Parallel synthesis of glycomimetic libraries: targeting a C-type lectin.

Article abstract of DOI:10.1021/ol0340383

We have developed methods for the parallel synthesis of two libraries of non-carbohydrate-based analogues of mannose on a solid support. The natural product shikimic acid was used as a key building block. The ability of the compounds to block the binding of the C-type lectin MBP-A to a mannosylated surface was assessed in a high-throughput assay. Ten library members with inhibitory activities equivalent to that of alpha-methyl mannopyranoside were identified. [reaction: see text]

Full text of DOI:10.1021/ol0340383

ORGANIC
LETTERS
2003
Vol. 5, No. 9
1407-1410
Parallel Synthesis of Glycomimetic
Libraries: Targeting a C-Type Lectin
Michael C. Schuster, David A. Mann, Tonia J. Buchholz, Kathryn M. Johnson,
William D. Thomas, and Laura L. Kiessling*
Departments of Chemistry and Biochemistry, UniVersity of Wisconsin-Madison,
Madison, Wisconsin 53706
kiessling@chem.wisc.edu
Received January 8, 2003
ABSTRACT
We have developed methods for the parallel synthesis of two libraries of non-carbohydrate-based analogues of mannose on a solid support.
The natural product shikimic acid was used as a key building block. The ability of the compounds to block the binding of the C-type lectin
MBP-A to a mannosylated surface was assessed in a high-throughput assay. Ten library members with inhibitory activities equivalent to that
of r-methyl mannopyranoside were identified.
Carbohydrate-protein interactions are critical in physiologi-
cal and pathological processes.1 Consequently, synthetic
glycomimetics and oligosaccharides designed to inhibit these
interactions have been pursued as potential therapeutic
agents.2 Effective inhibitors have been identified for some
carbohydrate-protein binding events, but most of these are
derived from carbohydrate scaffolds.3 The potential lability
of synthetic carbohydrates toward in vivo glycosidases has
prompted the search for stable carbohydrate analogues
(glycomimetics) with potent biological activities.
We sought to develop non-carbohydrate-based glycomi-
metics targeted toward the C-type lectin family. The C-type
lectins are a group of Ca2+-dependent carbohydrate-binding
proteins, many of which are found in mammals.4 Human
C-type lectins include the mannose-binding proteins (MBPs),
the selectins (E-, L- and P-selectin), and DC-SIGN.5 These
proteins are all involved in immune system regulation.6
Studies of ligand-bound MBP-A, MBP-C, E-selectin, and
P-selectin by X-ray crystallography have revealed the
importance of a vicinal axial-equatorial-equatorial display
of hydroxyl groups on the carbohydrate ligand (Figure 1A).7
We sought a core structure that was capable of providing a
collection of diverse ligands and that could give rise to
(4) (a) Drickamer, K.; Taylor, M. E. Annu. ReV. Cell Biol. 1993, 9, 237-
264. (b) Drickamer, K. Curr. Opin. Struct. Biol. 1999, 9, 585-590. (c)
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(d) Long, E. O.; Wagtmann, N. Curr. Opin. Immunol. 1997, 9, 344-350.
(e) Weis, W. I.; Taylor, M. E.; Drickamer, K. Immunol. ReV. 1998, 163,
19-34.
(5) Geijtenbeek, T. B. H.; Torensma, R.; van Vliet, S. J.; van Duijnhoven,
G. C. F.; Adema, G. J.; van Kooyk, Y.; Figdor, C. G. Cell 2000, 100, 575-
585.
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2001, 294, 2163-2166.
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2000, 103, 467-479. (b) Hakansson, K.; Reid, K. B. M. Protein Sci. 2000,
9, 1607-1617. (c) Graves, B. J.; Crowther, R. L.; Chandran, C.; Rumberger,
J. M.; Li, S.; Huang, K.-S.; Presky, D. H.; Familletti, P. C.; Wolitzky, B.
A.; Burns, D. K. Nature 1994, 367, 532-538. (d) Weis, W. I.; Drickamer,
K.; Hendrickson, W. A. Nature 1992, 360, 127-134. (e) Weis, W. I.;
Drickamer, K. Structure 1994, 2, 1227-1240. (f) Torgersen, D.; Mullin,
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(1) Varki, A. Glycobiology 1993, 3, 97-130.
(2) (a) Koeller, K. M.; Wong, C. H. Nat. Biotechnol. 2000, 18, 835-
841. (b) Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357-2364.
(c) Kaila, N.; Thomas, B. E. Med. Res. ReV. 2002, 22, 566-601. (d) Arya,
P.; Barkley, A.; Randell, K. D. J. Comb. Chem. 2002, 4, 193-198. (e)
Nilsson, U. J.; Fournier, E. J. L.; Fryz, E. J.; Hindsgaul, O. Comb. Chem.
High Throughput Screening 1999, 2, 335-352.
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10.1021/ol0340383 CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/08/2003

