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. 2021 Sep 22;6(39):25710–25719. doi: 10.1021/acsomega.1c03928

Chemoenzymatic and Protecting-Group-Free Synthesis of 1,4-Substituted 1,2,3-Triazole-α-d-glucosides with Potent Inhibitory Activity toward Lysosomal α-Glucosidase

Jaggaiah N Gorantla †,*, Santhi Maniganda †, Salila Pengthaisong †, Lukana Ngiwsara ‡, Phannee Sawangareetrakul ‡, Suwadee Chokchaisiri †, Prasat Kittakoop §, Jisnuson Svasti ‡, James R Ketudat Cairns †,‡,*
PMCID: PMC8495876  PMID: 34632227

Abstract

graphic file with name ao1c03928_0004.jpg

α-Glucosyl triazoles have rarely been tested as α-glucosidase inhibitors, partly due to inefficient synthesis of their precursor α-d-glucosylazide (αGA1). Glycosynthase enzymes, made by nucleophile mutations of retaining β-glucosidases, produce αGA1 in chemical rescue experiments. Thermoanaerobacterium xylanolyticus glucosyl hydrolase 116 β-glucosidase (TxGH116) E441G nucleophile mutant catalyzed synthesis of αGA1 from sodium azide and pNP-β-d-glucoside (pNPGlc) or cellobiose in aqueous medium at 45 °C. The pNPGlc and azide reaction product was purified by Sephadex LH-20 column chromatography to yield 280 mg of pure αGA1 (68% yield). αGA1 was successfully conjugated with alkynes attached to different functional groups, including aryl, ether, amine, amide, ester, alcohol, and flavone via copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reactions. These reactions afforded the 1,4-substituted 1,2,3-triazole-α-d-glucoside derivatives AGT2-14 without protection and deprotection. Several of these glucosyl triazoles exhibited strong inhibition of human lysosomal α-glucosidase, with IC50 values for AGT4 and AGT14 more than 60-fold lower than that of the commercial α-glucosidase inhibitor acarbose.

Introduction

Protein engineering has facilitated expansion of the roles of enzymes as green biocatalysts.1 Biocatalytic methods empower the pharmaceutical sector for the production of large-scale quantities of complex marketed drug intermediates, such as conversion of prositagliptin to stereopure sitagliptin, synthesis of the side chain of atorvastatin, semisynthesis of the antimalarial drug artemisinin and the blockbuster drug paclitaxel, as well as the synthesis of valuable chemicals in ton-scale quantities yearly.2,3 Thus, biocatalysts are well utilized in various fields.4−9

Carbohydrate-active enzymes (CaZymes, www.cazy.org), i.e., glycoside hydrolases, glycosyl transferases, and transglycosidases, are described as acting through either a retaining or inverting mechanism, depending on whether the stereochemistry at the bond that is broken is the same or different at the end of the reaction compared to at the beginning.10 The first (glycosylation) step of the most common retaining mechanism is displacement of a leaving group from the sugar with acid assistance of the catalytic acid/base to protonate the glycosidic bond oxygen and attack by the catalytic nucleophile from the opposite side as the departing bond. In the second (deglycosylation) step, an incoming nucleophile (such as water) is activated by deprotonation by the catalytic acid/base to attack the anomeric carbon of the sugar, displacing the catalytic nucleophile and resulting in a new bond with the same stereochemistry as the initial substrate.

The Withers group and others have demonstrated that retaining glycoside hydrolases in which the catalytic acid/base or nucleophile have been mutated to nonionizable amino acids can be rescued by small nucleophiles and can be used to transglycosylate substrates without hydrolysis of products.11−14 In fact, the rescue of mutant retaining β-glucosidases using small nucleophiles, such as azide, has become a diagnostic method to identify the catalytic acid/base and catalytic nucleophile.15−17 The rescue of the catalytic acid/base generally results in a product with the retained anomeric configuration since the nucleophile is simply taking the place of water in the deglycosylation step. In contrast, the rescue of the nucleophile mutant results in a product with inverted anomeric stereochemistry, since the small nucleophile takes the place of the mutated catalytic nucleophile and the product is released after the glycosylation step. Generally, such products have only been produced in analytical amounts for diagnosis of the role of the mutated residue in the reaction.17−19

