# Thio-β-D-glucosides: Synthesis and Evaluation as Glycosidase Inhibitors and Activators

Authors: Andrey V. Samoshin; Irina A. Dotsenko; Nataliya M. Samoshina; Andreas H. Franz; Vyacheslav V. Samoshin
Journal: International Journal of Carbohydrate Chemistry
 (2014)

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## Abstract

Structurally simple 1-thio-β-D-glucopyranosides were synthesized and tested as potential inhibitors toward several fungal glycosidases from Aspergillus oryzae and Penicillium canescens. Significant selective inhibition was observed for α- and β-glucosidases, while a weak to moderate activation for α- and β-galactosidases.


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## Body

## 1. Introduction

Thioglycosides are the hydrolysis- and metabolism-resistant syntheticS-analogs of naturalO-glycosides. They attracted recently a rapidly increasing attention as competitive inhibitors of glycosidases and other enzymes involved in a variety of biochemical processes [1, 2] related in particular to metabolic disorders and diseases, such as diabetes [1, 3], to inflammations [2, 4] and viral or bacterial infections [5–17], including tuberculosis [9, 10], and to cancer [2, 18–20]. However, surprisingly little is known about the inhibition of glucosidases by 1-thio-β-D-glucosides so far [21–24] (see the discussion). In a search for simple, readily accessible, and efficient glycosidase inhibitors [25–27], we designed, prepared, and assayed a series of structurally simple 1-thio-β-D-glucosides.


## 2. Results and Discussion

### 2.1. Synthesis of 1-Thio-β-D-glucopyranosides

A series of aryl and alkyl 1-thio-β-D-glucopyranosides2a–2i was synthesized according to Scheme 1. In addition to the model compounds2a–2f with simple aryl and alkyl aglycones, we included the phenanthroline derivative2g, which was found to possess activity towards some glycosidases [27]. Compounds2h–2i  were designed as disaccharide analogues in view of the significant inhibitory activity of the 1,2-cyclohexanedicarboxylic acid derivatives towards fungal glycosidases [25, 26].Scheme 1
Preparation of 1-thio-β-D-glucopyranosides2a–2i.The structures of all products and intermediates were determined from the data of1H NMR and 13C NMR, including COSY and HMQC techniques. Thanks to a bias of the conformational equilibria towards the all-equatorial form of the pyranose ring, the configurational assignment was rather straightforward: large spin-spin coupling constants (9–11 Hz) indicated atrans-diaxial orientation of the corresponding vicinal protons, while all other relative positions (axial-equatorial and equatorial-equatorial) resulted in small couplings between them (2–4 Hz).


### 2.2. Glycosidase Inhibitory Activity

The synthesized compounds have been assayed for enzyme inhibitory activity against several glycosidases in multienzyme complexes isolated from fungiPenicillium canescens andAspergillus oryzae [28–32]. The multienzyme complex fromP. canescenscontains α-D- and β-D-galactosidase, β-D-glucosidase, and α-L- and β-D-fucosidase [28–31]. The multienzyme complex fromA. oryzae contains α-D- and β-D-galactosidase, α-D- and β-D-glucosidase, and α-L- and β-D-fucosidase [32]. All assays have been performed in a standard way [33] by monitoring spectrophotometrically at 400 nm the release ofp-nitrophenol from the correspondingp-nitrophenyl glycosides. The inhibitory activity of studied compounds was found to depend on the structure of inhibitor, the type of enzyme, and the particular source of enzyme.Usually, the structural analogues of carbohydrates with certain configuration are expected to influence activity of the corresponding enzymes (“like inhibits like”). Thus, 4-nitrophenyl 1-thio-β-D-glucoside was used to study binding to β-D-glucosidase from barley and rice and was shown to be a competitive inhibitor [22]. As another example, a series of alkyl/aryl 1-thio-β-D-galactosides was designed and studied as inhibitors of β-D-galactosidase fromE. coli [17]. Similarly, we tried the 1-thio-β-D-glucosides2a–2i first of all as possible inhibitors against β-D-glucosidases. In the preliminary tests, all studied compounds demonstrated indeed a substantial inhibitory activity toward fungal β-D-glucosidases from bothP. canescens andA. oryzae strains (Figures 1 and 2). It is worth mentioning that compounds2a–2f were 10-fold more potent in the case of β-D-glucosidase fromA. oryzae than methyldeoxynojirimycin at 85% inhibition [34].Figure 1
Effect of 1-thio-β-D-glucopyranosides (1 mM) on the activity ofA. oryzae glycosidases.Figure 2
Effect of 1-thio-β-D-glucopyranosides (1 mM) on the activity ofP. canescens glycosidases.It was found previously that the inhibitors ofβ-D-glucosidase may also possess the α-D-glucosidase inhibitory activity [25, 26, 35]. For example,N-substituted aminomethyl-β-D-glucopyranosides inhibited yeast and rat intestinal α-D-glucosidases [35]. In this study, compounds2a–2h showed only weak inhibition of α-D-glucosidase fromA. oryzae (Figure 1).Quite unusually, 1-thio-β-D-glucosides revealed a weak to moderate ability to activate α- and β-D-galactosidases (Figures 1 and 2), especially α-D-galactosidase fromP. canescens(Figure 2). The strongest activation was observed in the case of phenanthrolyl-1-thio-β-D-glucoside2g (Figures 1 and 2). Some derivatives of 1,10-phenanthroline were found earlier to activate β-D-galactosidases, while other derivatives acted as inhibitors [27]. Towards the fungal β-D-glucosidases, all 1,10-phenanthrolines were activators, with exception of thioglucoside2g, which was an inhibitor [27]. Noteworthy, the nitrogen analog of2g,N-(1,10-phenanthrolin-5-yl)-β-D-glucopyranosylamine was recently synthesized, but no data on enzyme inhibition or activation were obtained yet [36]. The mechanism of activation is currently unknown. It may be a result of an allosteric effect in these enzymes [37] or of a change in their hydration [38]. The activation of α/β-D-galactosidases may be interesting for treatment of certain diseases and merits further studies [37, 38].The analysis of the Lineweaver-Burk plots indicated that compounds2a–2i inhibited the β-D-glucosidases competitively. Their inhibition parameters are presented in Table 1. The values of K
i are systematically larger for the enzyme fromA. oryzae as compared toP. canescens (by factors 32–35 for2a,2b,2d, and2g; and by factors 17–20 for2c,2e,2f, and2h). Similar ratios were obtained earlier for the inhibition of these enzymes by derivatives of cyclohexane-1,2-dicarboxylic acids [25]. This is apparently due to structural differences of the enzymes, which result also in different values of Michaelis constant: K
M
=
500 μM (A. oryzae) and K
M
=
40 μM (P. canescens) [25]. Interestingly, higher values of K
M were found also for β-D-galactosidase fromA. oryzae as compared to enzyme fromP. canescens[39].Table 1
Inhibition ofβ-D-glucosidases at pH 4.2 and 30°C.
Inhibitor (R)
β-D-Glucosidase
β-D-Glucosidase
A. oryzae
P. canescens
K
i, μM
K
i, μM
2a (Ph)a
129
4.02
2b (p-ClC6H4)
183
5.56
2c (p-MeC6H4)
218
11.1
2d (2′-naphthyl)
159
4.53
2e (PhCH2)
109
6.32
2f (n-C6H13)
80.7
3.45
2g (5′-phen)
91.7
2.84
2h (diester)
223
12.3
2i (diacid)
1500
27.5
a
K
i = 1530 μM (for β-D-glucosidase from fungusStachybotrys atra) [21].The previously available data on 1-thio-β-D-glucopyranosides as glycosidase inhibitors were limited to the derivatives with the following aglycones: phenyl (2a), K
i
=
1530 μM (Stachybotrys atra) [21]; 4-nitrophenyl, K
i
=
664 μM (rice) and K
i
=
95 μM (barley) [22]; benzo-1,4-quinone, K
i
=
60 μM (A. faecalis); chlorobenzo-1,4-quinone, K
i
=
400 μM (A. faecalis) [23]; and 2-(3-methyl-3H-diazirine-3-yl)-ethyl, K
i
=
1500 μM (sweet almond) [24]. Although thioglycosides are usually resistant to hydrolysis, it was found also that with some specific aglycones they may be hydrolysable and can serve as glycosidase substrates [40]. It is interesting to compare our results (Table 1) to the data for some known inhibitors of β-D-glucosidases: 1-deoxynojirimycin, K
i
=
0.3
–
2.7 μM (Aspergillus wentii); K
i
=
12 μM (Agrobacterium faecalis);N-methyl-1-deoxynojirimycin, K
i
=
43 μM (sweet almond); andN-hexyl-1-deoxynojirimycin, K
i
=
200 μM (calf liver cytosolic) and K
i
=
69 μM (calf spleen lysosomal) [41]. From this comparison, compounds2a–2h are moderately strong β-D-glucosidase inhibitors.The comparison of thioglucosides2a–2i allows a preliminary elucidation of the effect of aglycon upon their inhibitory activity. It is known that the presence of alkyl groups and other lipophilic moieties often increases the efficiency of glycosidase inhibitors. This effect, mostly towards β-glucosidases, was observed for derivatives of 1-deoxynojirimycin and other iminosugars, alkyl glycosides, carbasugars, and some other inhibitors. It was explained by additional stabilization of the alkyl chains or aromatic residues by the hydrophobic pocket or cleft within or in vicinity of the enzyme binding site (for the detailed discussion see [25, 42, 43] and references therein; in addition see [37, 44, 45]). The results of our study (Table 1) seem to confirm the general regularities observed earlier. Thus, the most potent inhibitors of β-D-glucosidases, compounds2f and2g, have large lipophilic aglycones, while the thioglucoside2i with the most polar aglycone in this series shows almost negligible inhibition. However, the dependence of inhibition on the aglycone’s lipophilicity is not straightforward: despite their large aromatic aglycones, the thioglucosides2a–2e produced much weaker inhibition than2f and2 g (see also discussion in [25]). The influence of the aglycone shape remains also uncertain. The best results were achieved both for the narrow alkyl chain (2f) and for the wide polyaromatic moiety (2g).


## 2.1. Synthesis of 1-Thio-β-D-glucopyranosides

A series of aryl and alkyl 1-thio-β-D-glucopyranosides2a–2i was synthesized according to Scheme 1. In addition to the model compounds2a–2f with simple aryl and alkyl aglycones, we included the phenanthroline derivative2g, which was found to possess activity towards some glycosidases [27]. Compounds2h–2i  were designed as disaccharide analogues in view of the significant inhibitory activity of the 1,2-cyclohexanedicarboxylic acid derivatives towards fungal glycosidases [25, 26].Scheme 1
Preparation of 1-thio-β-D-glucopyranosides2a–2i.The structures of all products and intermediates were determined from the data of1H NMR and 13C NMR, including COSY and HMQC techniques. Thanks to a bias of the conformational equilibria towards the all-equatorial form of the pyranose ring, the configurational assignment was rather straightforward: large spin-spin coupling constants (9–11 Hz) indicated atrans-diaxial orientation of the corresponding vicinal protons, while all other relative positions (axial-equatorial and equatorial-equatorial) resulted in small couplings between them (2–4 Hz).