Scheme 1. Solid-Supported Synthesis of Libraries 1 and 2a
Figure 1. (a) Mannose and fucose residues participate in binding
to C-type lectins via their axial-equatorial-equatorial vicinal
hydroxyl group. (b) Transformation of shikimic acid affords
products with the desired hydroxyl group arrangement.
a Conditions: (a) FMOC-(R1)-OH, HBTU, DIEA, DMF; (b)
piperidine, DMF; (c) shikimic acid, DIC, C6F5OH, HOBt, DMF;
(d) R2SH, KOt-Bu, t-BuOH, DMF; (e) TFA, Et3SiH, H2O,
HSCH2CH2SH; (f) HS-R3-SH, KOt-Bu, t-BuOH, DMF; (g) R4-Br,
KI, Et3N, DMF; (h) TFA, H2O.
ligands that target a range of lectins within the C-type lectin
superfamily. A key criterion for our scaffold was that it
should allow for the streamlined synthesis of libraries.8
We chose to build our library from the carbocycle shikimic
acid. Shikimic acid possesses three hydroxyl groups that are
displayed in a pseudoaxial-pseudoequatorial-pseudoequa-
torial configuration (Figure 1B). We envisioned that glyco-
mimetics could be generated by the stereoselective conjugate
addition of nucleophiles. Diversification could be achieved
by using different thiol building blocks and by elaboration
of the carboxylic acid. We chose to use thiolates as building
blocks because they are excellent nucleophiles, and the
corresponding conjugate addition reactions should proceed
under mild conditions. With proper stereochemical control,
conjugate addition could provide the requisite axial-
equatorial-equatorial display of vicinal hydroxyl groups
(Figure 1B).9 Multivalent derivatives of shikimic acid itself
have been used previously to inhibit a protein-carbohydrate
interaction.10 Because of the mode of C-type lectin ligand
binding, we surmised that the proposed transformations of
shikimic acid would yield more potent ligands.
the incoming nucleophile would approach the unsaturated
carbonyl compound on the si face opposite the allylic
hydroxyl group. The stereochemical outcome from proto-
nation of the incipient enolate, however, was more difficult
to predict. We found that when the addition was conducted
using a shikimic acid derivative with free hydroxyl groups,
the desired isomer was obtained. Under the optimized
conditions, thiolate addition occurs from the si face, followed
by axial protonation to afford the desired isomer (Figure 2).
With this knowledge, we set about to develop solid-phase
routes to the target compounds.
Figure 2. Desired stereochemical outcome was obtained from
thiolate conjugate addition to shikimic acid methyl ester. Protonation
of the incipient enolate on the other face affords a product that
would exist in a conformer in which the hydroxyl groups are
improperly oriented for C-type lectin binding.
To facilitate preparation of glycomimetic libraries, two
related solid-phase, parallel syntheses were envisioned
(Scheme 1). The key step in each strategy, the conjugate
addition of a thiolate to a shikimic acid derivative, was tested
in solution. At issue was the stereochemical outcome of the
conjugate addition reaction. Specifically, we anticipated that
In both of the synthetic routes developed, Rink amide
4-methylbenzhydrylamine polystyrene resin was utilized as
the synthetic support. It is stable toward a variety of reaction
conditions and delivers compounds with terminal amide
groups.11 To the immobilized amine was coupled either one
or two commercially available amino acids via standard
methods. Shikimic acid, which could be obtained via
fermentation12 or isolated from star anise, was then coupled
to the free N-terminal amine.13 Conveniently, shikimic acid
could be added without protection of its secondary hydroxyl
(8) (a) Bianco, A.; Brufani, M.; Manna, F.; Melchioni, C. Carbohydr.
Res. 2001, 332, 23-31. (b) Kim, C. U.; Lew, W.; Williams, M. A.; Liu,
H.; Zhang, L.; Swaminathan, S.; Bischofberger, N.; Chen, M. S.; Mendal,
D. B.; Tai, C. Y.; Laver, W. G.; Stevens, R. C. J. Am. Chem. Soc. 1997,
119, 681-690. (c) Kim, C. U.; Lew, W.; Williams, M. A.; Wu, H. W.;
Zhang, L. J.; Chen, X. W.; Escarpe, P. A.; Mendel, D. B.; Laver, W. G.;
Stevens, R. C. J. Med. Chem. 1998, 41, 2451-2460. (d) Lew, W.; Wu, H.
W.; Mendel, D. B.; Escarpe, P. A.; Chen, X. W.; Laver, W. G.; Graves, B.
J.; Kim, C. U. Bioorg. Med. Chem. Lett. 1998, 8, 3321-3324. (e) Williams,
M. A.; Lew, W.; Mendel, D. B.; Tai, C. Y.; Escarpe, P. A.; Laver, W. G.;
Stevens, R. C.; Kim, C. U. Bioorg. Med. Chem. Lett. 1997, 7, 1837-1842.
(9) Rich, R. H.; Lawrence, B. M.; Bartlett, P. A. J. Org. Chem. 1994,
59, 693-694.
(10) (a) Grandjean, C.; Rommens, C.; Gras-Masse, H.; Melnyk, O. J.
Chem. Soc., Perkin Trans. 1 1999, 2967-2975. (b) Grandjean, C.;
Angyalosi, G.; Loing, E.; Adriaenssens, E.; Melnyk, O.; Pancre, V.; Auriault,
C.; Gras-Masse, H. Chembiochem 2001, 2, 747-757.
(11) (a) Santini, R.; Griffith, M. C.; Qi, M. Tetrahedron Lett. 1998, 39,
8951-8954. (b) Adams, J. H.; Cook, R. M.; Hudson, D.; Jammalamadaka,
V.; Lyttle, M. H.; Songster, M. F. J. Org. Chem. 1998, 63, 3706-3716.
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Org. Lett., Vol. 5, No. 9, 2003

