Synthesis of galactofuranosyl-(1 → 5)-thiodisaccharide glycomimetics as inhibitors of a β-D-galactofuranosidase

Abstract

The first synthesis of methyl β-D-galactofuranosyl-(1 → 5)-thiofuranosides is reported. These molecules, which have the 6-deoxy-5-thio derivative of L-altrofuranose (16) or D-galactofuranose (18) as the reducing end, are mimetics of the motif β-D-Galf-(1 → 5)-D-Galf found in glycoconjugates of many pathogenic microorganisms. The conformational preferences of 16 and 18 in solution were assessed by means of molecular modeling and NMR techniques. These thiodisaccharides have been evaluated as inhibitors of the β-D-galactofuranosidase from Penicillium fellutanum. The kinetics of the inhibition showed that they behave as competitive inhibitors. As expected, compound 18 (K_i = 0.15 mM), with the same configuration for the reducing end as the natural substrate of the enzyme, was a stronger inhibitor than 16 (K_i = 2.23 mM).

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Introduction

Carbohydrate mimetics are usually prepared by replacing oxygen atoms in a sugar with carbon atoms or other heteroatoms. The modified molecules generally show altered binding properties or increased stability toward enzyme degradation, with respect to their natural counterparts.¹ Moreover, glycomimetics are useful tools for glycobiology, as they are employed to investigate recognition events that initiate immunological responses to bacterial and viral infections and in signaling processes that occur in inflammation and cancer metastasis.² The replacement of interglycosidic oxygen atoms in oligosaccharides by sulfur atoms leads to thiooligosaccharides. This structural modification usually induces resistance to enzymatic hydrolysis and, in many cases, inhibition of the activity of such enzymes, with the advantage that thiooligosaccharides are tolerated by most biological systems.³ The varied applications of thiooligosaccharides has stimulated investigations about their synthesis.^3,4 In this regard, we have contributed to the design and development of straightforward and stereoselective synthesis of thiooligosaccharides, and many of them have been evaluated as enzyme inhibitors.^5–7 The thioglycosidic linkage has been regio- and diastereoselective constructed using as key reactions the Michael addition⁵ and the epoxide⁶ or thiirane ring-opening.^6b,7 Among other topics we have focused our attention on the synthesis of thiooligosaccharides that possess a furanose sugar as constituent, since most methodologies reported are referred to the synthesis of thiooligosaccharides formed by pyranose units. Thus, we have described successful approaches to prepare S-disaccharides of 1-thiopentofuranose⁸ or 1-thiohexofuranose⁹ as non-reducing end; and the (1 → 6)-linked thiodisaccharide of galactofuranose (Galf) has also been prepared (Fig. 1).¹⁰ In addition, other researchers have reported the synthesis of analogues with the ring oxygen atom of Galf replaced by sulfur (4-thiohexofuranose, Fig. 1).¹¹

Fig. 1 Examples of sulfur-containing glycomimetics.

The interest in furanose-containing molecules arises from the fact that furanoses are widespread in nature, and particularly Galf occurs in glycoconjugates of bacteria, fungi, and protozoan parasites.¹² For example, the motif β-D-Galf-(1 → 5)-D-Galf is found in polyfuranosides of many pathogenic microorganisms, including Aspergillus and Mycobacterium.^12d,e The fact that Galf is present in structures that are considered to be essential for the survival or virulence of such microorganisms,^12,13 but is absent in higher eukaryotes, has attracted increasing interest on the biosynthetic pathways that involve this sugar,^13,14 the inhibition of the related enzymes as well as the synthesis of D-Galf containing molecules as biological probes.^12b,d,e,15

Degradation of Galf containing glycoconjugates is promoted in some microorganisms by a β-D-galactofuranosidase. Thus, a specific exo β-D-galactofuranosidase has been isolated from the culture medium of Penicillium fellutanum¹⁶ and later described in Helminthosporium sacchari¹⁷ and Penicillium and Apergillius species.¹⁸ This enzyme in P. fellutanum is responsible for the release of galactose by depolymerization of an extracellular glycopeptide that contains (1 → 5)-linked β-D-Galf units.¹⁶ In addition, the amount of Galf containing glycoconjugates is dramatically diminished during differentiation of Trypanosoma cruzi from the invasive to the infective stages¹⁹ and we have detected for the first time β-D-galactofuranosidase activity in this protozoo.²⁰ The inhibition of the enzymes involved in the metabolism of the polyfuranosides is expected to prevent the proliferation of T. cruzi, the agent of Chagas disease²⁰ and mycobacteria, including Mycobacterium tuberculosis, the agent of tuberculosis.^18b Furthermore, as to date the amino acid sequence for the catalytic site of the enzyme has not been identified, nor has the interactions with the substrate been determined, the development of new inhibitors can serve as tools for studying Galf processing enzymes.

We report here the synthesis of the methyl glycoside of the 6-deoxy-5-thio analogue of β-D-Galf-(1 → 5)-D-Galf, a rather common motif in polyfuranosides of pathogenic microorganisms. This thiodisaccharide was evaluated as inhibitor of the β-D-galactofuranosidase from P. fellutanum. The analogous thiodisaccharide which has opposite configuration for the stereocenter that carries the sulfur atom in the hexofuranose reducing end was also synthesized, in order to assess the influence of such a stereocenter on the inhibitory activity of the same enzyme.

Results and discussion

Synthesis of methyl 5-S-(β-D-galactofuranosyl)-6-deoxy-5-thio-α-L-altrofuranoside (16) and methyl 5-S-(β-D-galactofuranosyl)-6-deoxy-5-thio-β-D-galactofuranoside (18)

The target thiodisaccharides were designed using the 5-thiol derivatives 5 and 10 as key precursors. The synthetic route employed for the preparation of these two glycomimetics is depicted in Scheme 1. In the design of the target molecules we took into account that the deoxygenation at C-6 of galactofuranosyl derivatives increases the resistance to the hydrolytic activity of the β-D-galactofuranosidase from P. fellutanum.²¹ In addition, thiols 5 and 10 are expected to be readily prepared by reduction of the thiirane precursors 4 and 9, respectively, which have been previously synthesized in our laboratory starting from the α,β anomeric mixture of the uronate 1 via the diol 3.⁷ However, in this instance, we started from the anomerically pure glycoside 1, which was obtained from methyl(methyl β-D-galactofuranoside)uronate, the product of methanolysis of D-galacturonic acid.²² Compound 1 was silylated with an excess of tert-butyldimethylsilyl chloride (TBSCl) to give 2, which was treated with an excess of LiAlH₄ at −18 °C for 5 h and then the temperature was slowly increased until 0 °C. Under these conditions, the reduction of the methyl ester and the removal of the silyl ether at C-5 took place to give the diol 3 in 72% isolated yield. The regioselective tosylation of the primary hydroxyl group of 3, followed by epoxide formation and conversion of the epoxide into the thiirane group led to the 5,6-epithio-α-L-altrofuranoside derivative 4 (ref. 7) in 68% overall yield from 1. The ring-opening of the thiirane was accomplished by reduction of 4 with LiAlH₄ to afford the 5-thio derivative 5 (69%).

Scheme 1 Synthesis of 5-thiols 5 and 10 as glycosyl acceptors for the synthesis of the thiodisaccharides.