## 2.2. Glycosidase Inhibitory Activity

The synthesized compounds have been assayed for enzyme inhibitory activity against several glycosidases in multienzyme complexes isolated from fungiPenicillium canescens andAspergillus oryzae [28–32]. The multienzyme complex fromP. canescenscontains α-D- and β-D-galactosidase, β-D-glucosidase, and α-L- and β-D-fucosidase [28–31]. The multienzyme complex fromA. oryzae contains α-D- and β-D-galactosidase, α-D- and β-D-glucosidase, and α-L- and β-D-fucosidase [32]. All assays have been performed in a standard way [33] by monitoring spectrophotometrically at 400 nm the release ofp-nitrophenol from the correspondingp-nitrophenyl glycosides. The inhibitory activity of studied compounds was found to depend on the structure of inhibitor, the type of enzyme, and the particular source of enzyme.Usually, the structural analogues of carbohydrates with certain configuration are expected to influence activity of the corresponding enzymes (“like inhibits like”). Thus, 4-nitrophenyl 1-thio-β-D-glucoside was used to study binding to β-D-glucosidase from barley and rice and was shown to be a competitive inhibitor [22]. As another example, a series of alkyl/aryl 1-thio-β-D-galactosides was designed and studied as inhibitors of β-D-galactosidase fromE. coli [17]. Similarly, we tried the 1-thio-β-D-glucosides2a–2i first of all as possible inhibitors against β-D-glucosidases. In the preliminary tests, all studied compounds demonstrated indeed a substantial inhibitory activity toward fungal β-D-glucosidases from bothP. canescens andA. oryzae strains (Figures 1 and 2). It is worth mentioning that compounds2a–2f were 10-fold more potent in the case of β-D-glucosidase fromA. oryzae than methyldeoxynojirimycin at 85% inhibition [34].Figure 1
Effect of 1-thio-β-D-glucopyranosides (1 mM) on the activity ofA. oryzae glycosidases.Figure 2
Effect of 1-thio-β-D-glucopyranosides (1 mM) on the activity ofP. canescens glycosidases.It was found previously that the inhibitors ofβ-D-glucosidase may also possess the α-D-glucosidase inhibitory activity [25, 26, 35]. For example,N-substituted aminomethyl-β-D-glucopyranosides inhibited yeast and rat intestinal α-D-glucosidases [35]. In this study, compounds2a–2h showed only weak inhibition of α-D-glucosidase fromA. oryzae (Figure 1).Quite unusually, 1-thio-β-D-glucosides revealed a weak to moderate ability to activate α- and β-D-galactosidases (Figures 1 and 2), especially α-D-galactosidase fromP. canescens(Figure 2). The strongest activation was observed in the case of phenanthrolyl-1-thio-β-D-glucoside2g (Figures 1 and 2). Some derivatives of 1,10-phenanthroline were found earlier to activate β-D-galactosidases, while other derivatives acted as inhibitors [27]. Towards the fungal β-D-glucosidases, all 1,10-phenanthrolines were activators, with exception of thioglucoside2g, which was an inhibitor [27]. Noteworthy, the nitrogen analog of2g,N-(1,10-phenanthrolin-5-yl)-β-D-glucopyranosylamine was recently synthesized, but no data on enzyme inhibition or activation were obtained yet [36]. The mechanism of activation is currently unknown. It may be a result of an allosteric effect in these enzymes [37] or of a change in their hydration [38]. The activation of α/β-D-galactosidases may be interesting for treatment of certain diseases and merits further studies [37, 38].The analysis of the Lineweaver-Burk plots indicated that compounds2a–2i inhibited the β-D-glucosidases competitively. Their inhibition parameters are presented in Table 1. The values of K
i are systematically larger for the enzyme fromA. oryzae as compared toP. canescens (by factors 32–35 for2a,2b,2d, and2g; and by factors 17–20 for2c,2e,2f, and2h). Similar ratios were obtained earlier for the inhibition of these enzymes by derivatives of cyclohexane-1,2-dicarboxylic acids [25]. This is apparently due to structural differences of the enzymes, which result also in different values of Michaelis constant: K
M
=
500 μM (A. oryzae) and K
M
=
40 μM (P. canescens) [25]. Interestingly, higher values of K
M were found also for β-D-galactosidase fromA. oryzae as compared to enzyme fromP. canescens[39].Table 1
Inhibition ofβ-D-glucosidases at pH 4.2 and 30°C.
Inhibitor (R)
β-D-Glucosidase
β-D-Glucosidase
A. oryzae
P. canescens
K
i, μM
K
i, μM
2a (Ph)a
129
4.02
2b (p-ClC6H4)
183
5.56
2c (p-MeC6H4)
218
11.1
2d (2′-naphthyl)
159
4.53
2e (PhCH2)
109
6.32
2f (n-C6H13)
80.7
3.45
2g (5′-phen)
91.7
2.84
2h (diester)
223
12.3
2i (diacid)
1500
27.5
a
K
i = 1530 μM (for β-D-glucosidase from fungusStachybotrys atra) [21].The previously available data on 1-thio-β-D-glucopyranosides as glycosidase inhibitors were limited to the derivatives with the following aglycones: phenyl (2a), K
i
=
1530 μM (Stachybotrys atra) [21]; 4-nitrophenyl, K
i
=
664 μM (rice) and K
i
=
95 μM (barley) [22]; benzo-1,4-quinone, K
i
=
60 μM (A. faecalis); chlorobenzo-1,4-quinone, K
i
=
400 μM (A. faecalis) [23]; and 2-(3-methyl-3H-diazirine-3-yl)-ethyl, K
i
=
1500 μM (sweet almond) [24]. Although thioglycosides are usually resistant to hydrolysis, it was found also that with some specific aglycones they may be hydrolysable and can serve as glycosidase substrates [40]. It is interesting to compare our results (Table 1) to the data for some known inhibitors of β-D-glucosidases: 1-deoxynojirimycin, K
i
=
0.3
–
2.7 μM (Aspergillus wentii); K
i
=
12 μM (Agrobacterium faecalis);N-methyl-1-deoxynojirimycin, K
i
=
43 μM (sweet almond); andN-hexyl-1-deoxynojirimycin, K
i
=
200 μM (calf liver cytosolic) and K
i
=
69 μM (calf spleen lysosomal) [41]. From this comparison, compounds2a–2h are moderately strong β-D-glucosidase inhibitors.The comparison of thioglucosides2a–2i allows a preliminary elucidation of the effect of aglycon upon their inhibitory activity. It is known that the presence of alkyl groups and other lipophilic moieties often increases the efficiency of glycosidase inhibitors. This effect, mostly towards β-glucosidases, was observed for derivatives of 1-deoxynojirimycin and other iminosugars, alkyl glycosides, carbasugars, and some other inhibitors. It was explained by additional stabilization of the alkyl chains or aromatic residues by the hydrophobic pocket or cleft within or in vicinity of the enzyme binding site (for the detailed discussion see [25, 42, 43] and references therein; in addition see [37, 44, 45]). The results of our study (Table 1) seem to confirm the general regularities observed earlier. Thus, the most potent inhibitors of β-D-glucosidases, compounds2f and2g, have large lipophilic aglycones, while the thioglucoside2i with the most polar aglycone in this series shows almost negligible inhibition. However, the dependence of inhibition on the aglycone’s lipophilicity is not straightforward: despite their large aromatic aglycones, the thioglucosides2a–2e produced much weaker inhibition than2f and2 g (see also discussion in [25]). The influence of the aglycone shape remains also uncertain. The best results were achieved both for the narrow alkyl chain (2f) and for the wide polyaromatic moiety (2g).


## 3. Experimental Section

The chemicals used in this study were purchased from commercial sources (Sigma-Aldrich, TCI, and Across Organics) and used without additional purification. All solvents were purified by conventional techniques prior to use. Column chromatography was performed on silica gel (40–75μm, Sorbent Technologies) and aluminum oxide (activated basic, 58Å, Aldrich). The reactions were monitored by TLC on silica gel plates, Analtech Inc (visualization by staining with sulfuric acid followed by heating or with I
2).1H NMR and 13C NMR spectra were acquired on JEOL ECA-600 NMR-spectrometer (600 MHz for 1H and 150 MHz for 13C) with spinning at rt. 1H-1H-COSY and 1H-13C-HMQC techniques were used to assign the signals. High resolution mass spectra (HRMS) were obtained on a JEOL AccuTOF time-of-flight mass spectrometer (Peabody, MA) coupled with an Ionsense DART open-air ionization source (Saugus, MA). The instrument was tuned to a resolving power of 7,000 with reserpine directly infused into the electrospray ionization source; this provided a stable ion current to tune the time-of-flight parameters. Samples were introduced into the DART sample gap with a glass melting point capillary by first dipping the closed end of the capillary into the sample then immediately placing it into the helium metastable beam. The helium gas temperature was set to 250°C to aid in the desorption of the analyte from the capillary. The samples were held in the sample gap for 10–15 seconds to acquire several mass spectra to average for an accuratem/zassignment. Optical rotation was measured for solutions in a 5 cm cell with an automatic polarimeter Autopol I. A Beckman Du-65 spectrometer was used for the enzymatic hydrolysis studies.


### 3.1. General Procedure for the Synthesis of 1-Thio-β-D-glucopyranoside Tetraacetates (Adapted from [46])

α-D-Glucose pentaacetate (0.5–2.1 g and 1.3–5.3 mmol) was dissolved in CH2Cl2 (25 mL), followed by addition of a thiol (1.5 mol eq). The reaction mixture was stirred for 10 minutes in an ice bath. Then BF3
·Et2O (3 mol eq) was added slowly, and the mixture was allowed to come to room temperature and was stirred with gentle reflux overnight. The solvent was removed by evaporation and product was isolated by column chromatography (silica gel, hexane/EtOAc 5 : 1).


#### 3.1.1. Phenyl-1-thio-β-D-glucopyranoside Tetraacetate (1a)

The stirring overnight was done without reflux. Yield 1.01 g (45%) from 2.00 g (5.1 mmol)α-D-glucose pentaacetate. 1H NMR (CDCl3): δ 2.00 (s, CH3), 2.02 (s, CH3), 2.087 (s, CH3), 2.094 (s, CH3), 3.73 (ddd,J = 2.5, 5.1, 10.0 Hz; H5), 4.18 (dd,J = 2.5, 12.2 Hz; H6), 4.23 (dd,J = 5.1, 12.2 Hz; H6), 4.71 (d,J = 10.1 Hz; H1), 4.98 (dd,J = 9.3, 10.1 Hz; H2), 5.05 (dd,J = 9.6, 10.0 Hz; H4), 5.23 (t,J = 9.4 Hz; H3), 7.31–7.35 (m, 3H; Ph), 7.50 (m, 2H; Ph). 13C NMR (CDCl3): 20.60, 20.75 (CH3, Ac), 62.09 (C6), 68.10 (C4), 69.86 (C2), 73.91 (C3), 75.74 (C5), 85.71 (C1), 128.41, 128.92, 131.59, 133.10 (Ph), 169.26, 169.40, 170.18, 170.58 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C20H24O9S requiresm/z [M+NH4]+ 458.1485; observedm/z 458.1468.


