Synthesis of 1,2,3-triazole-linked galactohybrids and their inhibitory activities on galectins

Here a synthesis of novel galactose-1,2,3-triazole conjugates is described. The title compounds were obtained from 3-azido-3-deoxy-1,2:5,6-di-O -isopropylidene- α - D -galactofuranose via a copper catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction. It was demonstrated that the title compounds in their isopropylidene-protected form tend to chelate copper. The copper content can be diminished to 10 ppm by successive treatment with EDTA and Na 2 S followed by chromatographic purification. Acidic hydrolysis of the acetonide protecting groups provided water soluble galactohybrids that were tested for their affinity towards galectin-1 and galectin-3. The trimeric galactohybrid exhibited a 160-fold preference for galectin-3 binding with K d 50 μ M. One of the obtained disaccharides was characterized by X-ray analysis.


Introduction
Carbohydrate-protein interactions play many important roles in biochemical processes.These include lectin-carbohydrate recognition 1,2,3 and processes catalyzed by glycosidases, 4,5,6 glycosyltransferases 7 and glycogen phosphorylases. 8Very often modified carbohydrates are used to investigate these processes 9,10 and to create suitable inhibitors of the aforementioned enzymes.1,2,3-Triazole modified carbohydrates became easily available after the discovery of the Cu(I) catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction and quickly became a prominent class of non-natural sugars. 11The chemistry and biology of triazole modified sugars is dominated by triazolyl glycosides, 12,13 5-deoxy-5-triazolyl-furanoses 14 and 6-deoxy-6-triazolyl-pyranoses. 15evertheless, few 3-deoxy-3-triazolyl-and 3-deoxy-triazolylmetyl-derivatives of furanoses have been studied as glycosidase inhibitors, 16,17 but 3-deoxy-3-triazolyl-galactopyranose derivatives have proved to be excellent galectin inhibitors 18,19 with enhanced binding affinities if used as dimeric structures. 20ere we aimed to develop a user-friendly synthesis of novel C(3)-modified galactohybrids and to determine their affinities towards galectin-1 and galectin-3.The latter are involved in various pathological pathways (e.g.: metastasis, apoptosis, inflammatory response) and their selective inhibition might lead to the development of therapeutics. 21Since on many occasions multivalent ligands have shown enhanced protein binding affinities, 22 we designed the target structures as depicted in Figure 1.General formula A represents disaccharides with extended bistriazolyl-linkers that can be described as bivalent galectin inhibitors.General formula B corresponds to their higher order congeners.
Similarly, a combination of 1,3,5-triethynylbenzene (7) and penta-O-propargyl-β-D-glucose (9) 28 with a minimal excess of azidosugar 2 gave trivalent and pentavalent galacto-clusters 8 and 10 in 74 and 61% yields, respectively (Scheme 2).With di-, tri-and pentasaccharides in hand we were intrigued to determine their residual copper content.From a practical point of view it is much easier to analyze and if necessary repurify sufficiently lipophilic compounds 6a-h, 8 and 10 than their fully deprotected and watersoluble counterparts.There have been several reports dealing with specific removal of copper from click products.These include treatment of peptide-containing dendrimers with Na 2 S, 29 multiple extractions with solutions of EDTA, 30 dialysis against EDTA solution 31 and the use of copper chelating resins. 32Various flow techniques for elimination of copper traces have been developed as well. 30,33Careful control of copper content is necessitated by aspects such as limits on heavy metals in pharmaceutically active compounds, 30 product stability 34 and inhibition of enzymes by copper ions. 35,36Nevertheless, there are not many examples where the residual copper has been analyzed in the carbohydrate-triazole conjugates which were obtained via CuAAC reaction.In a few cases some of the aforementioned methods (e.g.EDTA wash) were used, albeit without any numeric data of the residual copper content in the final products. 37nalysis of our clicked products 6a-h, 8 and 10 revealed that after a simple extractive wash with brine the copper content in the solid material ranged from 20000 to 40000 ppm.Consecutive extractive EDTA washes diminished the copper content to levels ≤100 ppm in most cases.Only precipitation with Na 2 S followed by filtration through a silica gel pad and crystallization provided substances with copper content ≤5 ppm.In individual cases satisfactory results were achieved solely with the EDTA wash (entry 1, Table 2).In order to identify the main copper chelating structural motif, we prepared model compounds 13 and 14 and known substances 15 and 16 (Scheme 3). 17,38Compounds 13 (dimer) and 14 (monomer) contain an extra HO-group per monosaccharide unit.They were prepared via CuAAC reaction on azide 12.The latter was obtained in the diazotransfer reaction using amine 11 and trifluoromethanesulfonyl azide in the presence of catalytic amount of copper(II) sulfate. 24,39The molecular structure of disaccharide 13 was unambiguously established by its single crystal X-ray analysis (Figure 2).