For the synthesis of the thiol 10, which has opposite configuration at C-5 with respect to 5, a double inversion of the configuration of the C-5 stereocenter of the starting compound 3 was required. For this reason, the tosylate leaving group was installed in the secondary hydroxyl group of 3. This was accomplished by regioselective acetylation of HO-6 to give 6 (79% yield) and subsequent tosylation of HO-5, to afford 7 (81%). Treatment of 7 with NaOMe/MeOH produced the O-deacetylation and nucleophilic attack of the resulting alcoxyde to C-5 to afford the epoxide 8 (91%) by displacement of the tosylate leaving group and the inversion of the configuration at C-5. The second configurational inversion of this stereocenter was achieved on treatment of epoxide 8 with thiourea, to give the thiirane 9 (88%), which belongs to the D-galacto series.

The reduction of the thiirane group of 4 and 9 with LiAlH₄ to the respective 5-thiol derivatives 5 and 10 was confirmed using the ¹H NMR spectra. The diagnostic H-5 signal appeared at high field because it was geminal to the thiol group, and showed coupling with the vicinal protons of SH, C-4 and the C-6 methyl group, which appeared as a doublet in the region of 1.4 ppm. The ¹³C NMR spectra of 5 and 10 were also in agreement with the proposed structures.

Having the thiols 5 and 10 in hand, the next step was the synthesis of the target glycomimetics 16 and 18. We have reported that the thioglycosidic bond can be constructed from the sugar thiols, with exclusive diastereoselectivity in favour of the β anomer of galactofuranosides, using as glycosyl donor the per-O-benzoyl-β-D-galactofuranosyl trichloroacetimidate (11),^7,10 which can be readily prepared from per-O-benzoyl-α,β-D-galactofuranose.²³ The glycosylation of thiol 5 (Scheme 2) was attempted using 11 as glycosylating agent and trimethylsilyl triflate (TMSOTf) as catalyst. Under the usual reaction conditions,^7,10 a rather complex mixture was obtained, and their components were isolated by column chromatography. The less polar product was the expected fully substituted thiodisaccharide 12, but the following fractions from the column afforded partially protected thiodisaccharides. Thus, an inseparable mixture of the 2-O-TBS and 3-O-TBS derivatives of the thiodisaccharide (13a,b) was obtained together with the fully O-desilylated product 14. The location of the TBS substituent in 13a and 13b was tentatively assigned by comparison of the ¹³CNMR spectrum of the mixture with respect to that of 14, according to the carbon signal (C-2 or C-3) that showed a stronger downfield shifting on monosylilation of 14.²⁴ On this basis, the ratio of 13a : 13b was determined approximately as 1 : 3. The structure of 14 was also confirmed by 2,3-di-O-acetylation to give 15, which was fully characterized.

Scheme 2 Synthesis of methyl 5-S-(β-D-galactofuranosyl)-6-deoxy-5-thio-α-L-altrofuranoside (16).

Treatment of compounds 12 or 13a,b with tetrabutylammonium fluoride (TBAF) in THF produced the complete removal of the TBS groups to afford the thiodisaccharide 14. To avoid the isolation of the silyl derivatives 12, 13a,b by column chromatography, the crude mixture of thioglycosylation was subjected directly to de-O-silylation by treatment with TBAF/THF to afford 14, which was purified by filtration through a short column of silica gel. The removal of the benzoyl protecting groups of 14 was performed using sodium methoxide in methanol, to give the free thiodisaccharide 16 in 41% yield from 5, after three steps.

The same route was applied for the synthesis of the thiodisaccharide 18 formed by two furanosyl units of galacto configuration S-(1 → 5) linked (Scheme 3). The glycomimetic 18 was isolated in 36% yield from 10.

Scheme 3 Synthesis of methyl 5-S-(β-D-galactofuranosyl)-6-deoxy-5-thio-β-D-galactofuranoside (18).

Conformational analysis

The structure of the thiodisaccharides 16 and 18 and their synthetic precursors and intermediates was confirmed on the basis of their NMR spectra, which were fully assigned with assistance of 2D-experiments. Thus, the ¹³C NMR spectra of compounds 12–18 showed the signal of the anomeric carbon of the reducing O-furanosyl unit shifted downfield (107.6–109.6 ppm) with respect to the same signals of the 1-thiofuranosyl moiety (96.9–89.3 ppm) due to the shielding effect of the sulfur atom over the vicinal magnetic nucleus. Furthermore, the ¹H NMR spectra (recorded in CDCl₃) of the thiodisaccharide derivatives 12–15 and 17 showed small coupling constant values for the signal of the protons of both furanose rings as they possess the same relative configuration. This behavior is characteristic of β-galactofuranosides.^7,9,10 The small J values suggests a conformation of both the O- and S-furanoside rings centered in the ¹T₀ ⇌ ¹E ⇌ ¹T₂ segment of the pseudorotational ring. Such a conformation is stabilized by the quasiaxial disposition of the anomeric substituent (anomeric effect) and the quasiequatorial orientation of the bulky lateral chain at C-4. However, the ¹H NMR spectra of the free thiodisaccharide 16 and 18, recorded in D₂O, showed a significant increment of the magnitude of the coupling constants for the thiofuranoside ring protons, while those of the O-furanosides remind similar to the one measured for 12–15 and 17 (Table 1). The larger J values suggest a tendency for all the trans protons of the Galf rings of 16 and 18 to occupy quasiaxial orientations. Hence, the conformational equilibrium seems to be shifted towards the ⁴T₃ ⇌ E₃ ⇌ ²T₃ segment. The conformational change may be attributed to the strength of the anomeric effect, which is less intense for S- than for O-glycosides,²⁵ and it should be even more weakened as the spectra were recorded in D₂O.²⁶ In the resulting conformation all the substituents of the ring are quasiequatorially disposed. Other O- and S-galactofuranosyl derivatives have shown a similar behavior.²⁷

Table 1 Coupling constant (J) values for the ring protons of selected compounds (12, 15, 16 and 18)

	J1,2 (Hz)	J2,3 (Hz)	J3,4 (Hz)	J1′,2′ (Hz)	J2′,3′ (Hz)	J3′,4′ (Hz)
12	1.2	1.2	5.2	<1	1.2	5.2
15	<1	1.1	5.6	<1	<1	4.3
16	1.4	2.6	—	5.4	5.4	7.5
18	1.7	3.2	6.3	4.9	5.3	7.4

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It was also interesting to determine the conformation of the thiodisaccharides 16 and 18, as the thioglycosidic linkage is located in a flexible region of the molecule. The conformation of many thiodisaccharides constituted by two pyranosyl residues has been studied using NMR methods complemented with theoretical calculations.²⁸ Interresidue NOE interactions have been employed to characterize the minimum energy conformations determined by molecular modelling.^28d However, as there are practically no examples of thiodisaccharides formed by furanose units, no conformational studies, apart from our own previous report,¹⁰ are available on this type of molecules. Such a conformational analysis is rather difficult as three bonds (C1′–S–C5–C4) are required to link the two furanose rings. This situation resembles that of the pyranosyl (1 → 6)-linked disaccharides, which also involves three bonds, and is more complex than that in disaccharides with glycosidic linkages to other positions of the pyranose (C2 to C4). Because of the additional C5–C6 exocyclic bond, a third angle (ω) is needed to describe all the possible orientations between the pyranose rings.²⁹ Similarly, we have defined the three torsion angles around the S-glycosidic linkage, which are the following: Φ = H1′–C1′–S–C5, Ψ = C1′–S–C5–C4 and ω = S–C5–C4–H4. The usual convention¹⁰ has been employed to establish the sign of the rotation of the dihedral angles.³⁰ Also for this preliminary study we performed molecular mechanics (MM+) and selected structures which showed minimum energy values were refined using a semiempirical method (AM1).¹⁰ The contribution to the conformational equilibrium of such low energy structures was experimentally confirmed by the detection of interresidue NOE contacts characteristic of a given conformer. The magnitude of the J_4,5 value, which is a function of the angle ω, was also considered.