The catalytic transglycosylation reaction has been well studied and significantly utilized in the production of several useful glycosides, i.e., ester-glycosides,20 flavonoid-glycosides,21,22 and α/β-azido-glycosides.11,12,15,17,23 Several synthetic routes were reported for the synthesis of 1-azido-β-d-glucose (also called β-d-glucosylazide) and well utilized in the area of bioconjugation via click chemistry.24,25 Numerous copper- and organocatalytic-mediated click reactions were reported in the multidisciplinary chemistry research field.25−28 Recently, our group reported the gram-scale production of 1-azido-β-d-glucose via using a retaining β-glucosidse acid/base-mutant enzyme as the catalyst and utilized it for the synthesis of 1,2,3-triazole-β-d-glucosides, including a glucosyl Aza-BODIPY dye for enhanced photodynamic cancer therapy applications.29,30 However, the synthesis and bioactivities of α-d-glucosylazide and its triazole glycosides are less well explored, due to the lack of easy synthetic routes. Only a few synthetic methods have been developed for the synthesis of pure α-d-glucosylazide (Figure 1A) or its mixture with β-configured 1-azido-d-glucose via a protection strategy of several steps, and this was subsequently utilized for the synthesis of triazole-α-d-glucosides and α-d-glucosylamides.31−34 One set of such triazole-α-d-glucosides exhibited weak inhibition against yeast α-glucosidase35 and rabbit muscle glycogen phosphorylase b.36

Figure 1.

Figure 1

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(A) Previous and present methods for the synthesis of α-d-glucosyl-azide. (B) Structures of reported α-glucosidase and glycogen phosphorylase inhibitors.

Iminosugars, such as nojirimycin, deoxynojirimycin, and its N-alkyl derivatives, have been extensively reported for their α-glucosidase inhibitory activities.37,38 Well-known FDA-approved iminosugar drugs include Miglitol or Glyset and Zavesca, which are used for the treatment of type-II diabetes and the lysosomal storage disorder Gaucher disease, respectively (Figure 1B). 1-Deoxynojirimycin is a strong α-glucosidase inhibitor but poor glycogen phosphorylase inhibitor.39 Nojirimycin-tetrazole and triazole (Figure 1B) bind at the active site through ionic bonding to inhibit glycogen phosphorylase.40,41 Various nitrogen and other heteroatom-containing glycoconjugates have been reported for their inhibition of α-glucosidase activities in yeast,42 rice,43 mouse, rat, and human intestine and liver, including lysosomal and endoplasmic reticulum isoenzymes.44−48

Iminosugar, azasugar, tetrazole, and triazole glycosides provide strong evidence that the nitrogen-containing glycosides are potential α-glucosidase and glycogen phosphorylase (GP) inhibitors. This motivated us to design and assess glucose-conjugated, nitrogen-containing potential α-glucosidase inhibitors via short synthetic routes, which might help in treating diabetes and other diseases.

Here, we report the production of α-d-glucosylazide αGA1 (Figure 1A) at the hundreds of milligram scale using the Thermoanaerobacterium xylanolyticus glucosyl hydrolase 116 β-glucosidase (TxGH116) nucleophile mutant TxGH116 E441G as a catalyst. αGA1 was successfully conjugated with alkynes attached to different functional groups, including aryl, ether, amine, amide, ester, alcohol, uracil, and flavone via copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reactions. These reactions afforded the 1,4-substituted 1,2,3-triazole-α-d-glucoside derivatives AGT2-14 in good yields by avoiding the protection strategy. Although two of these (AGT2 and AGT11) were previously reported,35 the remaining ones, AGT3-10 and AGT12-14, have not previously been described, to our knowledge. Among the derivatives, two exhibited 60-fold lower IC50 values against human lysosomal α-glucosidase compared with the standard inhibitor acarbose (Figure 1B).

