Protein subtype-targeting through ligand epimerization: Talose-selectivity of galectin-4 and galectin-8

Article abstract of DOI:10.1016/j.bmcl.2008.05.066

A series of O2 and O3-derivatized methyl β-d-talopyranosides were synthesized and evaluated in vitro as inhibitors of the galactose-binding galectin-1, -2, -3, -4 (N- and C-terminal domains), 8 (N-terminal domain), and 9 (N-terminal domain). Galectin-4C and 8N were found to prefer the d-talopyranose configuration to the natural ligand d-galactopyranose configuration. Derivatization at talose O2 and/or O3 provided selective submillimolar inhibitors for these two galectins.

Full text of DOI:10.1016/j.bmcl.2008.05.066

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3691–3694
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Protein subtype-targeting through ligand epimerization: Talose-selectivity
of galectin-4 and galectin-8
Christopher T. Öberg a, Helen Blanchard b, Hakon Leffler c, Ulf J. Nilsson a,
*
a Organic Chemistry, Lund University, PO Box 124, SE-221 00 Lund, Sweden
b Institute for Glycomics, Gold Coast Campus, Griffith University, Qld 4222, Australia
c Section MIG, Department of Laboratory Medicine, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of O2 and O3-derivatized methyl b-D-talopyranosides were synthesized and evaluated in vitro as
Received 10 March 2008
Revised 15 May 2008
Accepted 16 May 2008
Available online 20 May 2008
inhibitors of the galactose-binding galectin-1, -2, -3, -4 (N- and C-terminal domains), 8 (N-terminal
domain), and 9 (N-terminal domain). Galectin-4C and 8N were found to prefer the -talopyranose con-
figuration to the natural ligand -galactopyranose configuration. Derivatization at talose O2 and/or O3
provided selective submillimolar inhibitors for these two galectins.
D
D
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Galectin
Talopyranose
Inhibitor
The family of about 15 different galectin proteins is character-
ized by high sequence homology and galactose-binding properties,
which in general are related to their functions.1,2 The functions of
galectins have been discovered to be mainly in regulating inflam-
matory processes3–5 and in cancer growth and metastasis.6–10
The last few years have witnessed coherent pictures emerging on
the mechanisms behind galectins’ apparently multi-faceted influ-
ences on inflammation and cancer through modulating apoptosis,
cell adhesion, angiogenesis, growth factor signaling, and differenti-
ation. Di- or multimerization of galectins allows for ligand cross-
linking and lattice formation,11 which is believed to orchestrate
receptor half-lives on cell surfaces.12–15 A recent important exam-
ple of this is that differences in cell-surface glycosylation patterns
are decisive for galectin-1 lattice formation and subsequent T-cell
apoptosis.16 Intracellularly, galectins have been shown to direct
raft-independent apical protein sorting17 and intracellular target-
ing.18 Altogether, a majority of the mechanisms depend on carbo-
hydrate ligand binding, which strongly suggest that the
carbohydrate-binding activity of galectins is an attractive thera-
peutic target.19 This hypothesis is supported by in vivo experimen-
tal observations that inhibition of galectin functions suppresses
cancer growth.20,21
side chains.22 Noteworthy is that a large number of these polar
amino acids are arginines that line up alongside the natural ligands
to form hydrogen bonds (Fig. 1). However, the fine-structure in
positioning these polar side chains differs between the galectins.
An additional characteristic feature of galectin/ligand complexes
is that the core galactose O2 does not interact with the protein,
but rather extends out into the surrounding environment.
Following the observation that galactose O2 is directed out from
the galectin perpendicular to the arginines that form polar interac-
tions with ligands,
fold for the design of novel galectin inhibitors. The inverted C2
configuration, relative to -galactose, offers possibilities for install-
D-talopyranose emerged as an attractive scaf-
D
ing affinity-enhancing talose O2 substituents engaging in previ-
ously inaccessible interactions with polar amino acid
functionalities. As the galectins typically have two or three argi-
nine residues interacting with ligands, talose O2-substituents cho-
sen should be electron-rich. The O2-substituted talosides should
preferably also carry aromatic O3-sustituents due to the proven
affinity-enhancing effects on several of the galectins by such sub-
stituents.25,28–32 Furthermore, because talosides appear not to be
naturally present in mammalians, and hence no endogenous ta-
lose-processing enzymes are present, they may offer desirable
hydrolytic stability in talose-based drug candidates—the hydrolytic
lability is an often-quoted drawback of carbohydrate-based drugs.