For the thiodisaccharide 16, a minimum energy conformation was found for the syn Φ/syn Ψ/syn ω arrangement (A), which is characterized by the H1′–H4 NOE contact (Fig. 2). Since in the ¹H NMR spectrum of 16 the signals of H3 and H4 are overlapped, the observed NOE was attributed to the interaction of H1′–H4 as no low energy structures showing the H1′–H3 NOE have been found. The less intense NOE interaction H1′–H6, shown by this conformer, was also detected in the NOESY spectrum of 16. The presence in the conformational equilibrium of the syn Φ/anti Ψ/anti ω form (B) was confirmed by the intense H1′–H5 NOE. This interaction should also be detected for the low energy arrangement syn Φ/syn Ψ/anti ω (C), which should be present in the conformational equilibrium.

Fig. 2 Low energy conformations of 16, according to theoretical calculations and experimental NOE contacts (interresidue in black, intraresidue in red).

The average value for J_4,5 (4.5 Hz) for 16 is in agreement with the relative disposition of H4 and H5, which are anti in A, but syn-oriented in B and C. In addition, the experimentally detected intraresidue NOE cross peak between H4 and H6 protons of the terminal methyl group is in accordance with the proposed structures.

A similar conformational analysis was applied to the thiodisaccharide 18 (Fig. 3). As for the analogue 16, the syn Φ/syn Ψ/syn ω (D) was the lowest energy arrangement, according to theoretical calculations. The observed NOE contacts between H1′–H4 and H1′–H5 support experimentally the contribution of D to the conformational equilibrium. The other low energy structure syn Φ/syn Ψ/anti ω (E), which was also confirmed by the detected NOE interactions of H1′ with H5 and with the CH₃−6 protons. The third low energy rotamer F (syn Φ/anti Ψ/syn ω) arises by rotation around of the S–C5 linkage (Ψ angle) from D. The arrangement F is also characterized by the intense H1′–H5 NOE cross peak.

Fig. 3 Low energy conformations of 18, according to theoretical calculations and experimental NOE contacts (interresidue in black, intraresidue in blue).

The averaged value for the H4–H5 coupling constant (J_4,5 4.6 Hz, H4 and H5 are gauche in D and E, and anti in F) and the observed intraresidue NOE cross peaks H3–H6 (in D) and H4–H6 (in E and F) are in agreement with the conformations proposed.

Interestingly, no conformations other than the syn Φ, with Φ having a positive value, have been experimentally confirmed. Similar to other thiodisaccharides, all the conformations around the Φ angle theoretically found, and experimentally confirmed, are stabilized by the exo anomeric effect. These results are in agreement with previous reports³¹ that indicated that the conformational preferences of the glycosidic fragments are governed by steric interactions as well as stereoelectronic effects, such as the anomeric and exo anomeric effects.

Evaluation of the inhibitory activity

The natural substrate for the exo β-D-galactofuranosidase is the peptide phosphogalactomannan (pPGM), an extracellular glycopeptide from P. fellutanum containing terminal (1 → 5)-linked β-D-Galf units, attached to an α-mannose core.¹⁶ The enzyme, which is not commercially available, has been isolated in our laboratory from the culture growth of the fungus.¹⁵ Interestingly, the enzyme activity showed to be highly dependent on both the glycone and the aglycone structures.²²

As thiodisaccharides 16 and 18 are mimetics of the natural terminal disaccharide of the pPGM, they have been evaluated as inhibitors of the enzyme. For the inhibition studies, 4-nitrophenyl β-D-galactofuranoside was employed as substrate and the protocol previously established in our laboratory was followed.^10,25 The inhibitory profile was compared with those of the known inhibitor aldono-1,4-lactone (19, K_i = 0.10 mM) and the parent thiodisaccharide D-Galf-(1 → 6)-1-S-D-Galf (20, K_i = 3.62 mM).¹⁰ Compounds 16 and 18–20 were subjected to the enzymatic reaction, in concentrations ranging from 0.1 to 1.6 mM. Releasing of 4-nitrophenol was employed as a measurement of galactofuranosidase activity. The effect of the concentration of such compounds on the activity of the enzyme is shown in Fig. 4. In addition, the Lineweaver–Burk plots indicated that 16 (K_i = 2.23 mM) and 18 (K_i = 0.15 mM) are competitive inhibitors, as shown in Fig. 5 and 6. Compound 18 (IC₅₀ 0.15 mM), with the same configuration as the natural substrate of the enzyme, is about 15-fold a stronger inhibitor of the β-D-galactofuranosidase than the thiodisaccharide 16, with a L-altro configuration for the reducing end, and even more potent (24-fold) than the S-(1 → 6) linked disaccharide 20. These results demonstrate that the configuration of the aglycone moiety (D-Galf versus L-Altf) of the thiodisaccharide and the site of linkage (S-(1 → 5) versus S-(1 → 6)) have an strong influence on the inhibitory activity.

Fig. 4 Effect of the concentration of thiodisaccharides 16 and 18 on the enzymatic activity of the exo β-D-galactofuranosidase from Penicillium fellutanum. 4-Nitrophenyl β-D-galactofuranoside was used as substrate and D-galactono-1,4-lactone (19) as reference inhibitor. For comparison, the inhibition by thiodisaccharide 20 (ref. 10) was also included. Each point is the mean of three replicate experiments.

Fig. 5 Lineweaver–Burk reciprocal plot for the inhibition of the exo β-D-galactofuranosidase from P. fellutanum by thiodisaccharide 16 at concentrations: () 0.00, () 0.25, () 0.50 and () 1.00 mM. Each point is the mean obtained from three replicate experiments.

Fig. 6 Lineweaver–Burk reciprocal plot for the inhibition of the exo β-D-galactofuranosidase from P. fellutanum by thiodisaccharide 18 at concentrations: () 0.00, () 0.20, () 0.40 and () 0.60 mM. Each point is the mean obtained from three replicate experiments.

Conclusions

The regioselective reduction of the methyl glycosides of 5,6-epithio-α-L-altro- (4) or β-D-galactofuranoside (9) afforded the corresponding 6-deoxy-5-thio- derivatives 5 and 10 with very good yields. The thiol group of these two key intermediates was glycosylated using the per-O-benzoyl-Galf trichloroacetimidate (11) as glycosyl donor to afford, after removal of the protective groups of the hydroxyl functions, the glycomimetics of β-D-Galf-(1 → 5)-D-Galf. This motif is found in many pathogenic microorganisms, including bacteria, fungi and protozoan parasites.