Scheme 3. Synthesis of copper chelating model compounds.
A comparison of residual copper content in two representative title compounds ( 6a and 8) and model compounds is shown in Table 2.Additional chelating groups (e.g.HO-group) enhance the residual copper content (compound 14 versus 15).One can also observe that the dimeric structural motif characterized by the bis-triazolyl linker is responsible for enchanced copper chelating properties (compound 13 versus 14; entries 3 and 4, Table 2).This effect is even more pronounced when the carbohydrate residues are replaced by more lipophilic benzyl groups.It was not possible to diminish the residual copper content in compound 16 below 70 ppm, most probably due to the formation of micelle-like structures.Purified intermediates 6a-h, 8 and 10 (copper content <10 ppm) were submitted to acidic hydrolysis of their isopropylidene protecting groups.Several hydrolytic methods were tried: HCl/MeOH, H 2 O/AcOH/110 o C, CF 3 COOH/H 2 O.The latter, employing an aqueous solution of trifluoroacetic acid at ambient temperature, gave the cleanest transformation and produced watersoluble target compounds in quantitative yields (Scheme 4).Simple evaporation of the acidic solution under reduced pressure followed by lyophilization from water provided products 17a-k as colorless amorphous powders.Deprotection of 6g gave a mixture of 17h and 17i, with the latter arising from the post-hydrolytic decarboxylation of the Meldrum's acid moiety.To the best of our knowledge, this is the first time where galactopyranose-1,2,3-triazole conjugates have been prepared from the corresponding isopropylidene-protected galactofuranoses.
As expected, deprotection induced furanose-pyranose tautomerism and compounds 17a-k were obtained as a mixture of their α-and β-pyranose forms.This was demonstrated by 2D HMBC spectra that clearly revealed the correlations H-C(5)↔C( 1) and H-C(1)↔C (5).Due to the extended linker each sugar residue gave an "independent" NMR spectrum and it was impossible to establish the ratio between the α,α'-, α,β-and β,β'-forms of disaccharides 17a-i.Instead, each structural motif (3-deoxy-3-triazolyl-α-or β-D-galactopyranose) was characterized separately and the virtual ratio between all α-pyranose and all β-pyranose forms is given (see Experimental Section). 1 H-and 13 C-NMR analysis of 17a is given in Figure 3.It represents the spectral properties of all galactohybrids 17a-k as the linkers apparently do not significantly influence the conformations of the galactopyranose moieties.The α-anomer is characterized by   The binding affinities of galectin-1 and -3 for selected compounds 17 were estimated by a fluorescent anisotropy assay. 40,41,42Compounds depicted in Table 3 were tested up to 5 mM concentrations and gave K d values in the range of 1-4 mM, except trisaccharide 17j.The latter exhibited enhanced galectin-3 binding with K d 50 μM and 160-fold preference for galectin-3.Galectins bind galactose with affinities of 5-20 mM. 43The observed K d value of galectin-3 for 17j is about 100-fold better than that of free galactose and can be regarded as remarkable for a molecule that does not possess a natural or pseudo-natural disaccharide structural motif.

Conclusions
We have developed a straightforward synthesis of novel divalent and multivalent galactopyranose hybrids.It uses the CuAAC reaction of 3-azido-3-deoxy-1,2:5,6-di-Oisopropylidene-α-D-galactofuranose to assemble the molecular skeletons of the target compounds.Acidic hydrolysis of the acetonides induces formation of the final products in their pyranose form.We have also demonstrated that the 1,2,3-triazole-linked sugars chelate copper.
In this context careful control of the residual copper content is recommended if one deals with carbohydrate-1,2,3-triazole conjugates.Galectin-1 and galectin-3 exhibited interesting levels of binding affinities for the title compounds.Nearly identical binding affinities were found for bivalent galactohybrids bearing various linkers and pentavalent galactohybrid.It is interesting to note that the relatively simple trivalent molecule 17j showed 100-fold better binding to galectin-3 than the parent galactopyranose.The fact that it differs from other investigated molecules with the 4-aryl-1H-1,2,3-triazol-moiety warrants further study.

Figure 1 .
Figure 1.General structures of bivalent galactohybrids (A) and their higher order congeners (B).

Table 1 .
Synthesis of disaccharides 6a-e according to Scheme 1

Table 2 .
Copper content in the carbohydrate-1,2,3-triazole conjugates depending on the purification procedure