Results and Discussion

Recently, we showed that TxGH116 E441G was more active in transglycosylation than TxGH116 E441A17 in the synthesis of α-d-glucosylazide and had the most robust catalysis among the TxGH116 nucleophile mutants tested in small-scale rescue and glycosynthase reactions.49 Therefore, purified TxGH116 E441G enzyme was used for the optimization of the transglucosylation reaction conditions for the formation of αGA1 in 24 h, by varying the enzyme, p-nitrophenyl β-d-glucopyranoside (pNPGlc) and NaN3 concentrations, buffer, pH, and temperature. At the enzyme/substrate ratio, E/S = 1:6, the highest transglucosylation product formation was observed in citrate-phosphate buffer pH 3.5 (final pH 4.6), at 45 or 50 °C, while the concentrations of 100–300 mM NaN3 showed similar results. So, the conditions of 100 mM NaN3 in 100 mM citrate-phosphate buffer, pH 3.5 (pH 4.6 after mixing with azide), and the incubation temperature of 45 °C for 24 h were selected as optimal. With the optimized reaction conditions, the E/S ratio was varied at 1:3, 1:6, and 1:9 (w/w). TLC (Figure S1A) indicated the complete conversion of pNPGlc to αGA1 in E/S (1:3, w/w), and the highest amount of product formation was observed in E/S (1:6, w/w), although some starting material was left unreacted. TxGH116 E441G could also synthesize α-d-glucosylazide using cellobiose as the substrate for transglucosylation in the same conditions as used with the pNPGlc substrate, but cellobiose gave less α-d-glucosylazide product than pNPGlc (Figure S1B).

Scale-Up Synthesis of α-d-Glucosylazide (αGA1) Using Enzyme Catalysis

When the optimized reaction conditions were scaled up to 100 mL with 100 mg of TxGH116 E441G as the catalyst to react 600 mg of pNPGlc with 100 mM NaN3, a white solid product was isolated by Sephadex LH-20 chromatography. However, the amount of white solid was more than expected (>1 g) due to the presence of sodium azide that eluted together with α-d-glucosylazide. Dissolving the product in methanol and filtering out the undissolved azide afforded 280 mg of αGA1 in 68% yield as a white solid. αGA1 was characterized by 1H NMR, and the α-anomeric proton chemical shift was observed at δ 5.51 ppm (H-1, d, J = 4 Hz, 1H). The coupling constant and the spectral values agreed well with those in the literature,11 as did the C-1 carbon peak, which appeared at δ 89 ppm in 13C NMR.

Recently, a similar approach was used to generate a 100 mg quantity of rutinosyl α-azide (α-l-rhamnopyranosyl (1 → 6) α-d-glucopyranosyl azide) in 44% yield from the flavonoid disaccharide rutin with a mutant rutinosidase.50 This indicates that retaining glycosidase nucleophile mutants have general usefulness for production of synthetically challenging glycosyl azides.

Synthesis of 1,4-Substituted 1,2,3-Triazole-α-d-glucoside Derivatives (AGT2-14)

Pure αGA1 was then conjugated with different alkynes attached with variable functional groups, i.e., alcohol, amine, amide, ester, and ether, by the well-known copper-catalyzed azide/alkyne cycloaddition (CuAAC) “click” reactions in H2O/CH3CN and H2O/t-BuOH, which afforded the set of 1,2,3-triazole-α-d-glucoside derivatives AGT2-14 (Figure 2). The reaction of propargyl alcohol (1a) with αGA1 and copper(I) iodide (CuI), N,N-diisopropylethylamine (DIPEA), in CH3CN/H2O (2:1) overnight gave AGT2(35) in 78% yield as a white solid. The pure alkynes 1e–1g and 1i–1l were reacted with αGA1 in click reactions using the above conditions (Method A) to afford AGT6, AGT7, AGT8, AGT10, AGT11, AGT12, and AGT13 in 26–81% yields (Figure 2). The ester-conjugated AGT8 yield was improved from 26 to 50%, by including the additives TBTA and TCEP instead of the base DIPEA. Protected N-Boc-propargylamine (1j) was synthesized from propargylamine by reacting with (Boc)2O and triethylamine (Et3N), while 1j combined with αGA1 by applying the above conditions (Method A) afforded AGT11(35) in 66% yield as a white solid. Our previous experience showing that phenyl acetylenes (1b, 1d) have poor reactivity with unprotected glucosylazide29 directed us not to follow up the above reaction conditions for these.