The conformation in solution of the thiodisaccharides was studied using theoretical calculations and confirmation of the minimum energy structures by the presence of characteristic NOE contacts. The exo-anomeric effect seems to play an important role in stabilizing the conformation of the thioglycosidic linkage of the 1-thio-β-D-Galf, as all the conformers found satisfied such an effect.

In the design of the thiodisaccharides we took into account that the presence of the non-reducing Galf unit is required for the recognition of the molecule by the β-galactofuranosidase of P. fellutanum, a model enzyme which has been isolated and purified in our laboratory. Therefore, the glycomimetics were evaluated as inhibitors of the enzyme. The kinetics of the inhibition indicated that they are competitive inhibitors. In addition, the inhibitory activity showed to be affected by the inversion of the configuration of the stereocenter linked to sulfur in the reducing end of the thiodisaccharides, being the one that contains the 5-thio-D-Galf unit (the same configuration as the natural substrate) a stronger inhibitor than that with the 5-thio-L-Altf moiety.

Experimental

General methods

The solvents used were distilled, dried and stored according to standard procedures. Analytical thin layer chromatography (TLC) was performed on Silica Gel 60 F254 (Merck) aluminum supported plates (layer thickness 0.2 mm) with solvent systems given in the text. Visualization of the spots was effected by exposure to UV light and charring with a solution of 5% (v/v) sulfuric acid in EtOH, containing 0.5% p-anisaldehyde. Column chromatography was carried out with Silica Gel 60 (230–400 mesh, Merck). Optical rotations were measured with a Perkin-Elmer 343 digital polarimeter. Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker AC 200 or with a Bruker AMX 500 instruments. Chemical shifts (δ) are reported in ppm, with residual chloroform (δ 7.27 for ¹H and δ 77.1 for ¹³C) or acetone (δ 2.16 for ¹H and δ 29.8 for ¹³C) as internal references. Assignments of ¹H and ¹³C NMR spectra were assisted by 2D ¹H COSY and HSQC experiments. High resolution mass spectra (HRMS) were obtained by Electrospray Ionization (ESI) and Q-TOF detection. Molecular mechanics (MM+) and semiempirical quantum chemical (AM1) calculations have been performed with Hyperchem Professional 8.0.

Methyl 2,3-di-O-tert-butyldimethylsilyl-β-D-galactofuranoside (3)

A solution of 2 (ref. 7) (603 mg, 1.07 mmol) in dry THF (25 mL) was cooled to −18 °C and LiAlH₄ (81 mg, 2.14 mmol) was added. The reaction mixture was stirred at −18 °C for 5 h and the temperature was gradually increased to 0 °C. Analysis by TLC (hexane/EtOAc 7 : 3) showed a single spot of R_f 0.30. To the mixture were sequentially added EtOAc (15 mL), MeOH (15 mL), and AcOH (to pH 7), and finally the mixture was concentrated. The residue was purified by column chromatography (hexane/EtOAc 3 : 2) to afford 3 (325 mg, 72%). Physical and spectroscopic data were in agreement with those previously reported.⁷

Methyl 6-deoxy-2,3-di-O-tert-butyldimethylsilyl-6-deoxy-5-thio-α-L-altrofuranoside (5)

To a solution of thiirane 4 (ref. 7) (214 mg, 0.509 mmol) in dry ethyl ether (10 mL) was added LiAlH₄ (32 mg, 0.865 mmol) and the mixture was stirred at room temperature for 3 h. Monitoring by TLC (hexane/EtOAc 97 : 3) revealed a major spot of R_f 0.36. After sequential addition of EtOAc (10 mL) and MeOH (10 mL) the mixture was concentrated. The resultant solid was purified by column chromatography (hexane/EtOAc 99 : 1) to afford 5 (148 mg, 69%); [α]²⁵_D −39.9 (c 0.9, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 4.71 (br s, 1H, H-1), 4.00 (dd, J_2,3 = 1.9, J_3,4 = 5.2 Hz, 1H, H-3), 3.96 (dd, J_1,2 = 0.9, J_2,3 = 1.9 Hz, 1H, H-2), 3.88 (t, J_3,4 = J_4,5 = 5.2 Hz, 1H, H-4), 3.35 (s, 3H, CH₃O), 3.11 (ddq, J_4,5 = 5.2, J_5,SH = 7.3, J_5,6 = 6.9 Hz, 1H, H-5), 1.71 (d, J_5,SH = 7.3 Hz, 1H, SH), 1.37 (d, J_5,6 = 6.9 Hz, 3H, H-6), 0.89, 0.88 [2s, 18H, (CH₃)₃CSiMe₂], 0.10 (×2), 0.09, 0.08 [4s, 12H, (CH₃)₂SiBu^t]; ¹³C NMR (125.7 MHz, CDCl₃) δ 109.7 (C-1), 89.8 (C-4), 83.9 (C-2), 80.7 (C-3), 54.8 (CH₃O), 36.5 (C-5), 25.9 [×2, (CH₃)₃CSiMe₂], 20.6 (C-6), 18.0 (×2, Me₃CSiMe₂), −3.9, −4.4 (×2), −4.7 [(CH₃)₂SiBu^t]; HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₉H₄₂NaO₄SSi₂ 445.2235, found 445.2243.

Methyl 6-O-acetyl-2,3-di-O-tert-butyldimethylsilyl-β-D-galactofuranoside (6)

To a solution of the diol 3 (262 mg, 0.623 mmol) in dry CH₂Cl₂ (5 mL) was added imidazole (53 mg, 0.807 mmol) and acetic anhydride (64 μL, 0.681 mmol). The mixture was stirred at room temperature for 24 h. TLC analysis (hexane/EtOAc 7 : 3) showed a main spot of R_f 0.57. The mixture was poured into ice/water (10 mL) and extracted with CH₂Cl₂ (3 × 10 mL). The extract was dried (MgSO₄), concentrated, and the residue was purified by column chromatography (hexane/EtOAc 9 : 1) to afford 6 (228 mg, 79%), [α]²⁵_D −28.6 (c 0.7, CHCl₃); ¹H NMR (500 MHz, CDCl₃ with a drop of D₂O) δ 4.71 (br s, 1H, H-1), 4.22 (dd, J_5,6a = 7.4, J_6a,6b = 11.3 Hz, 1H, H-6a), 4.12 (dd, J_5,6b = 5.0, J_6a,6b = 11.3 Hz, 1H, H-6b), 4.09 (dd, J_2,3 = 2.9, J_3,4 = 5.3 Hz, 1H, H-3), 3.99 (dd, J_1,2 = 1.1, J_2,3 = 2.9 Hz, 1H, H-2), 3.92 (dd, J_3,4 = 5.3, J_4,5 = 1.9 Hz, 1H, H-4), 3.87 (ddd, J_4,5 = 1.9, J_5,6a = 7.4, J_5,6b = 5.0 Hz, 1H, H-5), 3.34 (s, 3H, CH₃O), 2.09 (s, 3H, CH₃CO), 0.89, 087 [2s, 18H, (CH₃)₃CSiMe₂], 0.10, 0.09, 0.07 [3s, 12H, (CH₃)₂SiBu^t]; ¹³C NMR (126 MHz, CDCl₃) δ 171.2 (CO), 109.8 (C-1), 83.9 (C-4), 83.2 (C-2), 79.4 (C-3), 68.2 (C-5), 66.4 (C-6), 55.1 (CH₃O), 25.9, 25.8 [(CH₃)₃CSiMe₂], 21.1 (CH₃CO), 18.0 (×2, Me₃CSiMe₂), −4.2, −4.5 (×2), −4.8 [(CH₃)₂SiBu^t]; HRMS (ESI) m/z [M + Na]⁺ calcd for C₂₁H₄₄NaO₇Si₂ 487.2518, found 487.2528.