#### 3.1.2.p-Chlorophenyl-1-thio-β-D-glucopyranoside Tetraacetate (1b)

Yield 0.97 g (39%) from 2.07 g (5.3 mmol)α-D-glucose pentaacetate. 1H NMR (CDCl3): δ 1.99 (s, CH3), 2.02 (s, CH3), 2.09 (s, CH3), 2.10 (s, CH3), 3.72 (ddd,J = 2.6, 4.9, 10.1 Hz; H5), 4.18 (dd,J = 2.6, 12.3 Hz; H6), 4.22 (dd,J = 4.9, 12.3 Hz; H6), 4.65 (d,J = 10.1 Hz; H1), 4.94 (dd,J = 9.4, 10.1 Hz; H2), 5.02 (t,J= 9.8 Hz; H4), 5.22 (t,J = 9.4 Hz; H3), 7.30 (m, 2H; Ar), 7.44 (m, 2H; Ar). 13C NMR (CDCl3): 20.56, 20.72 (CH3, Ac), 61.96 (C6), 67.99 (C4), 69.71 (C2), 73.79 (C3), 75.81 (C5), 85.16 (C1), 129.04, 129.38, 134.97 (Ar), 169.21, 169.35, 170.13, 170.51 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C20H23ClO9S requiresm/z [M+NH4]+ 492.1095; observedm/z 492.1062.


#### 3.1.3.p-Tolyl-1-thio-β-D-glucopyranoside Tetraacetate (1c)

Yield 1.75 g (71%) from 2.05 g (5.3 mmol)α-D-glucose pentaacetate. 1H NMR (CDCl3): δ 1.99 (s, CH3), 2.02 (s, CH3), 2.09 (s, CH3), 2.10 (s, CH3), 2.35 (s, CH3,p-tolyl), 3.70 (ddd,J = 2.6, 4.9, 10.1 Hz; H5), 4.18 (dd,J = 2.6, 12.2 Hz; H6), 4.22 (dd,J = 4.9, 12.2 Hz; H6), 4.64 (d,J = 10.1 Hz; H1), 4.94 (dd,J = 9.4, 10.0 Hz; H2), 5.03 (t,J = 9.8 Hz; H4), 5.21 (t,J = 9.4 Hz; H3), 7.13 (d,J = 7.8 Hz, 2H; Ar), 7.39 (d,J = 8.0 Hz, 2H; Ar). 13C NMR (CDCl3): 20.68, 20.70, 20.85, 20.87 (CH3, Ac), 21.29 (CH3,p-tolyl), 62.08 (C6), 68.12 (C4), 69.85 (C2), 73.97 (C3), 75.69 (C5), 85.79 (C1), 127.49, 129.65, 133.81, 138.79 (Ar), 169.23, 169.38, 170.19, 170.58 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C21H26O9S requiresm/z [M+NH4]+ 472.1641; observedm/z 472.1590.


#### 3.1.4.  (2-Naphthyl)-1-thio-β-D-glucopyranoside Tetraacetate (1d)

Yield 0.23 g (37%) from 0.50 g (1.3 mmol)α-D-glucose pentaacetate dissolved in 10 mL CH2Cl2. 1H NMR (CDCl3): δ 1.99 (s, CH3), 2.02 (s, CH3), 2.04 (s, CH3), 2.12 (s, CH3), 3.74 (ddd,J = 2.4, 5.1, 10.1 Hz; H5), 4.18 (dd,J = 2.4, 12.2 Hz; H6), 4.24 (dd,J = 5.2, 12.3 Hz; H6), 4.79 (d,J = 10.1 Hz; H1), 5.02 (dd,J = 9.3, 10.0 Hz; H2), 5.05 (dd,J = 9.5, 10.0 Hz; H4), 5.24 (t,J = 9.4 Hz; H3), 7.51 (m, 2H; Ar), 7.56 (dd,J = 1.8, 8.6 Hz, 1H; Ar), 7.79 (d,J = 8.5 Hz, 1H; Ar), 7.82 (m, 2H; Ar), 8.00 (d,J = 1.7 Hz, 1H; Ar). 13C NMR (CDCl3): 20.58, 20.70, 20.79 (CH3, Ac), 62.10 (C6), 68.14 (C4), 69.98 (C2), 73.92 (C3), 75.82 (C5), 85.80 (C1), 126.65, 126.75, 127.68, 128.46, 128.70, 130.19, 132.74, 132.84, 133.39 (Ar), 169.29, 169.36, 170.18, 170.59 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C24H26O9S requiresm/z [M+NH4]+ 508.1641; observedm/z 508.1583.


#### 3.1.5. Benzyl-1-thio-β-D-glucopyranoside Tetraacetate (1e)

Yield 1.09 g (46%) from 2.05 g (5.3 mmol)α-D-glucose pentaacetate. 1H NMR (CDCl3): δ 1.99 (s, CH3), 2.01 (s, CH3), 2.02 (s, CH3), 2.12 (s, CH3), 3.57 (ddd,J = 2.3, 5.1, 10.0 Hz; H5), 3.81 (d,J = 12.9 Hz, 1H; CH2Ph), 3.91 (d,J = 12.9 Hz, 1H; CH2Ph) 4.11 (dd,J = 2.3, 12.4 Hz; H6), 4.21 (dd,J = 5.1, 12.4 Hz; H6), 4.26 (d,J = 10.0 Hz; H1), 5.04 (dd,J = 9.3, 10.0 Hz; H2), 5.05 (dd,J = 9.3, 10.0 Hz; H4), 5.11 (t,J = 9.3 Hz; H3), 7.27–7.34 (m, 5H; Ph). 13C NMR (CDCl3): 20.56, 20.58, 20.64, 20.76 (CH3, Ac), 33.77 (CH2Ph), 62.18 (C6), 68.31 (C4), 69.74 (C2), 73.78 (C3), 75.76 (C5), 81.89 (C1), 127.41, 128.59, 129.04, 136.76 (Ph), 169.40, 170.18, 170.63 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C21H26O9S requiresm/z [M+NH4]+ 472.1641; observedm/z 472.1596.


### 3.2. General Procedure for the Synthesis of 1-Thio-β-D-glucopyranosides (Adapted from [46])

The respective 1-thio-β-D-glucopyranoside tetraacetate1a–1e (100 mg) was mixed with MeOH (2 mL) in a 5 mL flask. The freshly prepared MeONa solution was added slowly until the pH of the mixture was 9-10 as measured by pH paper. The reaction mixture was allowed to stir until all starting tetraacetate was consumed (TLC; silica gel, hexane/EtOAc 5 : 1). The basic solution was then neutralized with Dowex 50W X8 ion exchange resin, filtered, and evaporated to yield 95–99% of pure thioglucoside.


#### 3.2.1. Phenyl-1-thio-β-D-glucopyranoside (2a)

100 mg (0.227 mmol)1a yielded 60 mg (97%)2a as a white solid, mp 131–133°C (Lit. 113°C [48]). 1H NMR (CD3OD): δ 3.21 (dd,J = 8.8, 9.8 Hz; H2), 3.28 (m,J = 8.5 Hz; H3), 3.32 (ddd,J = 2.3, 5.7, 9.7 Hz; H5), 3.38 (t,J = 8.6 Hz; H4), 3.66 (dd,J = 5.5, 12.1 Hz; H6), 3.86 (dd,J = 2.1, 12.1 Hz; H6), 4.59 (d,J = 9.8 Hz; H1), 7.22 (m, 1H; Ph), 7.29 (m, 2H; Ph), 7.55 (m, 2H; Ph). 13C NMR (CD3OD): 62.85 (C6), 71.32 (C4), 73.75 (C2), 79.68 (C3), 82.04 (C5), 89.41 (C1), 128.29, 129.87, 132.66, 135.32 (Ph); (Lit. 1H, 13C NMR [48]). HRMS: C12H16O5S requiresm/z [M+NH4]+ 290.1062; observedm/z 290.0993.


#### 3.2.2.p-Chlorophenyl-1-thio-β-D-glucopyranoside (2b)

100 mg (0.21 mmol)1b yielded 61 mg (94%)2b as a white solid, mp 169.5–171.5°C (Lit. 172–175°C [49]). 1H NMR (CD3OD): δ 3.16 (dd,J = 8.8, 9.7 Hz; H2), 3.25 (dd,J = 8.9, 9.6 Hz; H3), 3.32 (ddd,J = 2.2, 5.7, 9.7 Hz; H5), 3.35 (t,J = 8.7 Hz; H4), 3.63 (dd,J = 5.7, 12.1 Hz; H6), 3.85 (dd,J = 2.1, 12.1 Hz; H6), 4.55 (d,J = 9.8 Hz; H1), 7.28 (m, 2H; Ar), 7.52 (m, 2H; Ar). 13C NMR (CD3OD): 62.81 (C6), 71.29(C4), 73.67 (C2), 79.63 (C3), 82.10 (C5), 89.11 (C1), 129.88, 134.04, 134.28, 134.41 (C, Ar). HRMS: C12H15ClO5S requiresm/z [M+NH4]+ 324.0673; observedm/z 324.0619.


#### 3.2.3.p-Tolyl-1-thio-β-D-glucopyranoside (2c)

100 mg (0.22 mmol)1c yielded 62 mg (98%)2c as a white solid, mp 139.5–142°C (Lit. 148°C [48]). 1H NMR (CD3OD): δ 2.30 (s, CH3), 3.16 (dd,J = 8.8, 9.7 Hz; H2), 3.25 (t,J = 9.8 Hz; H3), 3.28 (ddd,J = 2.1, 5.5, 9.6 Hz; H5), 3.35 (t,J = 8.6 Hz; H4), 3.64 (dd,J = 5.4, 12.1 Hz; H6), 3.84 (dd,J = 1.9, 12.0 Hz; H6), 4.50 (d,J = 9.8 Hz; H1), 7.11 (m, 2H; Ar), 7.45 (m, 2H; Ar). 13C NMR (CD3OD): 21.10 (CH3), 62.87 (C6), 71.35 (C4), 73.70 (C2), 79.68 (C3), 81.96 (C5), 89.64 (C1), 130.53, 131.21, 133.48, 138.74 (C, Ar); (Lit. 1H, 13C NMR [48]). HRMS: C13H18O5S requiresm/z [M+NH4]+ 304.1219; observedm/z 304.1214.


#### 3.2.4.  (2-Naphthyl)-1-thio-β-D-glucopyranoside (2d)

100 mg (0.20 mmol)1d yielded 62 mg (94%)2d as a white solid, mp 122–123.5°C (Lit. 117–125°C [50]). 1H NMR (CD3OD): δ 3.26 (dd,J = 9.8, 8.8 Hz; H2), 3.29 (m, H3), 3.32 (ddd,J = 2.3, 6.0, 9.8 Hz; H5), 3.38 (t,J = 8.9 Hz; H4), 3.66 (dd,J = 6.0, 12.1 Hz; H6), 3.86 (dd,J = 2.2, 12.1 Hz; H6), 4.71 (d,J = 9.8 Hz; H1), 7.45 (m, 2H; Ar), 7.63 (m, 1H; Ar), 7.79 (m, 3H; Ar), 8.07 (m, 1H; Ar). 13C NMR (CD3OD): 62.89 (C6), 71.39 (C4), 73.82 (C2), 79.70 (C3), 82.16 (C5), 89.37 (C1), 127.18, 127.52, 128.53, 128.65, 129.23, 130.13, 131.16, 132.75, 133.81, 135.14 (Ar). HRMS: C16H18O5S requiresm/z [M+NH4]+ 340.1219; observedm/z 340.1218.