Herein, the synthesis of a series of methyl 3-O-(4-methylbenzoyl)-
The galectins are highly conserved and share a common recog-
nition motif in that the
a-face of the core galactoside residue of
natural ligands stack face-to-face with a tryptophane side chain,
while the b-face galactose hydroxyls form hydrogen bonds to polar
b-D-talopyranoside O2 derivatives and their in vitro evaluation as
inhibitors of galectin-1, -2, -3, -4N (N-terminal domain), -4C (C-ter-
minal domain), -8N (N-terminal domain), and -9N (N-terminal do-
main) are presented.
* Corresponding author. Tel.: +46 462228218; fax: +46 462228209.
E-mail address: ulf.nilsson@organic.lu.se (U.J. Nilsson).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.066

3692
C. T. Öberg et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3691–3694
from 6 by pivaloyl chloride-mediated coupling of benzyl alcohol
and methanol, respectively, followed by iodine oxidization and
water/pyridine hydrolysis.34 Deprotection of 1 with Dowex 50X8-
400 in methanol gave methyl b-D-talopyranoside 9, while treatment
of 2–8 using aqueous acetic acid gave 10–16.
Evaluation of talosides 9–16, together with methyl b-D-galacto-
pyranoside 17 as reference, against galectin-1, -2, -3, -4N, -4C, -8N,
and -9N was performed in a competitive in vitro fluorescence
35–37
polarization assay (Table 1).
In general, but for a few notable
exceptions, the galactose-binding galectins did not bind or bound
only weakly to the talosides 9–16.
Although galectin-1 neither bound the underivatized taloside 9
nor the reference galactoside 17, talose 3-O-toluolyation gave
weak but detectable binding when O2 was unsubstituted (10) or
carried an aromatic substituent (11 and 15). This shows that ta-
lose-binding by galectin-1 is sensitive to the structure of the O2
substituent, which suggests that optimization of this substituent
may provide an avenue towards improved galectin-1 inhibitors.
Galectin-2 recognized the galactoside 17 better than the corre-
sponding taloside 9. Furthermore, none of the 3-O-substituted talo-
sideswererecognizedbythisgalectin, exceptfor theH-phosphonate
14 and the benzyl phosphate 15, which were weakly bound.
Galectin-3 did indeed bind the talose derivatives, although the
talopyranose configuration (i.e., 9) was 2- to 3-fold worse than
the galactopyranose configuration (i.e., 17). Interestingly, the
galectin-3 preferences for the talose O2-substitutents parallels
those for the analogous O2-substituted galactosides37 in that the
O2-sulfate 13 bound significantly stronger than other O2-substitu-
ents. This observation most likely reflects that both galactose and
talose O2-substituents interact similarly with galectin-3 Arg144.
While galectin-4N did not bind any of the talosides 9–16, galec-
tin-4C bound the taloside 9 somewhat, but nevertheless signifi-
cantly, better than the galactoside 17. Furthermore, O3-
toluoylation (10) greatly improved the affinity, while O2-substitu-
tion had marginal effect except for the 2-O-toluoate 11. The affinity
Figure 1. The galactose-binding sites23 of human galectin-1,24 -3,25 -8N,26 and
-9N27 complexed with lactose or lacNAc (a–d). Galectin-8N is a homology model,
while the remaining galectins shown are crystal structures. Yellow arrows indicate
the core galactose C2–H2 bond direction, which corresponds to a talose C2–O2
bond direction. (e) Talose HO2 (bold-face) is directed towards basic amino acid side
chains of galectins.
The key 3-O-toluolyated intermediate 2 was obtained in a good
70% yield by a selective acylation on the equatorial O3 of the known
benzylidene-protected methyl b-D
-talopyranoside 133 (Scheme 1).
