Synthesis of heteroaryl 1-thio-β-D-galactofuranosides and evaluation of their inhibitory activity towards a β-D-galactofuranosidase

Heteroaryl 1-thio-β-D-galactofuranosides have been synthesized and evaluated as inhibitors of the exo-β-D-galactofuranosidase from Penicillium fellutanum. 2-Pyridinethiol, 4-pyridinethiol, 1methylimidazole-2-thiol, 5-methyl-1,3,4-thiadiazole-2-thiol, 2-pyrimidinethiol and 4,6-dimethyl2-pyrimidinethiol were employed as thiols, as such heteroaromatic aglycons are expected to display particular interactions with the active site of the enzyme. These thiols were condensed with per-O-benzoyl-D-galactofuranose, in the presence of SnCl4 or BF3.OEt2, followed by Odebenzoylation under mild conditions to afford the heteroaryl 1-thio-β-D-galactofuranosides in high yields. The enzymatic assays showed that 4,6-dimethyl-2-pyrimidyl 1-thio-β-Dgalactofuranoside was the best inhibitor (IC50 135 μM), considerable more potent than the analogue lacking the methyl groups in the aglycon moiety.


Introduction
The synthesis of thioglycosides has received considerable attention because of their activity as inhibitors1 and inducers of glycosidases,2 and also because of their use as glycosyl donors in the convergent synthesis of oligosaccharides.3 Our laboratory has long been interested in the enzymes related to galactofuranose glycobiology, as galactofuranosyl residues are found in glycoconjugates of pathogenic microorganisms, such as the bacteria Mycobacterium tuberculosis and M. leprae,4,5 trypanosomatids like Trypanosoma cruzi and Leishmania,6-8 and fungi like Paracoccidioides brasilensis,9 but it is absent in mammalian cells.Therefore, the metabolic pathways involved in the biosynthesis and degradation of these microbial glycoconjugates, are attractive targets for the development of antimicrobial agents.
The enzymes responsible for the incorporation of galactofuranosyl units are the UDPgalactopyranosylmutase (EC 5.4.99.9),10-12 which converts UDP-Galp into the donor UDP-Galf, and a UDP-galactofuranosyltransferase, which is responsible for the incorporation of the sugar into the glycoconjugates.13 The galactofuranosyl content of these glycoconjugates varies in vivo.For example the presence of β-D-Galf units in the mucins of the parasite T. cruzi depends on the strain.14Also in T. cruzi, the main difference between the epimastigote form (the divisible form in the midgut of the insect host) and the trypomastigote form (the invasive form) is the Galf content in the glycoinositolphospholipids (GIPLs), the most abundant cell-surface molecules.15 The degradation of the GIPLs could be involved in the parasite differentiation, what suggests that the inhibition of this metabolic pathway could prevent the evolution of the parasite to the infective form.These structural variations are attributed to the action of β-Dgalactofuranosidases.
Penicillium fellutanum is a non pathogenic fungus which produces an extracellular peptidophosphogalactomannan (pPGM) containing terminal β-D-Galf units.The percentage of this sugar decreases with the age of the culture as result of the action of an exo-β-Dgalactofuranosidase.16,17 The enzymes of this fungus related to galactofuranose glycoconjugates, have become model enzymes for us.We developed the synthesis of galactofuranose derivatives, as tools for their characterization, which once optimized could be used for the study of analogue enzymes in other microorganisms.Thus, we have synthesized chromogenic18,19 and radioactive substrates,20,21 and deoxygenated analogues of galactofuranosides.[22][23][24] We reported the synthesis of the alkyl, benzyl and aryl 1-thio-β-D-galactofuranosides 1-625 and nucleosides 7-826 (Figure 1), and their activities as exo-β-D-galactofuranosidase inhibitors have been proved.We also demonstrated that D-galactono-1,4-lactone (9) is and efficient inhibitor of such enzyme,25 and we developed an affinity chromatography system for the purification of β-D-galactofuranosidases by immobilization of 6, using lactone 9 for the elution of the enzyme.27 We studied the influence of the inhibitors on the culture of Penicillium fellutanum,28 which produces a galactofuranosidase.17 The development of such tools allowed us to detect β-D-galactofuranosidase activity in T. cruzi for the first time.29 The growing interest in galactofuranose glycobiology prompted us to explore the inhibitory activities of new thioglycosides, such as heteroaryl 1-thio-β-D-galactofuranosides.We expect that these compounds could present an increased inhibitory activity as result of different interactions of the aglycon heteroatoms with the active site of the enzyme.Heteroaryl 1-thio-β-D-glycofuranosides were also synthesized by Plusquellec30,31 and they were used as donors for the synthesis of D-glycofuranosyl 1-phosphates, according to a procedure based on the "remote activation concept".32 We now report the synthesis of heteroaryl 1-thio-β-Dgalactofuranosides and their activity as inhibitors of the exo-β-D-galactofuranosidase from P. fellutanum.