groups.14 The coupling was carried out using diisopropyl
carbodimide, hydroxybenzotriazole (HOBt), and penta-
fluorophenol. These conditions were employed to generate
an activated shikimic acid derivative soluble under the
reaction conditions. The key precursor for both synthetic
routes was readily accessible.
The two synthetic strategies diverged at this stage, although
both relied on nucleophilic addition of thiolate nucleophiles.
In the first strategy, monothiols served as building blocks.
Optimized reaction conditions involve incubation of the
resin-bound substrate with the thiol in the presence of KOt-
Bu. The stereochemical outcome for each addition reactions
carried out on the resin was identical to that obtained under
solution conditions.13 Six thiols, three commercially available
and three readily synthesized, were thus incorporated into
the library.13 Treatment of the resin-bound intermediates with
trifluoroacetic acid in the presence of carbocation scavengers
effected cleavage from the resin and removal of the protect-
ing groups from the amino acid side chains.15 A library of
72 compounds was synthesized using this route (Figure 3).
to those described above. The alkyl bromides used in the
synthesis of Library 2, or their direct precursors, were
available commercially.16 The thiolate alkylation reactions
were effected in the presence of triethylamine and potassium
iodide. Cleavage and amino acid side chain protecting group
removal afforded the isomeric products. A total of 120
compounds were generated through this approach (Figure
4).
Figure 4. Target library derived from synthetic route 2. DTE and
(()-DTT were used as dithiols. The resulting diastereomeric
mixtures were tested in inhibition assays.
We investigated the potency of the library members toward
the well-studied and structurally characterized C-type lectin
MBP-A. To this end, a bead-elution binding assay was
developed.17 This assay measures the ability of library
members to compete with fluorescein-labeled MBP-A from
a mannose-derivatized Sepharose resin. The MBP-A-bound
resin is exposed to varying concentrations of a library
member, and the amount of liberated fluorescein-MBP-A in
solution is assessed by fluorescence detection. A major
advantage of this method is that no radiolabels are required.
Active library members were identified by comparing IC50
values for the library components to that of the MBP-A
ligand, R-methyl mannopyranoside (R-MeMan).
Figure 3. Target library derived from synthetic route 1.
A second library was synthesized using a modification of
the original approach (Figure 2). Additional substituents were
incorporated by introducing dithiol building blocks via
conjugate addition. One thiol of the linker was used as the
nucleophile in the conjugate addition, and the other was
available for subsequent reaction with an alkyl bromide. The
reaction conditions for the conjugate addition of the dithiols,
dithiothreitol (DTT) or dithioerythritol (DTE), were identical
A screen of the 192-member collection was carried out
following cleavage of the compounds from the resin. Active
compounds were purified by HPLC. From these samples,
10 compounds were identified with potencies comparable
(12) Knop, D. R.; Draths, K. M.; Chandran, S. S.; Barker, J. L.; von
Daeniken, R.; Weber, W.; Frost, J. W. J. Am. Chem. Soc. 2001, 123, 10173-
10182 and the Supporting Information.
(13) See Supporting Information.
(14) (a) Tan, D. S.; Foley, M. A.; Shair, M. D.; Schreiber, S. L. J. Am.
Chem. Soc. 1998, 120, 8565-8566. (b) Tan, D. S.; Foley, M. A.; Stockwell,
B. R.; Shair, M. D.; Schreiber, S. L. J. Am. Chem. Soc. 1999, 121, 9073-
9087.
(15) Mehta, A.; Jaouhari, R.; Benson, T. J.; Douglas, K. T. Tetrahedron
Lett. 1992, 33, 5441-5444.
(16) For procedures for the synthesis of the electrophiles used that were
not commercially available, see Supporting Information.
(17) (a) Mann, D. A., Ph.D. Thesis, University of Wisconsin-Madison,
Madison, WI, 2000. (b) Buchholz, T. J. M. S. Thesis, University of
Wisconsin-Madison, Madison, WI, 2000.
Org. Lett., Vol. 5, No. 9, 2003
1409