#### 3.2.5. Benzyl-1-thio-β-D-glucopyranoside (2e)

100 mg (0.22 mmol)1c yielded 61 mg (97%)2c as a clear oil. 1H NMR (CD3OD): δ 3.19–3.29 (m, 4H; H2+H3+H4+H5), 3.67 (dd,J = 6.2, 12.1 Hz; H6), 3.85 (d,J = 12.7 Hz, 1H; CH2Ph), 3.89 (dd,J = 2.2, 12.1 Hz; H6), 4.03 (d,J = 12.7 Hz, 1H; CH2Ph), 4.17 (d,J = 9.3 Hz; H1), 7.22 (t,J = 7.4 Hz, 1H; Ph), 7.29 (t,J = 7.3 Hz, 2H; Ph), 7.37 (m, 2H; Ph). 13C NMR (CD3OD): 32.98 (CH2Ph), 61.67 (C6), 70.27 (C4), 73.02 (C2), 78.35(C3), 80.68 (C5), 83.78 (C1), 126.66, 128.11, 128.97, 138.10 (Ph); (Lit. 1H, 13C NMR [17]). HRMS: C13H18O5S requiresm/z [M+NH4]+ 304.1219; observedm/z 304.1202.


### 3.3. Hexyl-1-thio-β-D-glucopyranoside (2f)

1-Thio-β-D-glucopyranose sodium salt3 (100 mg, 0.46 mmol) was mixed with methanol (2 mL) followed by the addition of bromohexane (75 μL, 0.6 mmol). The reaction mixture was stirred at room temperature overnight and then evaporatedin vacuo, and the resulting white paste was rinsed with hexane and then redissolved in methanol. The resulting suspension was filtered, and the filtrate evaporated to yield clear oil2f (98 mg, 76%). 1H NMR (CD3OD): δ 0.90 (t,J = 11.4 Hz, 3H; CH3) 1.31 (m, 4H; CH2), 1.40 (m, 2H; CH2), 1.62 (m, 2H; CH2), 2.68 (dt,J = 12.5, 7.5 Hz, 1H; SCH2), 2.73 (dt,J = 12.4, 7.3 Hz, 1H; SCH2), 3.18 (dd,J = 8.7, 9.7 Hz; H2), 3.25 (ddd,J = 2.1, 5.5, 9.5 Hz; H5), 3.29 (m, H3), 3.33 (t,J = 8.9 Hz; H4), 3.64 (dd,J = 5.5, 12.0 Hz; H6), 3.84 (dd,J = 2.2, 12.0 Hz; H6), 4.33 (d,J = 9.8 Hz; H1). 13C NMR (CD3OD): 13.05 (CH3), 22.30 (CH2), 28.36 (CH2), 29.52 (CH2), 29.68 (CH2), 31.26 (CH2), 61.58 (C6), 70.17 (C4), 73.06 (C2), 78.27 (C3), 80.79 (C5), 85.81 (C1); (Lit. 1H, 13C NMR [51]). HRMS: C12H24O5S requiresm/z [M+NH4]+ 298.1688; observedm/z 298.1661.


### 3.4.  (1,10-Phenanthrolin-5-yl)-1-thio-β-D-glucopyranoside (2g) [27]

1-Thio-β-D-glucopyranose sodium salt3 (65 mg, 0.30 mmol) in dry ethanol (2 mL) was added to a solution of 5,6-epoxy-5,6-dihydro-1,10-phenanthroline (50 mg, 0.255 mmol) in dry ethanol (2 mL) and stirred at room temperature for 36 h until complete conversion of the epoxide as monitored by TLC (silica gel, EtOAc/CH3OH/NH4OH 7 : 3 : 1). The precipitate was collected and recrystalized from EtOH affording a yellowish solid product (61 mg, 64%): mp 154-155°C; [
α
]
D
24  −64.0° (c 0.38, DMSO). 1H NMR (DMSO): 3.08 (t,J= 9.2 Hz; H4), 3.17 (dd,J= 9.6, 8.8 Hz; H2), 3.23 (t,J= 8.7 Hz; H3), 3.30 (ddd,J= 9.6, 6.5, 2.0 Hz; H5), 3.41 (dd,J= 11.8, 6.4 Hz; H6), 3.69 (dd,J= 11.8, 1.9 Hz; H6), 4.79 (d,J= 9.7 Hz; H1), 7.74 (dd,J= 8.1, 4.3 Hz; H8′), 7.80 (dd,J= 8.3, 4.2 Hz; H3′), 8.29 (s, H6′), 8.36 (dd,J= 8.1, 1.6 Hz; H4′), 8.79 (dd,J= 8.4, 1.6 Hz; H7′), 9.02 (dd,J= 4.3, 1.6 Hz; H9′), 9.09 (dd,J= 4.2, 1.6 Hz; H2′). 13C NMR (DMSO): 61.57 (C6), 70.36 (C4), 73.17 (C2), 79.74 (C3), 81.75 (C5), 87.46 (C1), 123.91 (C3′), 124.29 (C8′), 128.73, 128.82 (C4a′/C6a′), 129.13 (C6′), 131.44 (C5′), 134.13 (C4′), 136.34 (C7′), 145.20, 145.86 (C10a′/C10b′), 150.48, 150.55 (C2′/C9′). HRMS: C30H26N4O2S2 requiresm/z [M+H]+ 375.1015; observedm/z 375.1022.


### 3.5. Diethyl 7-oxabicyclo[4.1.0]heptane-trans-3,4-dicarboxylate (4)

The epoxide4 was prepared as described before [25] and isolated by column chromatography (Al2O3, gradient washing with hexane/EtOAc 8 : 2 → 7 : 3) as a colorless liquid (60%). 1H NMR (CDCl3): δ 1.21 (t,J= 7.1 Hz, 3H; CH3), 1.22 (t,J= 7.1 Hz, 3H; CH3), 1.87 (ddd,J= 14.9, 10.8, 2.1 Hz; H2), 2.04 (dd,J= 15.5, 10.9 Hz; H5), 2.30 (ddd,J= 15.5, 6.6, 4.8 Hz; H5), 2.45 (ddd,J= 14.9, 4.8, 1.8 Hz; H2), 2.58 (dt,J= 10.7, 6.7 Hz; H4), 2.80 (dt,J= 10.7, 4.9 Hz; H3), 3.17 (t,J= 4.3 Hz; H6), 3.24 (m, H1), 4.11 (m, 4H; OCH2). 13C NMR (CDCl3): δ 14.20 (CH3), 26.44 (C5), 27.30 (C2), 37.80 (C3), 40.14 (C4), 50.40 (C6), 52.00 (C1), 60.84 (OCH2), 173.76, 174.82 (C=O). HRMS: C12H18O5 requires [M+H]+
m/z243.1233; observedm/z243.1240.


### 3.6.  [(1S*,2S*,4S*,5S*)-2-Hydroxy-4,5-bis(ethoxycarbonyl)-cyclohexyl]-1-thio-β-D-glucopyranoside (2h)

1-Thio-β-D-glucopyranose sodium salt3 (100 mg, 0.46 mmol) in 7 mL of ethanol was added to a suspension of epoxide4 (80 mg, 0.33 mmol) in ethanol (2 mL) at room temperature. Reaction mixture was stirred at 40°C for 12 h until complete conversion of the epoxide as monitored by TLC (silica gel, EtOAc/MeOH 4 : 1). The solvent was evaporated under vacuum and residue was purified by column chromatography (silica gel, EtOAc/MeOH 7 : 3) affording product2h (a mixture of diastereomers 1 : 1) as a white solid (55 mg, 38%). 1H NMR (CD3OD): δ 1.21 (t,J = 7.1 Hz, 6H; 2CH3), 1.89 (dd,J = 8.3, 3.4 Hz, 1H; H3′); 1.94−2.05 (m, 2H; H3′+H6′), 2.21, 2.24 (dt,J = 3.9, 11.6 Hz, 1H; H6′), 2.86, 2.89 (dt,J = 3.7, 11.3 Hz; H4′), 2.98 (m, H5′), 3.17 (t,J = 8.6 Hz; H2), 3.23–3.35 (m, 4H; H1′+H3+H4+H5), 3.62, 3.65 (dd,J = 12.1, 5.1 Hz, 1H; H6), 3.84 (dd,J = 12.1, 1.8 Hz, 1H; H6), 3.98, 4.02 (br dd,J = 6.4, 3.3 Hz; H2′), 4.06–4.14 (m, 4H; OCH2), 4.41, 4.42 (d,J = 9.8 Hz; H1). 13C NMR (CD3OD): δ 13.14 (CH3), 28.51, 28.92 (C6′), 30.29, 30.45 (C3′), 39.09, 39.14 (C5′), 40.22, 40.24 (C4′), 44.34, 44.89 (C1′), 60.53, 60.58 (OCH2), 61.53 (C6), 67.73, 68.36 (C2′), 69.92, 70.01 (C4), 73.12, 73.18 (C2), 78.21 (C3), 80.77 (C5), 85.91, 86.00 (C1), 174.88, 175.18 (C=O). HRMS: C18H30O10S requiresm/z [M+NH4]+ 456.1903, [M+H]+ 439.1638; observedm/z 456.1967, 439.1654.


### 3.7.  [(1S*,2S*,4S*,5S*)-2-Hydroxy-4,5-bis (hydroxycarbonyl)-cyclohexyl]-1-thio-β-D-glucopyranoside (2i)

1 mL of KOH (1 M) was added to a solution of diester2h (150 mg, 0.34 mmol) in 2 mL of methanol and mixture stirred for 72 h at room temperature. HCl (1 M) was added to reaction mixture till pH 6 and solvent was evaporated under vacuum. Residue was purified by column chromatography (silica gel, EtOAc/CH3OH 1 : 4) affording product2i (one diastereomer) as a white solid (40 mg, 31%; mp > 240°C). 1H NMR (CD3OD): δ 1.85 (dd,J= 13.4, 3.8 Hz; H3′), 1.93–2.05 (m, 2H; H6′+H3′), 2.26 (m, 1H; H6′), 2.85 (m, H5′), 2.95 (m, H4′), 3.19 (t,J= 9.1 Hz; H2), 3.21–3.35 (m, 4H; H1′+H3+H4+H5), 3.64 (m, 1H; H6), 3.84 (m, 1H; H6), 3.95 (m, H2′), 4.43 (d,J= 9.8 Hz; H1). 13C NMR (CD3OD): δ 29.23 (C6′), 29.66 (C3′), 31.02 (C4′), 31.18 (C5′), 48.53 (C1′), 61.46 (C6), 69.05 (C2′), 69.98 (C4), 73.17 (C2), 78.24, 80.72 (C3/C5), 85.99 (C1), 179.73 (C=O). HRMS: C14H22O10S requiresm/z [M+NH4]+ 400.1277; observedm/z 400.1289.