Methyl 6-O-acetyl-2,3-di-O-tert-butyldimethylsilyl-5-O-tosyl-β-D-galactofuranoside (7)

To a solution of 6 (196 mg, 0.422 mmol) in dry pyridine (10 mL) was added tosyl chloride (646 mg, 3.4 mmol). The reaction mixture was stirred at room temperature for 3 days. Analysis by TLC (hexane/EtOAc 4 : 1) showed a major product of R_f 0.54. The mixture was diluted with MeOH (15 mL) and the syrup obtained after evaporation of the solvent was dissolved in CH₂Cl₂ (15 mL). The organic layer was washed with water (20 mL), dried (MgSO₄), and concentrated. The resulting residue was purified by column chromatography (hexane/EtOAc 9 : 1) to afford 7 (211 mg, 81%), [α]²⁵_D −12.0 (c 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.82 (d, J = 8.2 Hz, 2H, H-aromatic), 7.31 (d, J = 8.2 Hz, 2H, H-aromatic), 4.93 (dt, J_5,6a = 3.7, J_5,6b = 7.5 Hz, 1H, H-5), 4.57 (d, J_1,2 = 1.1 Hz, 1H, H-1), 4.31 (dd, J_5,6a = 3.7, J_6a,6b = 12.3 Hz, 1H, H-6a), 4.16 (dd, J_5,6b = 7.5, J_6a,6b = 12.3 Hz, 1H, H-6b), 4.06–4.02 (m, 2H, H-3,4), 3.94 (dd, J_1,2 = 1.1, J_2,3 = 1.8 Hz, 1H, H-2), 3.26 (s, 3H, CH₃O), 2.43 (s, 3H, CH₃Ar), 1.94 (s, 3H, CH₃CO), 0.87 [2s, 18H, (CH₃)₃CSiMe₂], 0.10, 0.09, 0.08, 0.06 [4s, 13H, (CH₃)₂SiBu^t]; ¹³C NMR (126 MHz, CDCl₃) δ 170.5 (CO), 144.7, 134.4, 129.7, 128.2 (C-aromatic), 109.7 (C-1), 83.9 (C-2), 83.2 (C-4), 79.5 (C-3), 78.0 (C-5), 63.1 (C-6), 55.1 (CH₃O), 25.9, 25.8 [(CH₃)₃CSiMe₂], 21.75 (CH₃Ar), 20.75 (CH₃CO), 18.0, 17.9 (Me₃CSiMe₂), −4.20, −4.4, −4.7, −4.8 [(CH₃)₂SiBu^t]; HRMS (ESI) m/z [M + Na]⁺ calcd for C₂₈H₅₀NaO₉SSi₂ 641.2606, found 641.2591.

Methyl 5,6-anhydro-2,3-di-O-tert-butyldimethylsilyl-α-L-altrofuranoside (8)

A solution of 7 (211 mg, 0.341 mmol) in CH₂Cl₂ (8 mL) was cooled at 0 °C, and 2 M NaOMe/MeOH (1.9 mL) solution was added. The mixture was stirred at 0 °C for 2 h, when TLC analysis (hexane/EtOAc, 9 : 1) showed a main product of R_f 0.72. The mixture was diluted with CH₂Cl₂ (15 mL) and washed with water (2 × 20 mL). The organic extract was dried (MgSO₄) and concentrated and the residue was purified by column chromatography (hexane/EtOAc 95 : 5) to afford 8 (125 mg, 91%). Physical and spectroscopic data were in agreement with those previously reported.⁷

Methyl 6-deoxy-2,3-di-O-tert-butyldimethylsilyl-6-deoxy-5-thio-β-D-galactofuranoside (10)

The thiirane 9 (ref. 7) (113 mg, 0.269 mmol) was reduced as already described for 4, to afford 10 (87 mg, 77%), R_f 0.38 (hexane/EtOAc 97 : 3), [α]²⁵_D −64.0 (c 1, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 4.68 (d, J_1,2 = 1.7 Hz, 1H, H-1), 4.04 (dd, J_2,3 = 3.7, J_3,4 = 6.7 Hz, 1H, H-3), 3.99 (dd, J_1,2 = 1.7, J_2,3 = 3.7 Hz, 1H, H-2), 3.80 (dd, J_3,4 = 6.7, J_4,5 = 3.8 Hz, 1H, H-4), 3.35 (s, 3H, CH₃O), 3.04 (ddq, J_4,5 = 3.8, J_5,SH = 8.1, J_5,6 = 7.0 Hz, 1H, H-5), 1.74 (d, J_5,SH = 8.1 Hz, 1H, SH), 1.43 (d, J_5,6 = 7.0 Hz, 3H, H-6), 0.89, 0.87 [2s, 18H, (CH₃)₃CSiMe₂], 0.10, 0.09, 0.08 (×2) [4s, 12H, (CH₃)₂SiBu^t]; ¹³C NMR (125.7 MHz, CDCl₃) δ 109.2 (C-1), 86.4 (C-4), 84.4 (C-2), 80.8 (C-3), 55.0 (CH₃O), 36.5 (C-5), 26.0, 25.9 [(CH₃)₃CSiMe₂], 23.3 (C-6), 18.0 (×2, Me₃CSiMe₂), −3.9, −4.3, −4.4, −4.7 [(CH₃)₂SiBu^t]; HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₉H₄₂NaO₄SSi₂ 445.2235, found 445.2252.

Methyl 2,3-di-O-tert-butyldimethilsilyl-5-S-(2,3,5,6-tetra-O-benzoyl-β-D-galactofuranosyl)-6-deoxy-5-thio-α-L-altrofuranoside (12), methyl 2-O-tert-butyldimethilsilyl-5-S-(2,3,5,6-tetra-O-benzoyl-β-D-galactofuranosyl)-6-deoxy-5-thio-α-L-altrofuranoside (13a), methyl 3-O-tert-butyldimethilsilyl-5-S-(2,3,5,6-tetra-O-benzoyl-β-D-galactofuranosyl)-6-deoxy-5-thio-α-L-altrofuranoside (13b) and methyl 5-S-(2,3,5,6-tetra-O-benzoyl-β-D-galactofuranosyl)-6-deoxy-5-thio-α-L-altrofuranoside (14)

A suspension of the thiol 5 (66 mg, 0.16 mmol), the trichloroacetimidate 11 (ref. 23) (134 mg, 0.19 mmol), and freshly activated powdered molecular sieves (4 Å) in dry CH₂Cl₂ (7 mL) was stirred at room temperature for 1.5 h. The mixture was then cooled to −20 °C, and TMSOTf (12 μL, 0.07 mmol) was added. After 1 h, TLC analysis (toluene/EtOAc 93 : 7) showed complete consumption of the starting thiol 5 and the presence of three spots having R_f 0.60, 0.26 and 0.00. The latter exhibited R_f 0.58 in a more polar solvent (hexane/EtOAc 3 : 7). The Lewis acid was neutralized upon addition of Et₃N (5 μL) and the mixture was poured into water (10 mL) and extracted with CH₂Cl₂ (3 × 10 mL). The organic extract was dried (MgSO₄), concentrated, and the residue was purified by column chromatography (toluene/EtOAc 60 : 1) to afford the following thiodisaccharides.