Figure 2.

Figure 2

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Synthesis of 1,4-substituted 1,2,3-triazole-α-d-glucoside derivatives AGT2-14 from αGA1 under click reaction conditions. The amount of 15–30 mg (0.073–0.146 mmol) of αGA1 was used for each reaction.

Phenyl acetylenes (1b, 1d) were initially dissolved in t-BuOH/H2O (5:1)36 containing αGA1 by gentle heating in a water bath, and then, CuSO4·5H2O and sodium ascorbate were added and stirred for 10 days (method B), which afforded the conjugated products AGT3 and AGT5 as white solids in 48 and 30% yields, respectively (Figure 2). αGA1 reacted with alkyne 1c in the presence of sodium ascorbate and CuSO4·5H2O, resulting in fluorescein-triazole-glucoside (AGT4) in 47% yield (method B). Hydroquinone-dipropargylether (1h) was conjugated with azide (αGA1) under the above conditions in t-BuOH/H2O (2:1) (method B) and gave a homo-bis-triazole-glucoside ether AGT9 in 55% yield (Figure 2). The reactions were carried out for longer times to produce the 1,4-addition product in average to good yields. The longer times were required due to the effect of the long bond length of the azide functional group present in αGA1 compared with the bond length in β-d-glucosylazide.35,51 We further investigated the different click reaction conditions and improved the yields of AGT3, AGT4, AGT5, and AGT8 using the additive TBTA along with CuI and DIPEA in acetonitrile (method C).52 Chrysin-7-propargyl ether (1m) was reacted with αGA1 under the above reaction conditions (method C) to give a chrysin-conjugated triazole AGT14 in 47% yield (Figure 2).

Lysosomal α-Glucosidase Inhibition

The human lysosomal α-glucosidase enzyme has previously been shown to be inhibited by conduritol B epoxide, deoxynojirimycin, and acarbose.53−55 All of the derivatives were screened for their glucosidase inhibitory activity against human lysosomal α-glucosidase in comparison to acarbose. AGT4, AGT8, AGT9, AGT10, AGT12, and AGT14 showed higher inhibition than the 66% α-glucosidase inhibition exhibited by acarbose at 1.43 mM concentration (Table 1), so their IC50 values were determined. AGT8, AGT9, AGT10, and AGT12 exhibited IC50 values of 447, 227, 137, and 483 μM, respectively (Table 1). AGT4 and AGT14 had IC50 values of 18 and 17 μM, respectively, which is 60-fold lower than the standard inhibitor acarbose (Figure 1B (IC50 1.10 mM)). The strong inhibition of α-glucosidase activity by AGT14 correlates with that of the flavone chrysin itself for which an IC50 of 77 μM was previously reported, with its derivatives showing even better activity.56−59 The presence of the glucosyl-triazole moiety may improve the solubility and inhibition of this flavone.