By employing a small excess of acid chloride, the 2,3-O-di-toluoly-
ated 3 could be isolated from the same reaction. Acetylation of 2
gave 4 and sulfation of 2 to give 5 was uneventful as well. PCl3-med-
iated installation of the H-phosphonate 6 under strictly anhydrous
conditions went smoothly. Subsequently, 7 and 8 were obtained
of 11 for galectin-4C (Kd 160 lM) is indeed remarkable, because 11
is a monosaccharide that should be compared to the virtually non-
binding prototype galectin monosaccharide ligand 17. Hence, the
taloside 11 can be estimated to display more than two orders of
Scheme 1. Reagents and conditions: (a) 4-Toluoyl chloride, pyr. (2: 70% and 3: 30%), (b) AcCl, pyr., CH2Cl2. (c) SO3ÁNMe3, DMF (86%). (d) PCl3, CH2Cl2, MeCN, Imidazole, Et3N,
pyr., H2O. (e) i—BnOH, pivaloyl chloride, pyr.; ii—I2, H2O, pyr. (82% from 2). (f) i—MeOH, pivaloyl chloride, pyr.; ii—I2, H2O, pyr.; (g) Dowex 50X8-400, MeOH (80%); (h) AcOH
(aq 70%), 70 °C (10: 84%, 11: 67%, 12: 91% from 2, 13: 75%, 14: 89% from 2, 15: 57%, 16: 65% from 2).

C. T. Öberg et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3691–3694
3693
Table 1
Galectin dissociation constants (mM) for compounds methyl b-D-galactopyranoside 17 and 9–16 as measured by a fluorescence polarization assay35–37
Compound
Galectin
4N
1
2
3
4C
8N
6.3 1.5
1.5 0.5
0.40 0.13
3.6 0.1
>4
1.1 0.3
1.5 0.5
2.0 0.3
9N
17
9
>10b
>10
13 3a
>10
4.4 0.8
10 3.6
1.4 0.6
0.70 0.18
0.55 0.15
0.25 0.08
2.3 0.6
6.6 0.2
>10
>4
>4
>4
>4
>4
>4
>4
>10
10 2.0
1.2 0.2
4.6 0.1
>4
>4
>4
>4
>4
>4
>4
10
11
12
13
14
15
16
ꢀ2
>4c
1.4 0.7
0.16 0.06
1.6 0.1
3.0 0.2
>4
1.8 0.6
>4
>4
>4
>4
1.5 0.5
4.6 2.0
>4
>4
>4
4.4 1.2
>4
2.5 1.3
0.60 0.33
3.8 0.2
>4
1.0
0
a
Mean values and standard deviations are from 2 to 12 single-point measurements (nd, not determined).
Compounds 9 and 17 were evaluated at concentrations up to 5 mM and non-inhibitory compounds are estimated to have Kd > 10.
Compounds 10–16 were evaluated at concentrations up to 2 mM and non-inhibitory compounds are estimated to have Kd > 4.
b
c
magnitude-improved affinity for galectin-4C, as compared to the
galactoside 17. This result holds promise for the development of
efficient monosaccharide inhibitors toward galectin-4C. Such
inhibitors may find use in in vivo situations, because activity of a
full-length tandem-repeat galectin (galectin-4) can be expected
to depend on both individual domains being fully functional, as
seen for galectin-8.26
‘Chemistry for Life Sciences’ sponsored by the Swedish Strategic
Research Foundation for financial support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.05.066.
Galectin-8N perhaps provided the most interesting results, be-
cause this galectin bound the taloside 9 about fourfold tighter than
the galactoside 17. Toluoylation at O3 (10) resulted in the first
submillimolar inhibitory activity discovered for a monosaccharide
against galectin-8N. However, all of the O2-substituents (11–16)
conferred decreased affinities. As galectin-8N indeed binds the
taloside configuration well, an investigation of other O2 structures
can be expected to lead to improved inhibitors. Again, inhibitors
against one domain (galectin-8N) can be expected to block in vivo
activity of the full-length protein (galectin-8) because function of
both domains (galectin-8 N- and C-terminal domains) is required
for activity. This has indeed been demonstrated for galectin-8
binding to cell surfaces.26
Finally, galectin-9N did not prefer the talopyranose configura-
tion, because this galectin bound the galactoside 17 four times
tighter than the taloside 9. Substitutions at O3 and O2 (10–16) de-
pleted binding to galectin-9N. For galectin-9 inhibition by talosides
to be viable, presumably talosides inhibiting the C-terminal do-
main have to be identified.