Compound 12 was isolated as a syrup (6 mg, 4%); [α]²⁵_D −53.1 (c 1, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.10–7.87 (H-aromatic), 7.60–7.27 (H-aromatic), 6.10 (dt, J_4′,5′ = J_5′,6′a ∼4.5, J_5′,6′b = 7.0 Hz, 1H, H-5′), 5.80 (br s, 1H, H-1′), 5.64 (dd, J_2′,3′ = 1.2, J_3′,4′ = 5.2 Hz, 1H, H-3′), 5.52 (t, J_1′,2′ = J_2′,3′ = 1.2, 1H, H-2′), 4.87 (dd, J_3′,4′ = 5.2, J_4′,5′ = 4.5 Hz, 1H, H-4′), 4.74 (dd, J_5′,6′a = 4.8, J_6′a,6′b = 11.8 Hz, 1H, H-6′a), 4.72 (br s, 1H, H-1), 4.71 (dd, J_5′,6′b = 7.0, J_6′a,6′b = 11.8 Hz, 1H, H-6′b), 4.09 (dd, J_3,4 = 5.6, J_4,5 = 4.3 Hz, 1H, H-4), 4.01 (dd, J_2,3 = 2.6, J_3,4 = 5.6 Hz, 1H, H-3), 3.99 (dd, J_1,2 = 1.1, J_2,3 = 2.6 Hz, 1H, H-2), 3.34 (s, 3H, CH₃O), 3.31 (m, 1H, H-5), 1.38 (d, J_5,6 = 7.1 Hz, 3H, H-6), 0.87, 0.87 [2s, 19H, (CH₃)₃CSiMe₂], 0.1, 0.09, 0.08, 0.07 [4s, 14H, (CH₃)₂SiBu^t]; ¹³C NMR (126 MHz, CDCl₃) δ 166.2, 165.8, 165.7, 165.3 (PhCO), 133.6–128.5 (C-aromatic), 109.6 (C-1), 89.1 (C-1′), 86.7 (C-4), 84.3 (C-2), 83.1 (C-2′), 81.2 (C-4′), 80.8 (C-3), 78.1 (C-3′), 70.4 (C-5′), 63.6 (C-6′), 54.9 (CH₃O), 42.0 (C-5), 25.9 [ × 2, (CH₃)₃CSiMe₂], 18.0 (Me₃CSiMe₂), 17.1 (C-6), −4.0, −4.4 ( × 2), −4.7 [(CH₃)₂SiBu^t]; HRMS (ESI) m/z [M + Na]⁺ calcd for C₅₃H₆₈NaO₁₃SSi₂ 1023.3811, found 1023.3808.

Compound 13a and 13b were isolated as an inseparable mixture (27 mg, 20%, ratio 13a : 13b–1 : 3); they showed the following ¹H and ¹³C NMR spectra (only sugar signals are reported).

Compound 13a. ¹H NMR (500 MHz, CDCl₃), the following signals have been identified: δ 5.69 (d, J_3′,4′ = 5.6 Hz, 1H, H-3′), 5.37 (t, J_1′,2′ = J_2′,3′ = 1.3 Hz, 1H, H-2′), 4.14 (dd, 1H, J_3,4 = 5.3, J_4,5 = 2.9 Hz, 1H, H-4), 4.19 (dd, J_2,3 = 1.7, J_3,4 = 5.3 Hz, 1H, H-3), 3.98 (d, J_2,3 = 1.7, 1H, H-2), 3.53 (m, 1H, H-5), 3.30 (s, 3H, CH₃O), 1.49 (d, J_5,6 = 7.5 Hz, 3H, H-6); ¹³C NMR (126 MHz, CDCl₃) δ 109.1 (C-1), 90.0 (C-1′), 89.1 (C-4), 84.0 (C-2′), 83.1 (C-2), 80.8 (C-4′), 80.1 (C-3), 77.5 (C-3′), 70.2 (C-5′), 63.8 (C-6′), 54.8 (CH₃O), 41.2 (C-5), 18.1 (C-6).

Compound 13b. ¹H NMR (500 MHz, CDCl₃) δ 6.11 (m, 1H, H-5), 5.94 (s, 1H, H-1′), 5.65 (d, J_3,4 = 5.2 Hz, 1H, H-3′), 5.52 (t, J_1′,2′ = J_2′,3′ = 1.3 Hz, 1H, H-2′), 4.90 (dd, 1H, J_3′,4′ = 4.8, J_4′,5′ = 3.9 Hz, H-4′), 4.81–4.77 (m, 2H, H-1, H-6′), 4.71 (dd, J_6a,6b = 11.9, J_5,6a = 7.3 Hz, 1H, H-6a), 4.10 (m, 1H, H-3), 4.06–4.03 (m, 2H, H-2, H-4), 3.34 (s, 3H, CH₃O), 3.16 (dq, J_4,5 = 8.6, J_5,6 = 7.0 Hz, 1H, H-5), 1.49 (d, J_5,6 = 7.0 Hz, 3H, H-6); ¹³C NMR (126 MHz, CDCl₃) δ 109.4 (C-1), 89.6 (C-4), 87.7 (C-1′), 83.2 (C-2′), 81.6 (C-2), 81.4 (C-4′), 80.2 (C-3), 78.0 (C-3′), 70.4 (C-5′), 63.8 (C-6′), 54.9 (CH₃O), 43.5 (C-5), 18.3 (C-6).

Compound 14 was isolated as a syrup (22 mg, 18%); [α]²⁵_D −61.1 (c 1, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.04–7.84 (H-aromatic), 7.61–7.25 (H-aromatic), 6.11 (ddd, J_4′,5′ = 3.5, J_5′,6′a = 4.7, J_5′,6′b = 7.0 Hz, Hz, 1H, H-5′), 5.99 (br s, 1H, H-1′), 5.69 (d, J_3′,4′ = 5.4 Hz, 1H, H-3′), 5.36 (d, J_1′,2′ = 1.1 Hz, 1H, H-2′), 4.88 (br s, 1H, H-1), 4.86 (m, 1H, H-4′), 4.79 (dd, J_5′,6′a = 4.7, J_6′a,6′b = 11.8 Hz, 1H, H-6′a), 4.74 (dd, J_5′,6′b = 7.0, J_6′a,6′b = 11.8 Hz, 1H, H-6′b), 4.09 (dd, J_3,4 = 3.7, J_4,5 = 4.7 Hz, 1H, H-4), 4.06 (br d, J_2,3 = 1.0 Hz, 1H, H-2), 4.03 (dd, J_2,3 = 1.0, J_3,4 = 3.7 Hz, 1H, H3), 3.44 (dq, J_4,5 = 4.7, J_5,6 = 7.4 Hz, 1H, H-5), 3.34 (s, 3H, CH₃O), 1.37 (d, J_5,6 = 7.4 Hz, 3H, H-6); ¹³C NMR (126 MHz, CDCl₃) δ 166.3, 165.9, 165.9, 165.7 (PhCO), 133.8–128.5 (C-aromatic), 108.6 (C-1), 90.2 (C-4), 89.3 (C-1′), 83.5 (C-2′), 81.2 (C-2), 80.9 (C-4′), 78.8 (C-3), 77.6 (C-3′), 70.2 (C-5′), 63.5 (C-6′), 54.8 (CH₃O), 41.3 (C-5), 18.3 (C-6); HRMS (ESI) m/z [M + Na]⁺ calcd for C₄₁H₄₀NaO₁₃S 795.2082, found 795.2092.