### 3.8. Procedures for the Glycosidase Inhibition and Activation Assay

The enzyme activities (α/β-D-galactosidases and α/β-D-glucosidases) were assayed using multienzyme complexes isolated from fungiP. canescens andA. oryzae as described before [25–27]. All assays were performed in a standard way by monitoring spectrophotometrically (with Beckman Du-65 spectrometer) the release ofp-nitrophenol from the correspondingp-nitrophenyl glycosides at 30°C. One unit of enzyme activity was defined as the amount of enzyme that releases 1 μmol ofp-nitrophenol per minute. Enzyme and substrate concentrations were selected so that the degree of hydrolysis was never more than 20% and in most cases was less than 10%, over the course of the assay. The method used to measure the rate of the reaction assumes that the amount of the substrate is high enough, such that the disappearance over a given period is insignificant; that is, the rate of the reaction is close to linear for the first stage of the reaction.For the preliminary estimation, the enzyme solutions (100μL with activities 750
±
150 μU) were mixed with a set of inhibitor/activator solutions (100 μL, 10 mM) and then diluted with 700 μL of 0.2 M acetic buffer (pH 4.2), and the mixture was incubated for 1 h at 30°C. The reaction was initiated with addition of a proper substrate (100 μL of 20 mMp-nitrophenyl glycopyranoside), and aliquots were taken after 5 and 10 min. The reaction was terminated by addition of 1 mL of 1 M Na2CO3 to 0.5 mL of aliquot solution. The concentration of the releasedp-nitrophenol was determined at 400 nm using molar extinction coefficient 18.3 mM−1cm−1. The inhibition/activation was estimated as a loss/increase of enzymatic activity in % (Figures 1 and 2).The same protocol was used for estimation of the kinetic parameters with the following changes: 1 mL of enzyme solution, 0.5 mL inhibitor solution, and 3.0 mL of 0.2 M acetic buffer (pH 4.2) were incubated for 1 h at 30°C before addition of 0.5 mL of substrate (20 mM). Six substrate solutions were prepared with various concentrations up to saturation point for each enzyme. The final substrate concentration after all additions varied from 0.01 mM to 2.00 mM. The final inhibitor concentrations were 330μM forA. oryzae and 33 μM forP. canescens. Reaction rates were measured as described above. Control experiments with no enzyme were performed to exclude the errors due to the substrate spontaneous hydrolysis. Enzyme behavior (at pH 4.2 and 30°C) abided the Michaelis-Menten equation. Parameters K
M and K
M
app were calculated from Lineweaver-Burk (double-reciprocal) plot [52, 53]. The K
M values were used to calculate K
i by the following equation:
(1)
K
i
=
K
M
[
I
]
(
K
M
app
-
K
M
)
.
Three independent trials using freshly prepared substrate and enzyme solutions were performed to obtain each parameter. Values for K
M were reproducible within ±5% and ±10%, respectively, and the standard error was ±15% for the K
i values (Table 1).


## 3.1. General Procedure for the Synthesis of 1-Thio-β-D-glucopyranoside Tetraacetates (Adapted from [46])

α-D-Glucose pentaacetate (0.5–2.1 g and 1.3–5.3 mmol) was dissolved in CH2Cl2 (25 mL), followed by addition of a thiol (1.5 mol eq). The reaction mixture was stirred for 10 minutes in an ice bath. Then BF3
·Et2O (3 mol eq) was added slowly, and the mixture was allowed to come to room temperature and was stirred with gentle reflux overnight. The solvent was removed by evaporation and product was isolated by column chromatography (silica gel, hexane/EtOAc 5 : 1).


### 3.1.1. Phenyl-1-thio-β-D-glucopyranoside Tetraacetate (1a)

The stirring overnight was done without reflux. Yield 1.01 g (45%) from 2.00 g (5.1 mmol)α-D-glucose pentaacetate. 1H NMR (CDCl3): δ 2.00 (s, CH3), 2.02 (s, CH3), 2.087 (s, CH3), 2.094 (s, CH3), 3.73 (ddd,J = 2.5, 5.1, 10.0 Hz; H5), 4.18 (dd,J = 2.5, 12.2 Hz; H6), 4.23 (dd,J = 5.1, 12.2 Hz; H6), 4.71 (d,J = 10.1 Hz; H1), 4.98 (dd,J = 9.3, 10.1 Hz; H2), 5.05 (dd,J = 9.6, 10.0 Hz; H4), 5.23 (t,J = 9.4 Hz; H3), 7.31–7.35 (m, 3H; Ph), 7.50 (m, 2H; Ph). 13C NMR (CDCl3): 20.60, 20.75 (CH3, Ac), 62.09 (C6), 68.10 (C4), 69.86 (C2), 73.91 (C3), 75.74 (C5), 85.71 (C1), 128.41, 128.92, 131.59, 133.10 (Ph), 169.26, 169.40, 170.18, 170.58 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C20H24O9S requiresm/z [M+NH4]+ 458.1485; observedm/z 458.1468.


### 3.1.2.p-Chlorophenyl-1-thio-β-D-glucopyranoside Tetraacetate (1b)

Yield 0.97 g (39%) from 2.07 g (5.3 mmol)α-D-glucose pentaacetate. 1H NMR (CDCl3): δ 1.99 (s, CH3), 2.02 (s, CH3), 2.09 (s, CH3), 2.10 (s, CH3), 3.72 (ddd,J = 2.6, 4.9, 10.1 Hz; H5), 4.18 (dd,J = 2.6, 12.3 Hz; H6), 4.22 (dd,J = 4.9, 12.3 Hz; H6), 4.65 (d,J = 10.1 Hz; H1), 4.94 (dd,J = 9.4, 10.1 Hz; H2), 5.02 (t,J= 9.8 Hz; H4), 5.22 (t,J = 9.4 Hz; H3), 7.30 (m, 2H; Ar), 7.44 (m, 2H; Ar). 13C NMR (CDCl3): 20.56, 20.72 (CH3, Ac), 61.96 (C6), 67.99 (C4), 69.71 (C2), 73.79 (C3), 75.81 (C5), 85.16 (C1), 129.04, 129.38, 134.97 (Ar), 169.21, 169.35, 170.13, 170.51 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C20H23ClO9S requiresm/z [M+NH4]+ 492.1095; observedm/z 492.1062.


### 3.1.3.p-Tolyl-1-thio-β-D-glucopyranoside Tetraacetate (1c)

Yield 1.75 g (71%) from 2.05 g (5.3 mmol)α-D-glucose pentaacetate. 1H NMR (CDCl3): δ 1.99 (s, CH3), 2.02 (s, CH3), 2.09 (s, CH3), 2.10 (s, CH3), 2.35 (s, CH3,p-tolyl), 3.70 (ddd,J = 2.6, 4.9, 10.1 Hz; H5), 4.18 (dd,J = 2.6, 12.2 Hz; H6), 4.22 (dd,J = 4.9, 12.2 Hz; H6), 4.64 (d,J = 10.1 Hz; H1), 4.94 (dd,J = 9.4, 10.0 Hz; H2), 5.03 (t,J = 9.8 Hz; H4), 5.21 (t,J = 9.4 Hz; H3), 7.13 (d,J = 7.8 Hz, 2H; Ar), 7.39 (d,J = 8.0 Hz, 2H; Ar). 13C NMR (CDCl3): 20.68, 20.70, 20.85, 20.87 (CH3, Ac), 21.29 (CH3,p-tolyl), 62.08 (C6), 68.12 (C4), 69.85 (C2), 73.97 (C3), 75.69 (C5), 85.79 (C1), 127.49, 129.65, 133.81, 138.79 (Ar), 169.23, 169.38, 170.19, 170.58 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C21H26O9S requiresm/z [M+NH4]+ 472.1641; observedm/z 472.1590.


### 3.1.4.  (2-Naphthyl)-1-thio-β-D-glucopyranoside Tetraacetate (1d)

Yield 0.23 g (37%) from 0.50 g (1.3 mmol)α-D-glucose pentaacetate dissolved in 10 mL CH2Cl2. 1H NMR (CDCl3): δ 1.99 (s, CH3), 2.02 (s, CH3), 2.04 (s, CH3), 2.12 (s, CH3), 3.74 (ddd,J = 2.4, 5.1, 10.1 Hz; H5), 4.18 (dd,J = 2.4, 12.2 Hz; H6), 4.24 (dd,J = 5.2, 12.3 Hz; H6), 4.79 (d,J = 10.1 Hz; H1), 5.02 (dd,J = 9.3, 10.0 Hz; H2), 5.05 (dd,J = 9.5, 10.0 Hz; H4), 5.24 (t,J = 9.4 Hz; H3), 7.51 (m, 2H; Ar), 7.56 (dd,J = 1.8, 8.6 Hz, 1H; Ar), 7.79 (d,J = 8.5 Hz, 1H; Ar), 7.82 (m, 2H; Ar), 8.00 (d,J = 1.7 Hz, 1H; Ar). 13C NMR (CDCl3): 20.58, 20.70, 20.79 (CH3, Ac), 62.10 (C6), 68.14 (C4), 69.98 (C2), 73.92 (C3), 75.82 (C5), 85.80 (C1), 126.65, 126.75, 127.68, 128.46, 128.70, 130.19, 132.74, 132.84, 133.39 (Ar), 169.29, 169.36, 170.18, 170.59 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C24H26O9S requiresm/z [M+NH4]+ 508.1641; observedm/z 508.1583.


### 3.1.5. Benzyl-1-thio-β-D-glucopyranoside Tetraacetate (1e)

Yield 1.09 g (46%) from 2.05 g (5.3 mmol)α-D-glucose pentaacetate. 1H NMR (CDCl3): δ 1.99 (s, CH3), 2.01 (s, CH3), 2.02 (s, CH3), 2.12 (s, CH3), 3.57 (ddd,J = 2.3, 5.1, 10.0 Hz; H5), 3.81 (d,J = 12.9 Hz, 1H; CH2Ph), 3.91 (d,J = 12.9 Hz, 1H; CH2Ph) 4.11 (dd,J = 2.3, 12.4 Hz; H6), 4.21 (dd,J = 5.1, 12.4 Hz; H6), 4.26 (d,J = 10.0 Hz; H1), 5.04 (dd,J = 9.3, 10.0 Hz; H2), 5.05 (dd,J = 9.3, 10.0 Hz; H4), 5.11 (t,J = 9.3 Hz; H3), 7.27–7.34 (m, 5H; Ph). 13C NMR (CDCl3): 20.56, 20.58, 20.64, 20.76 (CH3, Ac), 33.77 (CH2Ph), 62.18 (C6), 68.31 (C4), 69.74 (C2), 73.78 (C3), 75.76 (C5), 81.89 (C1), 127.41, 128.59, 129.04, 136.76 (Ph), 169.40, 170.18, 170.63 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C21H26O9S requiresm/z [M+NH4]+ 472.1641; observedm/z 472.1596.