In conclusion, evaluation of the synthetic O2- and O3-substituted
talosidesfor galectin bindingprovidedinterestingclues to the devel-
opment of selective inhibitors. Galectin-4C and -8N did prefer the
talopyranose to the galactopyranose configuration, suggesting that
optimized and selective inhibitors of these two galectins may be ob-
tained based on a talopyranose scaffold. In this context, it is particu-
larly noteworthy that galectin-4C and -8N display submillimolar
affinity for talopyranosides 11 and 10, respectively, which is more
than one order of magnitude better compared to the prototype
galectin ligand 17. In light of the importance of galectin-8N in intra-
cellular sorting,18 neutrophile activation,38 and cancer39 and of
galectin-4 in cancer,40 the discovery of routes towards efficient
inhibitors of these galectins is particularly promising. Replacing gal-
actose by optimized talopyranoside residues in disaccharide mole-
cules (e.g., lacNAc,25,28 thiodigalactoside,31,32 or lactose, 41) and/or
attaching additional affinity enhancing structural elements can be
expected to provide improved inhibitory potencies.
References and notes
1. Barondes, S. H.; Castronovo, V.; Cooper, D. N. W.; Cummings, R. D.; Drickamer,
K.; Feizi, T.; Gitt, M. A.; Hirabayashi, J.; Hughes, C.; Kasai, K.; Leffler, H.; Liu, F. T.;
Lotan, R.; Mecurio, A. M.; Monsigny, M.; Pillai, S.; Poirer, F.; Raz, A.; Rigby, P. W.
J.; Rini, J. M.; Wang, J. L. Cell 1994, 76, 597.
2. Leffler, H.; Carlsson, S.; Hedlund, M.; Qian, Y. Glycoconjugate J. 2004, 19, 433.
3. Liu, F. T.; Hsu, D. K. Drug News Perspect. 2007, 20, 455.
4. Rhaleb, N. E.; Pokharel, S.; Harding, P.; Rasoul, S.; Peng, H.; Carretero, O. A. Am. J.
Pathol. 2008, 172, 247.
5. Henderson, N. C.; Mackinnon, A. C.; Farnworth, S. L.; Kipari, T.; Haslett, C.;
Iredale, J. P.; Liu, F. T.; Hughes, J.; Sethi, T. Am. J. Pathol. 2008, 172, 288.
6. Liu, F.-T.; Rabinovich, G. A. Nat. Rev. Cancer 2005, 5, 29.
7. Camby, I.; Le Mercier, M.; Lefranc, F.; Kiss, R. Glycobiology 2006, 16, 137R.
8. Lahm, H.; Andre, S.; Hoeflich, A.; Kaltner, H.; Siebert, H. C.; Sordat, B.; von der
Lieth, C.-W.; Wolf, E.; Gabius, H.-J. Glycoconjugate J. 2004, 20, 227.
9. Rabinovich, G. A.; Liu, F.-T.; Anderson, A. Scand. J. Immunol. 2007, 66, 143.
10. Saussez, S.; Kiss, R. Cell. Mol. Life Sci. 2006, 63, 686.
11. Nieminen, J.; Kuno, A.; Hirabayashi, J.; Sato, S. J. Biol. Chem. 2007, 282,
1374.
12. Demetriou, M.; Granovsky, M.; Quaggin, S.; Dennis, J. W. Nature 2001, 409, 733.
13. Partridge, E. A.; Le Roy, C.; Di Guglielmo, G. M.; Pawling, J.; Cheung, P.;
Granovsky, M.; Nabi, I. R.; Wrana, J. L.; Dennis, J. W. Science 2004, 306, 120.
14. Lau, K. S.; Partridge, E. A.; Grigorian, A.; Silvescu, C. I.; Reinhold, V. N.;
Demetriou, M.; Dennis, J. W. Cell 2007, 129, 123.
15. Rabinovich, G. A.; Toscano, M. A.; Jackson, S. S.; Vasta, G. R. Curr. Opin. Struct.
Biol. 2007, 17, 513.
16. Toscano, M. A.; Bianco, G. A.; Ilarregui, J. M.; Croci, D. O.; Correale, J.;
Hernandez, J. D.; Zwirner, N. W.; Poirer, F.; Riley, E. M.; Baum, L.; Rabinovich, G.