An alternative procedure for the synthesis of 14. The crude mixture of 12, 13a,b and 14, obtained as described in the previous item, starting from the thiol 5 (66 mg, 0.156 mmol) and the trichloroacetimidate 11 (134 mg, 0.187 mmol), was dissolved in THF (6 mL) and TBAF (300 mg, 1.1 mmol) was added. The mixture was stirred overnight at room temperature. Monitoring by TLC (hexane/EtOAc 3 : 7) showed a main spot of R_f 0.58. The mixture was concentrated and the residue was dissolved in CH₂Cl₂; the resulting solution was washed with water, dried (MgSO₄) and the residue was purified by column chromatography (hexane/EtOAc 3 : 2) to afford syrupy 14 (54 mg, 45% from 5). The thiodisaccharide 14 showed the same physical and spectroscopic as the compound previously described.

Methyl 2,3-di-O-acetyl-5-S-(2,3,5,6-tetra-O-benzoyl-β-D-galactofuranosyl)-6-deoxy-5-thio-α-L-altrofuranoside (15)

To a solution of the thiodisaccharide 14 (60 mg, 0.078 mmol) in dry pyridine (0.5 mL) was added acetic anhydride (0.5 mL). The mixture was stirred at room temperature for 18 h, and then diluted with MeOH (5 mL) and, after stirring for 1 h, it was concentrated. The syrup obtained was dissolved in CH₂Cl₂ (5 mL), washed with water (10 mL), dried (MgSO₄) and concentrated. The resulting residue was purified by column chromatography (toluene/EtOAc 9 : 1) to afford 15 (49 mg, 75%); R_f 0.54 (toluene/EtOAc 85 : 15); [α]²⁵_D −84.7 (c 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.10–7.87 (m, H-aromatic), 7.61–7.27 (m, H-aromatic), 6.11 (dt, J_4′,5′ = J_5′,6′a∼ 4.3, J_5′,6′b = 7.1 Hz, 1H, H-5′), 5.90 (s, 1H, H-1′), 5.65 (d, J_3′,4′ = 4.3 Hz, 1H, H-3′), 5.53 (br s, 1H, H-2′), 5.21 (dd, J_2,3 = 1.1, J_3,4 = 5.6 Hz, 1H, H-3), 4.98 (d, J_2,3 = 1.1 Hz, 1H, H-2), 4.88 (s, 2H, H-1), 4.85 (t, J_3′,4′ = J_4′,5′ = 4.3 Hz, 1H, H-4′), 4.78 (dd, J_6′a,6′b = 11.8, J_5′,6′a = 4.4 Hz, 1H, H-6′a), 4.73 (dd, J_6′a,6′b = 11.8, J_5′,6′b = 7.1 Hz, 1H, H-6′b), 4.29 (dd, J_3,4 = 5.6, J_4,5 = 4.5 Hz, 1H, H-4), 3.47 (qd, J_4,5 = 4.5, J_5,6 = 7.2 Hz, 1H, H-5), 3.35 (s, 3H, CH₃O), 2.08 (s, 3H, CH₃CO), 2.06 (s, 3H, CH₃CO), 1.33 (d, J_5,6 = 7.2 Hz, 3H, H-6); ¹³C NMR (126 MHz, CDCl₃) δ 170.2, 170.1 (CH₃CO), 166.2, 165.9, 165.6, 165.3 (PhCO), 133.8–128.5 (C-aromatic), 106.4 (C-1), 89.2 (C-1′), 85.0 (C-4), 83.0 (C-2′), 82.2 (C-2), 81.5 (C-4′), 78.0 (C-3′), 77.7 (C-3), 70.3 (C-5′), 63.6 (C-6′), 54.9 (CH₃O), 41.6 (C-5), 21.0, 20.9 (CH₃CO), 17.3 (C-6); HRMS (ESI) m/z [M + Na]⁺ calcd for C₄₅H₄₄NaO₁₅S 879.2293, found 879.2293.

Methyl 5-S-(β-D-galactofuranosyl)-6-deoxy-5-thio-α-L-altrofuranoside (16)

Compound 14 (54 mg, 0.07 mmol) was dissolved in CH₂Cl₂ (12 mL) and 10 mM NaOMe/MeOH solution (6 mL) was added. The mixture was stirred at room temperature for 45 min, when TLC (nBuOH/EtOH/H₂O 2.5 : 1 : 1) showed a main spot of R_f 0.63. The reaction mixture was concentrated and the residue was dissolved in H₂O (1 mL), was passed through a column filled with Dowex 50×, which washed with water. The eluate was concentrated, dissolved in H₂O (1 mL) and filtered through an octadecyl C18 minicolumn, to afford 16 (23 mg, 41% from 5); [α]²⁵_D −182.7 (c 1, CH₃OH); ¹H NMR (500 MHz, D₂O) δ 5.28 (d, J_1′,2′ = 5.4 Hz, 1H, H-1′), 4.93 (d, J_1,2 = 1.4 Hz, 1H, H-1), 4.12 (dd, J_2′,3′ = 5.4, J_3′,4′ = 7.5 Hz, 1H, H-3′); 4.10–4.07 (m, 2H, H-3, H-4), 4.05 (dd, J_1,2 = 1.4, J_2,3 = 2.6 Hz, 1H, H-2), 4.02 (t, J_1′,2′ = J_2′,3′ = 5.4 Hz, 1H, H-2′), 3.99 (dd, J_3′,4′ = 7.5, J_4′,5′ = 3.5 Hz, 1H, H-4′), 3.83 (ddd, J_4′,5′ = 3.5, J_5′,6′a = 4.7, J_5′,6′b = 7.5 Hz, 1H, H-5′), 3.68 (dd, J_5′,6′a = 4.7, J_6′a,6′b = 11.7 Hz, 1H, H-6′a), 3.64 (dd, J_5′,6′b = 7.5, J_6′a,6′b = 11.7 Hz, 1H, H-6′b), 3.40 (s, 3H, CH₃O), 3.34 (dq, J_4,5 = 4.5, J_5,6 = 7.2 Hz, 1H, H-5), 1.37 (d, J_5,6 = 7.2 Hz, 3H, H-6); ¹³C NMR (126 MHz, D₂O) δ 107.7 (C-1), 87.6 (C-1′), 86.3 (C-4), 80.9 (C-4′), 80.7 (C-2′), 80.5 (C-2), 77.2 (C-3), 75.5 (C-3′), 70.0 (C-5′), 62.3 (C-6′), 54.4 (CH₃O), 41.2 (C-5), 16.5 (C-6); HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₃H₂₄NaO₉S 379.1033, found 379.1041.

