Synthesis of multivalent lactose derivatives by 1,3-dipolar cycloadditions: selective galectin-1 inhibition

doi:10.1016/j.carres.2006.04.028

Carbohydrate Research

Volume 341, Issue 10, 24 July 2006, Pages 1353-1362

https://doi.org/10.1016/j.carres.2006.04.028 Get rights and content

Abstract

Acetylene derivatives of phenylalanine, phenethylamine and the multifunctional unnatural amino acids, phenyl-bis-alanine and phenyl-tris-alanine, were synthesized and functionalized with 2-azidoethyl β-d-galactopyranosyl-(1→4)-β-d-glucopyranoside via regioselective copper(I)-mediated 1,3-dipolar cycloaddition to give a panel of mono-, di- and trivalent lactoside derivatives. Evaluation of the compounds as inhibitors against the tumour- and inflammation-related galectin-1, -3, -4N, -4C, -4, -7, -8N and -9N revealed a divalent compound with a K_d value as low as 3.2 μM for galectin-1, which corresponded to a relative potency of 30 per lactose unit as compared to the natural disaccharide ligand lactose. This divalent compound had at least one order of magnitude higher affinity for galectin-1 than for any of the other galectins investigated.

Graphical abstract

Introduction

Galectins are defined by two criteria established in 1994:¹ ‘affinity for β-galactosides and significant sequence similarity in the carbohydrate-binding site …’, and now include about 14 proteins in mammals and many in other species.² Galectins are implicated in different biological events such as cancer3, 4, 5, 6, 7 and the regulation of immunity and inflammation.8, 9, 10, 11 In several of these biological events, glycoconjugate binding is crucial, which makes the development of galectin inhibitors important.

The recognition of glycoconjugates by lectins is typically characterized by low affinity and the biological systems use multivalent interactions to circumvent these low affinities, a phenomenon referred to as ‘the glycoside clustering effect’12, 13, 14 Several mechanisms may operate to enhance the relative potency of multivalent ligands. These processes include the intramolecular chelate effect, the intermolecular aggregative process and statistical effects. The intramolecular chelate effect appears when multivalent ligands occupy multiple binding sites within a protein. In contrast, when multivalent ligands bind to multivalent receptors, large cross-linked aggregates may form.¹⁵ The statistical effect comes from slower off rates due to higher local concentrations of the binding moiety (in our case lactose) around a single binding site.

Several research groups have exploited the idea of using multivalent compounds to find high affinity galectin ligands/inhibitors. Multivalent enhancement has been seen for galectin-1,16, 17 galectin-3,14, 16, 17, 18 galectin-4 (N-terminal)¹⁹ and galectin-5.¹⁷ In these studies, a large array of scaffolds have been examined for creating multivalent carbohydrate ligands including glycopolymers and glycodendrimers.20, 21, 22

To optimize and study the glycoside cluster effect between multivalent ligands and galectins, we have developed new scaffolds between the linked carbohydrate units based on polyfunctional unnatural amino acids like phenyl-bis-alanine (PBA) and phenyl-tris-alanine (PTA). These scaffolds have previously been used for the preparation of amino-alcohol ligands, cage molecules and dendrimers.23, 24, 25 Typically, the unnatural PBA and PTA amino acids have been further functionalized by peptide couplings to furnish more complex structures. An important advantage with the choice of PBA and PTA as scaffolds is that the two orthogonal functionalities (amine and ester) enable derivatization with two different molecular entities (e.g., a galectin ligand and a fluorescent tag). A drawback of using standard peptide coupling reactions for PBA and PTA functionalizations is that efficiencies and yields may vary significantly with the choice of coupling partner. In particular, carbohydrate carboxylic acid or amino derivatives that carry unprotected hydroxyl groups are often difficult to couple directly via amide bond formations. To avoid these problems, we turned our attention to other methods of attachment of unprotected galectin ligands to PBA and PTA.