## 3.1.1. Phenyl-1-thio-β-D-glucopyranoside Tetraacetate (1a)

The stirring overnight was done without reflux. Yield 1.01 g (45%) from 2.00 g (5.1 mmol)α-D-glucose pentaacetate. 1H NMR (CDCl3): δ 2.00 (s, CH3), 2.02 (s, CH3), 2.087 (s, CH3), 2.094 (s, CH3), 3.73 (ddd,J = 2.5, 5.1, 10.0 Hz; H5), 4.18 (dd,J = 2.5, 12.2 Hz; H6), 4.23 (dd,J = 5.1, 12.2 Hz; H6), 4.71 (d,J = 10.1 Hz; H1), 4.98 (dd,J = 9.3, 10.1 Hz; H2), 5.05 (dd,J = 9.6, 10.0 Hz; H4), 5.23 (t,J = 9.4 Hz; H3), 7.31–7.35 (m, 3H; Ph), 7.50 (m, 2H; Ph). 13C NMR (CDCl3): 20.60, 20.75 (CH3, Ac), 62.09 (C6), 68.10 (C4), 69.86 (C2), 73.91 (C3), 75.74 (C5), 85.71 (C1), 128.41, 128.92, 131.59, 133.10 (Ph), 169.26, 169.40, 170.18, 170.58 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C20H24O9S requiresm/z [M+NH4]+ 458.1485; observedm/z 458.1468.


## 3.1.2.p-Chlorophenyl-1-thio-β-D-glucopyranoside Tetraacetate (1b)

Yield 0.97 g (39%) from 2.07 g (5.3 mmol)α-D-glucose pentaacetate. 1H NMR (CDCl3): δ 1.99 (s, CH3), 2.02 (s, CH3), 2.09 (s, CH3), 2.10 (s, CH3), 3.72 (ddd,J = 2.6, 4.9, 10.1 Hz; H5), 4.18 (dd,J = 2.6, 12.3 Hz; H6), 4.22 (dd,J = 4.9, 12.3 Hz; H6), 4.65 (d,J = 10.1 Hz; H1), 4.94 (dd,J = 9.4, 10.1 Hz; H2), 5.02 (t,J= 9.8 Hz; H4), 5.22 (t,J = 9.4 Hz; H3), 7.30 (m, 2H; Ar), 7.44 (m, 2H; Ar). 13C NMR (CDCl3): 20.56, 20.72 (CH3, Ac), 61.96 (C6), 67.99 (C4), 69.71 (C2), 73.79 (C3), 75.81 (C5), 85.16 (C1), 129.04, 129.38, 134.97 (Ar), 169.21, 169.35, 170.13, 170.51 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C20H23ClO9S requiresm/z [M+NH4]+ 492.1095; observedm/z 492.1062.


## 3.1.3.p-Tolyl-1-thio-β-D-glucopyranoside Tetraacetate (1c)

Yield 1.75 g (71%) from 2.05 g (5.3 mmol)α-D-glucose pentaacetate. 1H NMR (CDCl3): δ 1.99 (s, CH3), 2.02 (s, CH3), 2.09 (s, CH3), 2.10 (s, CH3), 2.35 (s, CH3,p-tolyl), 3.70 (ddd,J = 2.6, 4.9, 10.1 Hz; H5), 4.18 (dd,J = 2.6, 12.2 Hz; H6), 4.22 (dd,J = 4.9, 12.2 Hz; H6), 4.64 (d,J = 10.1 Hz; H1), 4.94 (dd,J = 9.4, 10.0 Hz; H2), 5.03 (t,J = 9.8 Hz; H4), 5.21 (t,J = 9.4 Hz; H3), 7.13 (d,J = 7.8 Hz, 2H; Ar), 7.39 (d,J = 8.0 Hz, 2H; Ar). 13C NMR (CDCl3): 20.68, 20.70, 20.85, 20.87 (CH3, Ac), 21.29 (CH3,p-tolyl), 62.08 (C6), 68.12 (C4), 69.85 (C2), 73.97 (C3), 75.69 (C5), 85.79 (C1), 127.49, 129.65, 133.81, 138.79 (Ar), 169.23, 169.38, 170.19, 170.58 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C21H26O9S requiresm/z [M+NH4]+ 472.1641; observedm/z 472.1590.


## 3.1.4.  (2-Naphthyl)-1-thio-β-D-glucopyranoside Tetraacetate (1d)

Yield 0.23 g (37%) from 0.50 g (1.3 mmol)α-D-glucose pentaacetate dissolved in 10 mL CH2Cl2. 1H NMR (CDCl3): δ 1.99 (s, CH3), 2.02 (s, CH3), 2.04 (s, CH3), 2.12 (s, CH3), 3.74 (ddd,J = 2.4, 5.1, 10.1 Hz; H5), 4.18 (dd,J = 2.4, 12.2 Hz; H6), 4.24 (dd,J = 5.2, 12.3 Hz; H6), 4.79 (d,J = 10.1 Hz; H1), 5.02 (dd,J = 9.3, 10.0 Hz; H2), 5.05 (dd,J = 9.5, 10.0 Hz; H4), 5.24 (t,J = 9.4 Hz; H3), 7.51 (m, 2H; Ar), 7.56 (dd,J = 1.8, 8.6 Hz, 1H; Ar), 7.79 (d,J = 8.5 Hz, 1H; Ar), 7.82 (m, 2H; Ar), 8.00 (d,J = 1.7 Hz, 1H; Ar). 13C NMR (CDCl3): 20.58, 20.70, 20.79 (CH3, Ac), 62.10 (C6), 68.14 (C4), 69.98 (C2), 73.92 (C3), 75.82 (C5), 85.80 (C1), 126.65, 126.75, 127.68, 128.46, 128.70, 130.19, 132.74, 132.84, 133.39 (Ar), 169.29, 169.36, 170.18, 170.59 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C24H26O9S requiresm/z [M+NH4]+ 508.1641; observedm/z 508.1583.


## 3.1.5. Benzyl-1-thio-β-D-glucopyranoside Tetraacetate (1e)

Yield 1.09 g (46%) from 2.05 g (5.3 mmol)α-D-glucose pentaacetate. 1H NMR (CDCl3): δ 1.99 (s, CH3), 2.01 (s, CH3), 2.02 (s, CH3), 2.12 (s, CH3), 3.57 (ddd,J = 2.3, 5.1, 10.0 Hz; H5), 3.81 (d,J = 12.9 Hz, 1H; CH2Ph), 3.91 (d,J = 12.9 Hz, 1H; CH2Ph) 4.11 (dd,J = 2.3, 12.4 Hz; H6), 4.21 (dd,J = 5.1, 12.4 Hz; H6), 4.26 (d,J = 10.0 Hz; H1), 5.04 (dd,J = 9.3, 10.0 Hz; H2), 5.05 (dd,J = 9.3, 10.0 Hz; H4), 5.11 (t,J = 9.3 Hz; H3), 7.27–7.34 (m, 5H; Ph). 13C NMR (CDCl3): 20.56, 20.58, 20.64, 20.76 (CH3, Ac), 33.77 (CH2Ph), 62.18 (C6), 68.31 (C4), 69.74 (C2), 73.78 (C3), 75.76 (C5), 81.89 (C1), 127.41, 128.59, 129.04, 136.76 (Ph), 169.40, 170.18, 170.63 (C=O); (Lit. 1H, 13C NMR [47]). HRMS: C21H26O9S requiresm/z [M+NH4]+ 472.1641; observedm/z 472.1596.


## 3.2. General Procedure for the Synthesis of 1-Thio-β-D-glucopyranosides (Adapted from [46])

The respective 1-thio-β-D-glucopyranoside tetraacetate1a–1e (100 mg) was mixed with MeOH (2 mL) in a 5 mL flask. The freshly prepared MeONa solution was added slowly until the pH of the mixture was 9-10 as measured by pH paper. The reaction mixture was allowed to stir until all starting tetraacetate was consumed (TLC; silica gel, hexane/EtOAc 5 : 1). The basic solution was then neutralized with Dowex 50W X8 ion exchange resin, filtered, and evaporated to yield 95–99% of pure thioglucoside.


### 3.2.1. Phenyl-1-thio-β-D-glucopyranoside (2a)

100 mg (0.227 mmol)1a yielded 60 mg (97%)2a as a white solid, mp 131–133°C (Lit. 113°C [48]). 1H NMR (CD3OD): δ 3.21 (dd,J = 8.8, 9.8 Hz; H2), 3.28 (m,J = 8.5 Hz; H3), 3.32 (ddd,J = 2.3, 5.7, 9.7 Hz; H5), 3.38 (t,J = 8.6 Hz; H4), 3.66 (dd,J = 5.5, 12.1 Hz; H6), 3.86 (dd,J = 2.1, 12.1 Hz; H6), 4.59 (d,J = 9.8 Hz; H1), 7.22 (m, 1H; Ph), 7.29 (m, 2H; Ph), 7.55 (m, 2H; Ph). 13C NMR (CD3OD): 62.85 (C6), 71.32 (C4), 73.75 (C2), 79.68 (C3), 82.04 (C5), 89.41 (C1), 128.29, 129.87, 132.66, 135.32 (Ph); (Lit. 1H, 13C NMR [48]). HRMS: C12H16O5S requiresm/z [M+NH4]+ 290.1062; observedm/z 290.0993.


### 3.2.2.p-Chlorophenyl-1-thio-β-D-glucopyranoside (2b)

100 mg (0.21 mmol)1b yielded 61 mg (94%)2b as a white solid, mp 169.5–171.5°C (Lit. 172–175°C [49]). 1H NMR (CD3OD): δ 3.16 (dd,J = 8.8, 9.7 Hz; H2), 3.25 (dd,J = 8.9, 9.6 Hz; H3), 3.32 (ddd,J = 2.2, 5.7, 9.7 Hz; H5), 3.35 (t,J = 8.7 Hz; H4), 3.63 (dd,J = 5.7, 12.1 Hz; H6), 3.85 (dd,J = 2.1, 12.1 Hz; H6), 4.55 (d,J = 9.8 Hz; H1), 7.28 (m, 2H; Ar), 7.52 (m, 2H; Ar). 13C NMR (CD3OD): 62.81 (C6), 71.29(C4), 73.67 (C2), 79.63 (C3), 82.10 (C5), 89.11 (C1), 129.88, 134.04, 134.28, 134.41 (C, Ar). HRMS: C12H15ClO5S requiresm/z [M+NH4]+ 324.0673; observedm/z 324.0619.


