversion of 4
to the primary triflate 8, followed by anionic displacements,
yielded the target malonate 9 and phosphonate 11.
Schemes 3 and 4 illustrate the successful application of a
CM approach to first generation bivalent M6P-mimetic
ligands. The requisite alkene was installed by methyl to allyl
glycoside interconversion under acidic conditions from 4.
Pleasingly, the R-anomeric stereochemistry was cleanly
obtained, probably due to a combination of the usual
anomeric effect and the ∆2 effect in mannose. Using the
Grubbs I catalyst, the cross-metathesis reaction proceeded
efficiently, whether the 6-position carried a free alcohol
(67%) or a protected malonate (84%). For the bivalent
phosphonate, not only could we cleanly obtain the bis-triflate
19, but its double displacement with dibenzyl lithiomethyl-
phosphonate proceeded very efficiently (75% ) 87% per
triflate displacement!).
Scheme 2. Installation of Phosphate Surrogate Functionalities
upon an R-Mannopyranosyl Core
Relative binding affinities to the CI-MPR were determined
by ligand displacement assays vs both the synthetic ligand
PMP-BSA and native receptor ligand hGUS (Table 1).
Analysis of the resulting displacement curves (see Supporting
(18) (a) Shen, Q.; Sloss, D. G.; Berkowitz, D. B. Synth. Commun. 1994,
24, 1519-1530. (b) Berkowitz, D. B.; Eggen, M.; Shen, Q.; Shoemaker,
R. K. J. Org. Chem. 1996, 61, 4666-4675. (c) Berkowitz, D. B.; Bose,
M.; Asher, N. G. Org. Lett. 2001, 3, 2009-2012.
(19) Compound 12 has been independently synthesized by others and
reported to show affinity comparable to M6P (both elute the receptor at 10
mM concentrations) for the CI-MPR via an affinity chromatography
assay: (a) Khanjin, N. A.; Montero, J.-L. Tetrahedron Lett. 2002, 43, 4017-
4020. (b) Vidil, C.; More`re, A.; Garcia, M.; Barragan, V.; Hamdaoui, B.;
Rochefort, H.; Montero, J.-L. Eur. J. Org. Chem. 1937, 2, 447-450.
Org. Lett., Vol. 6, No. 26, 2004
4923

Scheme 4 Bivalent M6P-Type Ligand via the Reverse
Sequence: Alkene Cross-Metathesis/Bistriflate Displacement
Table 2. Docking to CI-MPR Repeats 1-3
AutoDock-predicted ∆Gbinding (kcal/mol)
conformationa
M6P
G6P
7
10
12
1b
2c
-7.02
-7.14
-6.83
-6.51
-6.74
-6.74
-6.82
-6.48
-7.13
-7.18
a Conformation to which the ligand set was docked. b Conformation 1
denotes the coordinates of chain A from pdb 1SZ0. c Conformation 2 denotes
the receptor coordinates obtained upon performing a 500 ps molecular
dynamics simulation, with 1SZ0 and a terminally phosphorylated R-1,2-
linked mannobiose sugar (see Supporting Information for more details).
Significantly, our RBA results with the model bivalent
ligands, 17 and 21 (Table 1), show little to no effect of
bivalency here, suggesting that they cannot access two M6P
binding pockets. This is notable since these ligands map onto
specific trimannose sectors of a canonical N-linked high-
mannose glycosylation site (Figure 1 and Scheme 1).
Specifically, if one considers the three most prevalent patterns
of bisphosphorylation seen by Kornfeld in a classic early
study (Figure 3),13a our results suggest that pattern “I” (at
least insofar as it implies an Mc-Mf-Mh biantennary
bisphosphate), is not a high-affinity binding element for the
CI-MPR. This illustrates how this new bioorganic “measuring
tool” might be used to explore other potential bivalent
binding modes for mannose-rich glycoprotein ligands to this
receptor and others.
Information) allows for rather precise quantitation of both
IC50 and RBA. Several observations emerge from the data.
First, the RBAs obtained are quite consistent regardless of
radioligand, lending confidence in the data. Second, survey-
ing the phosphate surrogate functionalities examined, one
sees a significant SAR, with affinity increasing in roughly
order of magnitude steps as one goes from the malonyl ether
(7) to the malonate (10) to the phosphonate (12). The
deleterious effect of the insertion of the C6-oxygen (i.e.,
10f7) is reminiscent of observations made by Frost in
examining binding to DHQ synthase.20
Given that crystallographic coordinates are now available
describing the conformation of a domain 1-3 construct that
binds M6P,12a attempts were made to examine this same set
of M6P analogues, in silico, using AutoDock.21 While the
superiority of the phosphonate functionality is reproduced
(Table 2), neither of these computations sees the malonate
ether effect, nor do they predict the magnitude of the SAR
seen. Perhaps, this is an indication that binding to repeat 9
is important.
Figure 3. Three possible bis-M6P-presenting motifs for mannose-
rich glycoproteins from mouse lymphocyte (ref 13a).
Acknowledgment. The authors thank the Nebraska State
Department of Health for a seed grant (D.B.B.) and the NIH
(CA 91885; RGM) for support. D.B.B. thanks the Alfred P.
Sloan Foundation for a fellowship. B.D.C. acknowledges a
UCARE Undergraduate Research Fellowship. Mohua Bose
is thanked for early studies. This research was facilitated by
shared instrumentation grants for NMR (NIH SIG-1-510-
RR-06301, NSF CHE-0091975, NSF MRI-0079750) and
GC/MS (NSF CHE-9300831).
Table 1. Relative Binding Affinities for the M6P/IGF2R
radioligand
PMP-BSA
IC50 (n)a
hGUS
IC50 (n)a
ligand
RBAb
RBAb
M6P
G6P
7
0.049 ( 0.015 (6) 1.0
0.033 ( 0.005 (6) 1.0
>10 (6)
1.06 ( 0.22 (5)
>10 (6)
2.14 ( 1.19 (4)
nac
nac
0.041
0.024
Supporting Information Available: NMR spectra, char-
acterization data, and experimental procedures; binding
curves and protocols for the radioligand displacement assay;
and a description of docking methods and docked images.
This material is available free of charge via the Internet at
http://pubs.acs.org.
10
12
17
21
0.166 ( 0.090 (4) 0.30
0.025 ( 0.008 (5) 1.82
0.145 ( 0.083 (4) 0.39
0.020 ( 0.005 (6) 2.79
0.126 ( 0.032 (4) 0.35
0.015 ( 0.007 (4) 3.52
0.151 ( 0.019 (6) 0.226
0.014 ( 0.003 (4) 3.16
a The mM concentration of each compound that displaced 50% of the
indicated radioligand tracers in a binding assay utilizing bovine soluble
M6P/IGF2R immobilized on Sepharose 4B was calculated. Values in
parentheses denote the number of replicate experiments performed yielding
mean ( SEM for each compound. b Relative binding affinity was calculated
by dividing the IC50 value for each compound by that of mannose
6-phosphate (M6P). c Not applicable. Values for RBA of the negative
control, glucose 6-phosphate (G6P), cannot be calculated from these data.
OL0479444
(20) Tian, R.; Montchamp, J.-L.; Frost, J. W. J. Org. Chem. 1996, 61,
7373-7381.
(21) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W.
E.; Belew, R. K.; Olson, A. J. J. Comput. Chem. 1998, 19, 1639-1662.
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