A. Nat. Immun. 2007, 8, 825.
17. Delacour, D.; Greb, C.; Koch, A.; Salomonsson, E.; Leffler, H.; Le Bivic, A.; Jacob,
R. Traffic 2007, 8, 379.
18. Carlsson, S.; Carlsson, M. C.; Leffler, H. Glycobiology 2007, 17, 906.
19. Pieters, R. J. ChemBioChem 2006, 7, 721.
20. John, C. M.; Leffler, H.; Kahl-Knutsson, B.; Svensson, I.; Jarvis, G. A. Clin. Cancer
Res. 2003, 9, 2374.
21. Thijssen, V. L.; Poirer, F.; Baum, L. G.; Griffioen, A. W. Blood 2007, 110, 2819.
22. Sujatha, M. S.; Balaji, P. V. Proteins: Struct., Funct., Bioinform. 2004, 55, 44.
23. DeLano, W. L.; DeLano ScientificLLC, Palo Alto, The figures were generated with
the PyMol software.
´
24. López-Lucendo, M. F.; Solıs, D.; André, S.; Hirabayashi, J.; Kasai, K.-i.; Kaltner,
H.; Gabius, H.-J.; Romero, A. J. Mol. Biol. 2004, 343, 957.
25. Sörme, P.; Arnoux, P.; Kahl-Knutsson, B.; Leffler, H.; Rini, J. M.; Nilsson, U. J. J.
Am. Chem. Soc. 2005, 127, 1737.
26. Carlsson, S.; Öberg, C. T.; Carlsson, M. C.; Sundin, A.; Nilsson, U. J.; Smith, D.;
Cummings, R. D.; Almkvist, J.; Karlsson, A.; Leffler, H. Glycobiology 2007, 17,
663.
Acknowledgments
27. Nagae, M.; Nishi, N.; Nakamura-Tsuruta, S.; Hirabayashi, J.; Wataksuki, S.; Kato,
R. J. Mol. Biol. 2007, 375, 119.
28. Sörme, P.; Qian, Y.; Nyholm, P.-G.; Leffler, H.; Nilsson, U. J. ChemBioChem 2002,
3, 183.
29. Salameh, B. A.; Leffler, H.; Nilsson, U. J. Bioorg. Med. Chem. Lett. 2005, 15, 3344.
We thank Lund University, the Swedish Research Council, and
the programs ‘Glycoconjugates in Biological Systems’ (GLIBS) and

3694
C. T. Öberg et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3691–3694
30. Salameh, B. A.;Sundin, A.; Leffler, H.;Nilsson, U. J. Bioorg. Med. Chem. 2006, 14, 1215.
31. Cumpstey, I.; Sundin, A.; Leffler, H.; Nilsson, U. J. Angew. Chem., Int. Ed. 2005, 44,
5110.
32. Cumpstey, I.; Salomonsson, E.; Sundin, A.; Leffler, H.; Nilsson, U. J. Chem. Eur. J.
2008, 14, 4233.
36. Cumpstey, I.; Carlsson, S.; Leffler, H.; Nilsson, U. J. Org. Biomol. Chem. 2005, 3,
1922.
37. Öberg, C. T.; Leffler, H.; Nilsson, U. J. J. Med. Chem. 2008, 51, 2297.
38. Nishi, N.; Shoji, H.; Seki, M.; Itoh, A.; Miyanaka, H.; Yuube, K.; Hirashima, M.;
Nakamura, T. Glycobiology 2003, 13, 755.
33. Knapp, S.; Kukkola, P. J. S. S.; Murali Dhar, T. G.; Naughton, A. B. J. J. Org. Chem.
1990, 55, 5700.
39. Zick, Y.; Eisenstein, M.; Goren, R. A.; Hadari, Y. R.; Levy, Y.; Ronen, D.
Glycoconjugate J. 2004, 19, 517.
34. Lindh, I.; Stawinski, J. J. Org. Chem. 1989, 54, 1338.
35. Sörme, P.; Kahl-Knutsson, B.; Huflejt, M.; Nilsson, U. J.; Leffler, H. Anal. Biochem.
2004, 334, 36.
40. Huflejt, M.; Leffler, H. Glycoconjugate J. 2004, 20, 247.
41. Cumpstey, I.; Salomonsson, E.; Sundin, A.; Leffler, H.; Nilsson, U. J.
ChemBioChem 2007, 8, 1389.