### 3.2.3.p-Tolyl-1-thio-β-D-glucopyranoside (2c)

100 mg (0.22 mmol)1c yielded 62 mg (98%)2c as a white solid, mp 139.5–142°C (Lit. 148°C [48]). 1H NMR (CD3OD): δ 2.30 (s, CH3), 3.16 (dd,J = 8.8, 9.7 Hz; H2), 3.25 (t,J = 9.8 Hz; H3), 3.28 (ddd,J = 2.1, 5.5, 9.6 Hz; H5), 3.35 (t,J = 8.6 Hz; H4), 3.64 (dd,J = 5.4, 12.1 Hz; H6), 3.84 (dd,J = 1.9, 12.0 Hz; H6), 4.50 (d,J = 9.8 Hz; H1), 7.11 (m, 2H; Ar), 7.45 (m, 2H; Ar). 13C NMR (CD3OD): 21.10 (CH3), 62.87 (C6), 71.35 (C4), 73.70 (C2), 79.68 (C3), 81.96 (C5), 89.64 (C1), 130.53, 131.21, 133.48, 138.74 (C, Ar); (Lit. 1H, 13C NMR [48]). HRMS: C13H18O5S requiresm/z [M+NH4]+ 304.1219; observedm/z 304.1214.


### 3.2.4.  (2-Naphthyl)-1-thio-β-D-glucopyranoside (2d)

100 mg (0.20 mmol)1d yielded 62 mg (94%)2d as a white solid, mp 122–123.5°C (Lit. 117–125°C [50]). 1H NMR (CD3OD): δ 3.26 (dd,J = 9.8, 8.8 Hz; H2), 3.29 (m, H3), 3.32 (ddd,J = 2.3, 6.0, 9.8 Hz; H5), 3.38 (t,J = 8.9 Hz; H4), 3.66 (dd,J = 6.0, 12.1 Hz; H6), 3.86 (dd,J = 2.2, 12.1 Hz; H6), 4.71 (d,J = 9.8 Hz; H1), 7.45 (m, 2H; Ar), 7.63 (m, 1H; Ar), 7.79 (m, 3H; Ar), 8.07 (m, 1H; Ar). 13C NMR (CD3OD): 62.89 (C6), 71.39 (C4), 73.82 (C2), 79.70 (C3), 82.16 (C5), 89.37 (C1), 127.18, 127.52, 128.53, 128.65, 129.23, 130.13, 131.16, 132.75, 133.81, 135.14 (Ar). HRMS: C16H18O5S requiresm/z [M+NH4]+ 340.1219; observedm/z 340.1218.


### 3.2.5. Benzyl-1-thio-β-D-glucopyranoside (2e)

100 mg (0.22 mmol)1c yielded 61 mg (97%)2c as a clear oil. 1H NMR (CD3OD): δ 3.19–3.29 (m, 4H; H2+H3+H4+H5), 3.67 (dd,J = 6.2, 12.1 Hz; H6), 3.85 (d,J = 12.7 Hz, 1H; CH2Ph), 3.89 (dd,J = 2.2, 12.1 Hz; H6), 4.03 (d,J = 12.7 Hz, 1H; CH2Ph), 4.17 (d,J = 9.3 Hz; H1), 7.22 (t,J = 7.4 Hz, 1H; Ph), 7.29 (t,J = 7.3 Hz, 2H; Ph), 7.37 (m, 2H; Ph). 13C NMR (CD3OD): 32.98 (CH2Ph), 61.67 (C6), 70.27 (C4), 73.02 (C2), 78.35(C3), 80.68 (C5), 83.78 (C1), 126.66, 128.11, 128.97, 138.10 (Ph); (Lit. 1H, 13C NMR [17]). HRMS: C13H18O5S requiresm/z [M+NH4]+ 304.1219; observedm/z 304.1202.


## 3.2.1. Phenyl-1-thio-β-D-glucopyranoside (2a)

100 mg (0.227 mmol)1a yielded 60 mg (97%)2a as a white solid, mp 131–133°C (Lit. 113°C [48]). 1H NMR (CD3OD): δ 3.21 (dd,J = 8.8, 9.8 Hz; H2), 3.28 (m,J = 8.5 Hz; H3), 3.32 (ddd,J = 2.3, 5.7, 9.7 Hz; H5), 3.38 (t,J = 8.6 Hz; H4), 3.66 (dd,J = 5.5, 12.1 Hz; H6), 3.86 (dd,J = 2.1, 12.1 Hz; H6), 4.59 (d,J = 9.8 Hz; H1), 7.22 (m, 1H; Ph), 7.29 (m, 2H; Ph), 7.55 (m, 2H; Ph). 13C NMR (CD3OD): 62.85 (C6), 71.32 (C4), 73.75 (C2), 79.68 (C3), 82.04 (C5), 89.41 (C1), 128.29, 129.87, 132.66, 135.32 (Ph); (Lit. 1H, 13C NMR [48]). HRMS: C12H16O5S requiresm/z [M+NH4]+ 290.1062; observedm/z 290.0993.


## 3.2.2.p-Chlorophenyl-1-thio-β-D-glucopyranoside (2b)

100 mg (0.21 mmol)1b yielded 61 mg (94%)2b as a white solid, mp 169.5–171.5°C (Lit. 172–175°C [49]). 1H NMR (CD3OD): δ 3.16 (dd,J = 8.8, 9.7 Hz; H2), 3.25 (dd,J = 8.9, 9.6 Hz; H3), 3.32 (ddd,J = 2.2, 5.7, 9.7 Hz; H5), 3.35 (t,J = 8.7 Hz; H4), 3.63 (dd,J = 5.7, 12.1 Hz; H6), 3.85 (dd,J = 2.1, 12.1 Hz; H6), 4.55 (d,J = 9.8 Hz; H1), 7.28 (m, 2H; Ar), 7.52 (m, 2H; Ar). 13C NMR (CD3OD): 62.81 (C6), 71.29(C4), 73.67 (C2), 79.63 (C3), 82.10 (C5), 89.11 (C1), 129.88, 134.04, 134.28, 134.41 (C, Ar). HRMS: C12H15ClO5S requiresm/z [M+NH4]+ 324.0673; observedm/z 324.0619.


## 3.2.3.p-Tolyl-1-thio-β-D-glucopyranoside (2c)

100 mg (0.22 mmol)1c yielded 62 mg (98%)2c as a white solid, mp 139.5–142°C (Lit. 148°C [48]). 1H NMR (CD3OD): δ 2.30 (s, CH3), 3.16 (dd,J = 8.8, 9.7 Hz; H2), 3.25 (t,J = 9.8 Hz; H3), 3.28 (ddd,J = 2.1, 5.5, 9.6 Hz; H5), 3.35 (t,J = 8.6 Hz; H4), 3.64 (dd,J = 5.4, 12.1 Hz; H6), 3.84 (dd,J = 1.9, 12.0 Hz; H6), 4.50 (d,J = 9.8 Hz; H1), 7.11 (m, 2H; Ar), 7.45 (m, 2H; Ar). 13C NMR (CD3OD): 21.10 (CH3), 62.87 (C6), 71.35 (C4), 73.70 (C2), 79.68 (C3), 81.96 (C5), 89.64 (C1), 130.53, 131.21, 133.48, 138.74 (C, Ar); (Lit. 1H, 13C NMR [48]). HRMS: C13H18O5S requiresm/z [M+NH4]+ 304.1219; observedm/z 304.1214.


## 3.2.4.  (2-Naphthyl)-1-thio-β-D-glucopyranoside (2d)

100 mg (0.20 mmol)1d yielded 62 mg (94%)2d as a white solid, mp 122–123.5°C (Lit. 117–125°C [50]). 1H NMR (CD3OD): δ 3.26 (dd,J = 9.8, 8.8 Hz; H2), 3.29 (m, H3), 3.32 (ddd,J = 2.3, 6.0, 9.8 Hz; H5), 3.38 (t,J = 8.9 Hz; H4), 3.66 (dd,J = 6.0, 12.1 Hz; H6), 3.86 (dd,J = 2.2, 12.1 Hz; H6), 4.71 (d,J = 9.8 Hz; H1), 7.45 (m, 2H; Ar), 7.63 (m, 1H; Ar), 7.79 (m, 3H; Ar), 8.07 (m, 1H; Ar). 13C NMR (CD3OD): 62.89 (C6), 71.39 (C4), 73.82 (C2), 79.70 (C3), 82.16 (C5), 89.37 (C1), 127.18, 127.52, 128.53, 128.65, 129.23, 130.13, 131.16, 132.75, 133.81, 135.14 (Ar). HRMS: C16H18O5S requiresm/z [M+NH4]+ 340.1219; observedm/z 340.1218.


## 3.2.5. Benzyl-1-thio-β-D-glucopyranoside (2e)

100 mg (0.22 mmol)1c yielded 61 mg (97%)2c as a clear oil. 1H NMR (CD3OD): δ 3.19–3.29 (m, 4H; H2+H3+H4+H5), 3.67 (dd,J = 6.2, 12.1 Hz; H6), 3.85 (d,J = 12.7 Hz, 1H; CH2Ph), 3.89 (dd,J = 2.2, 12.1 Hz; H6), 4.03 (d,J = 12.7 Hz, 1H; CH2Ph), 4.17 (d,J = 9.3 Hz; H1), 7.22 (t,J = 7.4 Hz, 1H; Ph), 7.29 (t,J = 7.3 Hz, 2H; Ph), 7.37 (m, 2H; Ph). 13C NMR (CD3OD): 32.98 (CH2Ph), 61.67 (C6), 70.27 (C4), 73.02 (C2), 78.35(C3), 80.68 (C5), 83.78 (C1), 126.66, 128.11, 128.97, 138.10 (Ph); (Lit. 1H, 13C NMR [17]). HRMS: C13H18O5S requiresm/z [M+NH4]+ 304.1219; observedm/z 304.1202.


## 3.3. Hexyl-1-thio-β-D-glucopyranoside (2f)

1-Thio-β-D-glucopyranose sodium salt3 (100 mg, 0.46 mmol) was mixed with methanol (2 mL) followed by the addition of bromohexane (75 μL, 0.6 mmol). The reaction mixture was stirred at room temperature overnight and then evaporatedin vacuo, and the resulting white paste was rinsed with hexane and then redissolved in methanol. The resulting suspension was filtered, and the filtrate evaporated to yield clear oil2f (98 mg, 76%). 1H NMR (CD3OD): δ 0.90 (t,J = 11.4 Hz, 3H; CH3) 1.31 (m, 4H; CH2), 1.40 (m, 2H; CH2), 1.62 (m, 2H; CH2), 2.68 (dt,J = 12.5, 7.5 Hz, 1H; SCH2), 2.73 (dt,J = 12.4, 7.3 Hz, 1H; SCH2), 3.18 (dd,J = 8.7, 9.7 Hz; H2), 3.25 (ddd,J = 2.1, 5.5, 9.5 Hz; H5), 3.29 (m, H3), 3.33 (t,J = 8.9 Hz; H4), 3.64 (dd,J = 5.5, 12.0 Hz; H6), 3.84 (dd,J = 2.2, 12.0 Hz; H6), 4.33 (d,J = 9.8 Hz; H1). 13C NMR (CD3OD): 13.05 (CH3), 22.30 (CH2), 28.36 (CH2), 29.52 (CH2), 29.68 (CH2), 31.26 (CH2), 61.58 (C6), 70.17 (C4), 73.06 (C2), 78.27 (C3), 80.79 (C5), 85.81 (C1); (Lit. 1H, 13C NMR [51]). HRMS: C12H24O5S requiresm/z [M+NH4]+ 298.1688; observedm/z 298.1661.