Inspired by a multitude of successful copper catalyzed 1,3-dipolar cycloaddition reactions between terminal alkynes and azides, under mild conditions, for the synthesis of intricate carbohydrate structures and the ready availability of 2-azidoethyl β-d-galactopyranosyl-(1→4)-β-d-glucopyranoside, we set out to synthesize suitable alkyne precursors derived from the above PBA and PTA. Acetylene derivatives of phenylalanine and of phenethylamine were also prepared for comparative purposes. In the presence of copper(I), the acetylene derivatives were coupled with 1,4-regioselectivity20, 26, 27 with 2-azidoethyl β-d-galactopyranosyl-(1→4)-β-d-glucopyranoside. The ligands were evaluated as inhibitors in a competitive fluorescence polarization assay against galectin-1, -3 and -7. They were also evaluated against intact galectin-4, the C-terminal domain of galectin-4, as well as the N-terminal domains of galectin-4, -8 and -9.28, 29 In this paper, we report the synthesis of these ligands and the relative potency, as galectin inhibitors, of the multivalent ligands as compared with their monovalent homologues.

Section snippets

Synthesis of acetylenes

Amine precursors phenethyl amine, l-phenylalanine and d,l-phenylalanine methyl esters, and PBA and PTA methyl esters 6 and 7 were coupled with either of the two types of acetylenic precursors (Table 1). In the first case, propiolic amides 1,³⁰3, 4 and 10 were formed by coupling propiolic acid with the corresponding amine using 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) in dichloromethane (method A), as this activating agent has in our hands been found to give particularly

Conclusions

The copper-mediated 1,3-dipolar cycloaddition proved to be a convenient mode of attachment for the synthesis of unprotected carbohydrate ligands to PBA and PTA. The inhibitor multivalency proved to have a significant influence on the affinity of the ligands against galectin-1 and -4N. Galectin-1 showed a preference for ligands with a carbamate linker and a moderate, but significant, glycoside cluster effect with all multivalent ligands 16, 17 and 19 suggesting the formation of cross-linked

General methods

All commercial chemicals were used without further purification. Thin layer chromatography (TLC) was carried out on 60F₂₅₄ silica (Merck) and visualization was made by UV light followed by heating with aqueous sulfuric acid. Column chromatography was performed on silica gel (Amicon Matrex 35–70 μm, 60 Å). Reversed phase chromatography was performed on Waters Sep-Pack Vac 35cc C₁₈ columns. NMR experiments were recorded with Bruker ARX 300 MHz or Bruker DRX 400 MHz spectrometers at ambient

Acknowledgements

The authors would like to thank Barbro Kahl-Knutson for excellent help with fluorescence polarization analysis and galectin production, Susanne Carlsson for providing galectins 8N and 9N, and Christopher Öberg for providing the probe used for galectin-4. Support from the Lund University Research School of Medicinal Sciences, the Swedish Research Council, the programs ‘Glycoconjugates in Biological Systems’ and ‘Chemistry for the Life Sciences’ sponsored by the Swedish Strategic Research

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^†: Contributed equally to this article.

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Synthesis of multivalent lactose derivatives by 1,3-dipolar cycloadditions: selective galectin-1 inhibition

Abstract

Graphical abstract

Introduction

Section snippets

Synthesis of acetylenes

Conclusions

General methods

Acknowledgements

Cell

Biochimie

Tetrahedron: Asymmetry

Anal. Biochem.

Tetrahedron

Carbohydr. Res.

Tetrahedron: Asymmetry

Tetrahedron: Asymmetry

Glycoconjugate J.

Glycoconjugate J.

Glycoconjugate J.

Glycoconjugate J.

Glycoconjugate J.

Nat. Rev. Cancer

Glycoconjugate J.

Glycoconjugate J.

Glycoconjugate J.

Glycoconjugate J.

Acc. Chem. Res.