Results and Discussion
In contrast to glycosyltransferases, glycosidases are characterized for a broad substrate specificity.Compounds with considerable structural differences with respect to the natural substrates, can be hydrolysed with a characteristic kinetics,33 or act as inhibitors of the glycosidase.1However, the exo-β-D-galactofuranosidase from P. fellutanum is highly specific, and its activity strongly depends on the size and polarity of the aglycons, as well as the glycon structure.For example, 4-nitrophenyl β-D-galactofuranoside (10, Figure 1)18 is a good substrate for this enzyme (KM 0.31 mM),27 whereas 4-methylcoumarin-7-yl β-D-galactofuranoside (11) 19, with similar polarity but considerably bulkier, was not hydrolyzed by the enzyme.34On the other hand, compound 6 was more potent than 5 as a galactofuranosidase inhibitor,25 proving the polarity dependence.The nucleoside 8 (IC50 0.10 mM) was ten times more active than the analogue 7, suggesting that the change in the flexibility of the imidazolidine ring facilitates the interaction with the active site of the enzyme.26 The importance of the glycon structure was first evidenced by the fact that α-L-arabinofuranosides, the pentosyl homologues, were not hydrolyzed by the enzyme,17 and later we found that galactofuranosyl analogues deoxygenated at positions 2, 3 or 6 were resistant to the hydrolytic activity of the exo-β-D-galactofuranosidase, indicating that the hydroxyl groups at these positions are essential for the interaction with the enzyme.[22][23][24] Considering the specificity of this enzyme, we selected thiols with certain size for the synthesis of new thiogalactofuranosides as potential inhibitors.Thus, glycosylations of 2pyridinethiol, 4-pyridinethiol, 1-methylimidazole-2-thiol, 5-methyl-1,3,4-thiadiazole-2-thiol, 2pyrimidinethiol and 4,6-dimethyl-2-pyrimidinethiol were conducted.
The 13C NMR spectra of compounds 14a-19a, 14b-16b, 18b and 19b (Table 1), showed the characteristic β-D-galactofuranosyl pattern of signals, with those corresponding to C-2 and C-4 appearing over 80 ppm, and the anomeric signals shifted upfield in comparison with the analogue O-glycofuranosides, due to the shielding effect of sulfur atom.36The 1H NMR spectra of 14a and 16a-18a (Table 2) were similar to those previously described for per-O-benzoylated 1-thio-β-D-galactofuranosides,25 but with the anomerical signals significantly shielded downfield as effect of the heteroaryl aglycons.Compounds 14a and 16a-18a, showed vicinal coupling constants characteristic of conformations of the 1E-1T0-E0 segment of the pseudorotational itinerary, having the anomeric substituent in a quasi-axial position.37 In contrast, compounds 15a, 19a and the free thioglycosides 14b-16b, 18b and 19b, showed vicinal coupling constants suggesting a conformational shift towards the 3E-2T3-E3 region of the circle, with the anomeric sulfur atom in a quasi equatorial disposition.Previous protocols for the evaluation of the thioglycosides as inhibitors of β-Dgalactofuranosidase, were followed.4-Nitrophenyl β-D-galactofuranoside (10) was used as substrate, and lactone 9 was tested as a reference inhibitor (IC50 0.10 mM). 25 The enzymatic reaction was performed in the presence of thioglycosides 14b-16b, 18b and 19b at concentrations ranging from 0.15 to 1.25mM.We found that compounds 14b-16b, 18b are weak inhibitors (Figure 2), whereas 19b is a stronger inhibitor (IC50 135 µM).Interestingly, the structural difference between pyrimidyl derivatives 18b and 19b is the presence of the 4'-and 6'-methyl groups in the latter.These subtituents should favor particular interactions in the active site of the enzyme, resulting in an enhancement of the inhibitory activity.Although the IC50 value is moderate, there are not reports about more potent inhibitors of this enzyme.Recent progresses in the tridimensional analysis of glycosidases and their crystalline structures have contributed to the rational design of enzyme inhibitors.Unfortunately, none of the β-D-galactofuranosidases have been yet sequenced nor crystallized.Therefore, the synthesis and evaluation of 1-thio-β-D-galactofuranosides as those described here can contribute to the understanding of how the enzyme works.Furthermore, evaluation of these compounds as inhibitors of galactofuranosyltransferases, and of their benzoylated precursors as glycosyl donors, are in progress.

De-O-benzoylation. General procedure
To a solution of 14a-16a, 18a or 19a (0.7 mmol) in dried CH2Cl2 (10.0 mL) at 0 ºC, 0.5 N NaOMe/MeOH (5.0 mL) was added.The solution was stirred under argon during 30 min.Then, the solution was diluted with methanol, concentrated under vacuum in order to eliminate the CH2Cl2, passed through a column of Dowex 50W (H+) and eluted with methanol.The solvent was removed under vacuum and the remaining methyl benzoate was eliminated by several coevaporations with water.The following compounds were obtained.1 and  2) in agreement with reported data.314,6-Dimethyl-2-pyrimidyl 1-thio-β-D-galactofuranoside (19b).0.20 g, 95 %, RF 0.66 (solvent d).An analytical sample was obtained by column chromatography purification (solvent

Figure 2 .
Figure 2. Effect of concentration of heteroaryl 1-thio-β-D-galactofuranosides on the enzymatic activity of β-D-galactofuranosidase from Penicillium fellutanum.Incubations were performed as described in the Experimental Section.The amount of 4-nitrophenol released from 4-nitrophenyl β-D-galactofuranoside was determined as a measure of galactofuranosidase activity.The numbers indicate the heteroaryl 1-thio-β-D-galactofuranoside added.