## 3.4.  (1,10-Phenanthrolin-5-yl)-1-thio-β-D-glucopyranoside (2g) [27]

1-Thio-β-D-glucopyranose sodium salt3 (65 mg, 0.30 mmol) in dry ethanol (2 mL) was added to a solution of 5,6-epoxy-5,6-dihydro-1,10-phenanthroline (50 mg, 0.255 mmol) in dry ethanol (2 mL) and stirred at room temperature for 36 h until complete conversion of the epoxide as monitored by TLC (silica gel, EtOAc/CH3OH/NH4OH 7 : 3 : 1). The precipitate was collected and recrystalized from EtOH affording a yellowish solid product (61 mg, 64%): mp 154-155°C; [
α
]
D
24  −64.0° (c 0.38, DMSO). 1H NMR (DMSO): 3.08 (t,J= 9.2 Hz; H4), 3.17 (dd,J= 9.6, 8.8 Hz; H2), 3.23 (t,J= 8.7 Hz; H3), 3.30 (ddd,J= 9.6, 6.5, 2.0 Hz; H5), 3.41 (dd,J= 11.8, 6.4 Hz; H6), 3.69 (dd,J= 11.8, 1.9 Hz; H6), 4.79 (d,J= 9.7 Hz; H1), 7.74 (dd,J= 8.1, 4.3 Hz; H8′), 7.80 (dd,J= 8.3, 4.2 Hz; H3′), 8.29 (s, H6′), 8.36 (dd,J= 8.1, 1.6 Hz; H4′), 8.79 (dd,J= 8.4, 1.6 Hz; H7′), 9.02 (dd,J= 4.3, 1.6 Hz; H9′), 9.09 (dd,J= 4.2, 1.6 Hz; H2′). 13C NMR (DMSO): 61.57 (C6), 70.36 (C4), 73.17 (C2), 79.74 (C3), 81.75 (C5), 87.46 (C1), 123.91 (C3′), 124.29 (C8′), 128.73, 128.82 (C4a′/C6a′), 129.13 (C6′), 131.44 (C5′), 134.13 (C4′), 136.34 (C7′), 145.20, 145.86 (C10a′/C10b′), 150.48, 150.55 (C2′/C9′). HRMS: C30H26N4O2S2 requiresm/z [M+H]+ 375.1015; observedm/z 375.1022.


## 3.5. Diethyl 7-oxabicyclo[4.1.0]heptane-trans-3,4-dicarboxylate (4)

The epoxide4 was prepared as described before [25] and isolated by column chromatography (Al2O3, gradient washing with hexane/EtOAc 8 : 2 → 7 : 3) as a colorless liquid (60%). 1H NMR (CDCl3): δ 1.21 (t,J= 7.1 Hz, 3H; CH3), 1.22 (t,J= 7.1 Hz, 3H; CH3), 1.87 (ddd,J= 14.9, 10.8, 2.1 Hz; H2), 2.04 (dd,J= 15.5, 10.9 Hz; H5), 2.30 (ddd,J= 15.5, 6.6, 4.8 Hz; H5), 2.45 (ddd,J= 14.9, 4.8, 1.8 Hz; H2), 2.58 (dt,J= 10.7, 6.7 Hz; H4), 2.80 (dt,J= 10.7, 4.9 Hz; H3), 3.17 (t,J= 4.3 Hz; H6), 3.24 (m, H1), 4.11 (m, 4H; OCH2). 13C NMR (CDCl3): δ 14.20 (CH3), 26.44 (C5), 27.30 (C2), 37.80 (C3), 40.14 (C4), 50.40 (C6), 52.00 (C1), 60.84 (OCH2), 173.76, 174.82 (C=O). HRMS: C12H18O5 requires [M+H]+
m/z243.1233; observedm/z243.1240.


## 3.6.  [(1S*,2S*,4S*,5S*)-2-Hydroxy-4,5-bis(ethoxycarbonyl)-cyclohexyl]-1-thio-β-D-glucopyranoside (2h)

1-Thio-β-D-glucopyranose sodium salt3 (100 mg, 0.46 mmol) in 7 mL of ethanol was added to a suspension of epoxide4 (80 mg, 0.33 mmol) in ethanol (2 mL) at room temperature. Reaction mixture was stirred at 40°C for 12 h until complete conversion of the epoxide as monitored by TLC (silica gel, EtOAc/MeOH 4 : 1). The solvent was evaporated under vacuum and residue was purified by column chromatography (silica gel, EtOAc/MeOH 7 : 3) affording product2h (a mixture of diastereomers 1 : 1) as a white solid (55 mg, 38%). 1H NMR (CD3OD): δ 1.21 (t,J = 7.1 Hz, 6H; 2CH3), 1.89 (dd,J = 8.3, 3.4 Hz, 1H; H3′); 1.94−2.05 (m, 2H; H3′+H6′), 2.21, 2.24 (dt,J = 3.9, 11.6 Hz, 1H; H6′), 2.86, 2.89 (dt,J = 3.7, 11.3 Hz; H4′), 2.98 (m, H5′), 3.17 (t,J = 8.6 Hz; H2), 3.23–3.35 (m, 4H; H1′+H3+H4+H5), 3.62, 3.65 (dd,J = 12.1, 5.1 Hz, 1H; H6), 3.84 (dd,J = 12.1, 1.8 Hz, 1H; H6), 3.98, 4.02 (br dd,J = 6.4, 3.3 Hz; H2′), 4.06–4.14 (m, 4H; OCH2), 4.41, 4.42 (d,J = 9.8 Hz; H1). 13C NMR (CD3OD): δ 13.14 (CH3), 28.51, 28.92 (C6′), 30.29, 30.45 (C3′), 39.09, 39.14 (C5′), 40.22, 40.24 (C4′), 44.34, 44.89 (C1′), 60.53, 60.58 (OCH2), 61.53 (C6), 67.73, 68.36 (C2′), 69.92, 70.01 (C4), 73.12, 73.18 (C2), 78.21 (C3), 80.77 (C5), 85.91, 86.00 (C1), 174.88, 175.18 (C=O). HRMS: C18H30O10S requiresm/z [M+NH4]+ 456.1903, [M+H]+ 439.1638; observedm/z 456.1967, 439.1654.


## 3.7.  [(1S*,2S*,4S*,5S*)-2-Hydroxy-4,5-bis (hydroxycarbonyl)-cyclohexyl]-1-thio-β-D-glucopyranoside (2i)

1 mL of KOH (1 M) was added to a solution of diester2h (150 mg, 0.34 mmol) in 2 mL of methanol and mixture stirred for 72 h at room temperature. HCl (1 M) was added to reaction mixture till pH 6 and solvent was evaporated under vacuum. Residue was purified by column chromatography (silica gel, EtOAc/CH3OH 1 : 4) affording product2i (one diastereomer) as a white solid (40 mg, 31%; mp > 240°C). 1H NMR (CD3OD): δ 1.85 (dd,J= 13.4, 3.8 Hz; H3′), 1.93–2.05 (m, 2H; H6′+H3′), 2.26 (m, 1H; H6′), 2.85 (m, H5′), 2.95 (m, H4′), 3.19 (t,J= 9.1 Hz; H2), 3.21–3.35 (m, 4H; H1′+H3+H4+H5), 3.64 (m, 1H; H6), 3.84 (m, 1H; H6), 3.95 (m, H2′), 4.43 (d,J= 9.8 Hz; H1). 13C NMR (CD3OD): δ 29.23 (C6′), 29.66 (C3′), 31.02 (C4′), 31.18 (C5′), 48.53 (C1′), 61.46 (C6), 69.05 (C2′), 69.98 (C4), 73.17 (C2), 78.24, 80.72 (C3/C5), 85.99 (C1), 179.73 (C=O). HRMS: C14H22O10S requiresm/z [M+NH4]+ 400.1277; observedm/z 400.1289.


## 3.8. Procedures for the Glycosidase Inhibition and Activation Assay

The enzyme activities (α/β-D-galactosidases and α/β-D-glucosidases) were assayed using multienzyme complexes isolated from fungiP. canescens andA. oryzae as described before [25–27]. All assays were performed in a standard way by monitoring spectrophotometrically (with Beckman Du-65 spectrometer) the release ofp-nitrophenol from the correspondingp-nitrophenyl glycosides at 30°C. One unit of enzyme activity was defined as the amount of enzyme that releases 1 μmol ofp-nitrophenol per minute. Enzyme and substrate concentrations were selected so that the degree of hydrolysis was never more than 20% and in most cases was less than 10%, over the course of the assay. The method used to measure the rate of the reaction assumes that the amount of the substrate is high enough, such that the disappearance over a given period is insignificant; that is, the rate of the reaction is close to linear for the first stage of the reaction.For the preliminary estimation, the enzyme solutions (100μL with activities 750
±
150 μU) were mixed with a set of inhibitor/activator solutions (100 μL, 10 mM) and then diluted with 700 μL of 0.2 M acetic buffer (pH 4.2), and the mixture was incubated for 1 h at 30°C. The reaction was initiated with addition of a proper substrate (100 μL of 20 mMp-nitrophenyl glycopyranoside), and aliquots were taken after 5 and 10 min. The reaction was terminated by addition of 1 mL of 1 M Na2CO3 to 0.5 mL of aliquot solution. The concentration of the releasedp-nitrophenol was determined at 400 nm using molar extinction coefficient 18.3 mM−1cm−1. The inhibition/activation was estimated as a loss/increase of enzymatic activity in % (Figures 1 and 2).The same protocol was used for estimation of the kinetic parameters with the following changes: 1 mL of enzyme solution, 0.5 mL inhibitor solution, and 3.0 mL of 0.2 M acetic buffer (pH 4.2) were incubated for 1 h at 30°C before addition of 0.5 mL of substrate (20 mM). Six substrate solutions were prepared with various concentrations up to saturation point for each enzyme. The final substrate concentration after all additions varied from 0.01 mM to 2.00 mM. The final inhibitor concentrations were 330μM forA. oryzae and 33 μM forP. canescens. Reaction rates were measured as described above. Control experiments with no enzyme were performed to exclude the errors due to the substrate spontaneous hydrolysis. Enzyme behavior (at pH 4.2 and 30°C) abided the Michaelis-Menten equation. Parameters K
M and K
M
app were calculated from Lineweaver-Burk (double-reciprocal) plot [52, 53]. The K
M values were used to calculate K
i by the following equation:
(1)
K
i
=
K
M
[
I
]
(
K
M
app
-
K
M
)
.
Three independent trials using freshly prepared substrate and enzyme solutions were performed to obtain each parameter. Values for K
M were reproducible within ±5% and ±10%, respectively, and the standard error was ±15% for the K
i values (Table 1).


## 4. Conclusions

The results of our studies show that simple and readily available 1-thio-β-D-glucopyranosides can be potent inhibitors for α- and β-D-glucosidases. The efficiency of inhibition strongly depends on the structure of aglycon and generally increases for β-glucosidases with increase of its lipophilicity. Another interesting feature of the studied compounds is an activation of α- and β-galactosidases.



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*Source: 941059-2014-08-21.xml*