position has only a minor influence on binding. The charged
amino acid side chains in the active ligands, however, are
all anionic; thus, they may engage in a Coulombic interaction
or hydrogen bond. With regard to the thiol substituent, all
of the active ligands possess a hydrophobic group at their
terminus. In both libraries, none of the compounds bearing
an anionic functional group at this position were found to
have activity. The identification of MBP-A ligands that
possess hydrophobic groups at this position suggests the
presence of a hydrophobic pocket near the carbohydrate
binding site. Significantly, ligands that bind as well as the
carbohydrates that bind MBP-A have been identified from
these libraries of modest size. These results indicate that
shikimic acid is useful in glycomimetic synthesis.
The glycomimetics described here are effective inhibitors
of a C-type lectin. Shikimic acid is a valuable building block
because its carboxylic acid group and conjugated alkene
allow for the incorporation of diversifying elements. These
elements may be used to distinguish between members of
the C-type lectin family. Thus, we anticipate that the
synthetic strategy outlined here may serve as a general
method for producing selective inhibitors of C-type lectins.
Acknowledgment. This research was supported by the
NIH (GM49975) and the Mizutani and W. M. Keck
Foundations. We thank Prof. J. Frost for suggesting shikimic
acid as a template and Prof. J. Frost and Dr. K. Draths for
a donation of shikimic acid. We thank Prof. K. Drickamer
for the plasmids used for MBP-A production and S. Reddy
for contributions to the assay. M.C.S. and T.J.B. were
supported by the NIH (Chemistry-Biology Interface Train-
ing Grant, GM08505) and the Molecular Biosciences Train-
ing Grant (GM07215). K.M.J. was supported by a Hilldale
Research Fellowship.
Figure 5. Compounds with activity in the MBP-A bead elution
assay from Libraries 1 and 2. The reference IC50 for R-MeMan in
this assay is 10 mM. An asterisk (*) indicates where compounds
synthesized with DTT or DTE afforded diastereomers that were
not separated.
Supporting Information Available: Experimental pro-
cedures for the synthesis of library members, description and
synthesis of thiol- and alkyl bromide library building blocks,
and NMR spectra for select compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
to that of R-MeMan for MBP-A (Figure 5). Shikimic acid
alone was inactive in the assay even at a concentration of
100 mM. These results indicate that members of the designed
library have the key structural features required for interaction
with the C-type lectins.
Analysis of the data reveals several trends. The amino acid
substituent of the active ligands varies, suggesting that this
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