Methyl 5-S-(2,3,5,6-tetra-O-benzoyl-β-D-galactofuranosyl)-6-deoxy-5-thio-β-D-galactofuranoside (17)

The glycosylation of the thiol 10 (60 mg, 0.14 mmol) with the trichloroacetimidate 11 (120 mg, 0.17 mmol) was conducted as already described for the preparation of 15. The crude mixture containing the analogues of 12–14 was treated with TBAF (300 mg, 1.1 mmol) in THF (6 mL). The usual workup and purification afforded 17 (48 mg, 39% from 10); [α]²⁵_D −87.8 (c 1.1, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.13–7.82 (H-aromatic), 7.60–7.17 (H-aromatic), 6.06 (ddd, J_4′,5′ = 3.6, J_5′,6′a = 4.5, J_5′,6′b = 6.4 Hz, 1H, H-5′), 5.82 (br s, 1H, H-1′), 5.67 (dd, J_2′,3′ = 1.3, J_3′,4′ = 5.2 Hz, 1H, H-3′), 5.51 (t, J_1′,2′ = J_2′,3′ = 1.3 Hz, 1H, H-2′), 4.90 (dd, J_3′,4′ = 5.2, J_4′,5′ = 3.6 Hz, 1H, H-4′), 4.85 (br s, 1H, H-1), 4.81 (dd, J_5′,6′a = 4.5, J_6′a,6′b = 11.9 Hz, 1H, H-6′a), 4.74 (dd, J_5′,6′b = 6.4, J_6′a,6′b = 11.9 Hz, 1H, H-6′b), 4.07 (dd, J_3,4 = 4.4, J_4,5 = 3.2 Hz, 1H, H-4), 4.03 (br s, 1H, H-3), 3.99 (br s, 1H, H-2), 3.36 (s, 3H, CH₃O), 3.31 (dq, J_4,5 = 3.2, J_5,6 = 7.1 Hz, 1H, H-5), 1.54 (d, J_5,6 = 7.1 Hz, 3H, H-6); ¹³C NMR (126 MHz, CDCl₃) δ 166.6, 165.9, 165.7, 165.7 (PhCO), 133.9–128.5 (C-aromatic), 108.9 (C-1), 89.1 (C-4), 88.1 (C-1′), 83.1 (C-2′), 81.7 (C-4′), 81.4 (C-2), 80.3 (C-3), 77.9 (C-3′), 70.5 (C-5′), 63.6 (C-6′), 55.0 (CH₃O), 43.3 (C-5), 20.3 (C-6); HRMS (ESI) m/z [M + Na]⁺ calcd for C₄₁H₄₀NaO₁₃S 795.2081, found 795.2092.

Methyl 5-S-(β-D-galactofuranosyl)-6-deoxy-5-thio-β-D-galactofuranoside (18)

The thiol 10 (50 mg, 0.12 mmol) was subjected to glycosylation with the trichloroacetimidate 11 (107 mg, 0.145 mmol) and subsequent deprotection reactions, as already described for 16, to afford 18 (15 mg, 36%); R_f 0.60 (nBuOH/EtOH/H₂O 2.5 : 1 : 1); [α]²⁵_D −104.8 (c 1.0, CH₃OH); ¹H NMR (500 MHz, D₂O) δ 5.26 (d, J_1′,2′ = 4.9 Hz, 1H, H-1′), 4.90 (d, J_1,2 = 1.7 Hz, 1H, H-1), 4.12 (dd, J_2′,3′ = 5.3, J_3′,4′ = 7.4 Hz, 1H, H-3′), 4.10 (dd, J_2,3 = 3.2, J_3,4 = 6.3 Hz, 1H, H-3), 4.06-4.03 (m, H-2, H-2′), 4.03 (dd, J_3,4 = 6.3, J_4,5 = 4.6 Hz, 1H, H-4), 4.00 (dd, J_3′,4′ = 7.4, J_4′,5′ = 3.5 Hz, 1H, H-4′), 3.85 (ddd, J_4′,5′ = 3.5, J_5′,6′a = 4.6, J_5′,6′b = 7.5 Hz, 1H, H-5′), 3.69 (dd, J_5′,6′a = 4.6, J_6′a,6′b = 11.6 Hz, 1H, H-6′a), 3.64 (dd, J_5′,6′b = 7.5, J_6′a,6′b = 11.6 Hz, 1H, H-6′b), 3.41 (s, 3H, CH₃O), 3.25 (dq, J_4,5 = 4.6, J_5,6 = 7.1 Hz, 1H, H-5), 1.46 (d, J_5,6 = 7.1 Hz, 3H, H-6); ¹³C NMR (126 MHz, D₂O) δ 107.6 (C-1), 86.9 (C-1′), 85.6 (C-4), 81.1 (C-2), 80.8 (C-4′), 80.6 (C-2′), 77.5 (C-3), 75.8 (C-3′), 69.9 (C-5′), 62.3 (C-6′), 54.5 (CH₃O), 41.4 (C-5), 18.1 (C-6); HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₃H₂₄NaO₉S 379.1033, found 379.1034.

Enzymatic assays

The enzymatic activity was assayed using the filtered medium of a stationary culture of P. fellutanum as source of exo β-D-galactofuranosidase and 4-nitrophenyl β-D-galactofuranoside as substrate.²⁵ The standard assay was conducted with 50 μL of 66 mM NaOAc buffer (pH4.6), 20 μL of a 5 mM solution of 4-nitrophenyl β-D-galactofuranoside and 20 μL (4 μg protein) of the enzyme medium, in a final volume of 250 μL. Compound 16 and 18 were incorporated in the amounts required to obtain a final concentration of 0.1 to 1.6 mM. The enzymatic reaction was stopped after 1.5 h of incubation at 37 °C by addition of 1 mL of 0.1 M Na₂CO₃ buffer (pH 9.0). The 4-nitrophenol released was measured spectrophotometrically at 410 nm. K_m and K_i values were determined by the Lineweaver–Burk plot.

Acknowledgements

The authors are indebted to the University of Buenos Aires (UBACYT 2002010030100571BA and 20020100100164), National Agency for Promotion of Science and Technology (ANPCyT-PICT-2012 no. 0717) and the National Research Council of Argentina (CONICET-PIP 2012 no. 112 201101 00370CO and no. 2012 11220110100699) for financial support. C.M. and O.V. are Research Members of CONICET. M.L.F. was supported by a fellowship from CONICET.

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Footnotes

† Electronic supplementary information (ESI) available: ¹H and ¹³C NMR spectra of compounds 5–7, 10, 12–18 and NOESY spectra of compound 16 and 18. See DOI: 10.1039/c5ra06899a

‡ Present address: INQUISUR-CONICET, Departamento de Química, Universidad Nacional del Sur, Avenida Alem 1253, Bahía Blanca (8000), Argentina.

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