Table 1. Relative α-Glucosidase Inhibitory Activities and IC50 Values of α-d-Glucosyl-triazole Derivatives.

entry compound α-glucosidase inhibition (%)a IC50 (μM)b
1 acarbose 66 1100 ± 0.10
2 αGA1 09 ND
3 AGT2 12 ND
4 AGT3 46 ND
5 AGT4 100c 18 ± 0.002
6 AGT5 57 ND
7 AGT6 56 ND
8 AGT7 47 ND
9 AGT8 72 447 ± 0.035
10 AGT9 87 227 ± 0.025
11 AGT10 80 137 ± 0.015
12 AGT11 52 ND
13 AGT12 77 483 ± 0.021
14 AGT13 18 ND
15 AGT14 96 17 ± 0.001
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a

Percentage inhibition of lysosomal α-glucosidase in comparison to extracts incubated with 1.43 mM acarbose, αGA1, and AGT2–AGT14.

b

IC50 values were determined by measuring human lysosomal α-glucosidase activity at different concentrations of the best inhibitors, AGT4, AGT8, AGT9, AGT10, AGT12, and AGT14.

c

The absorbance and fluorescence of AGT4 interfered with activity detection at the initial concentration, but AGT4 did show high inhibition at lower concentrations, which did not interfere with the assay.

In conclusion, enzyme TxGH116 E441G was successfully used for the multi-milligram-scale synthesis of αGA1 in aqueous medium, thereby avoiding the chemical protection strategy. αGA1 was further joined with different types of alkynes via a 1,2,3-triazole as a linker by click chemistry. A lack of safe and easy methods for preparation has hindered the azide (αGA1) utilization and its applications. The present process might be a useful method for the synthesis of αGA1 at the industrial level if it can be scaled up effectively. Future improvement of the synthesis of αGA1 from cellobiose and recovery of residual azide may lead to an ecologically friendly αGA1 synthesis process. The utility of this convenient method for the production of αGA1 was demonstrated by its application in the synthesis of some novel 1,4-substituted 1,2,3-triazole-a-d-glucoside derivatives (AGT2-14) in a short route through protecting-group-free synthesis. We also demonstrated the lysosomal α-glucosidase inhibitory activities of all of the derivatives and found that AGT4 and AGT14 exhibited 60-fold lower IC50 than acarbose on human lysosomal α-glucosidase. In the future, the use of these compounds as molecular chaperones for mutant lysosomal α-glucosidase found in Pompe disease and as inhibitors to reduce glycemic effects in diabetes will be explored.

Experimental Section

General Methods and Materials

Silica gel 60 F254 aluminum TLC plates were used to monitor the reactions with short-wavelength ultraviolet light and by charring the TLC plate after spraying with 10% sulfuric acid in ethanol to visualize the spots. Column chromatography was performed on silica gel 60–200 mesh and Sephadex LH-20 resin. Distilled water was used for the reactions and purification. Lyophilization was used for drying the water samples. 1H NMR spectra were recorded at 500 MHz and 13C NMR spectra were recorded at 125 MHz on a Bruker Avance III 500 MHz instrument with a Cryoprobe Prodigy. Either deuterated water (D2O) or deuterated DMSO (CD6SO) was used as the solvent. Chemical shifts are given in parts per million and coupling constants are in hertz. LC-ToF-MS analysis was performed on a Bruker MicrOTOF-Q ESI-MS or a Thermo Scientific Exactive mass spectrometer with ion mass data given in m/z.

Production of α-d-Glucosylazide (αGA1)

TxGH116 E441G was prepared according to the previously described method for wild-type and mutant TxGH116 enzymes.17 Four-liter cultures were used to prepare 100–200 mg of purified TxGH116 E441G enzyme. The αGA1 yield was optimized in small-scale (0.1–10 mL) reactions in 100 mM citrate-phosphate at different pH values, 100–300 mM sodium azide, and varying concentrations of pNPGlc. The optimized reaction conditions were scaled up to 100 mL with 100 mg of TxGH116 E441G as the catalyst to react 600 mg of pNPGlc with 100 mM NaN3 in 100 mM citrate-phosphate buffer, pH 3.5 (final pH 4.6 after addition of azide). The reaction was mixed in a fume hood, and the reaction bottle was tightly sealed to prevent exposure to hydrazoic acid vapor. After 24 h incubation at 45 °C, the pH was adjusted to pH 8 with 2 M Na2CO3 to minimize hydrazoic acid and the yellow reaction mixture was frozen and lyophilized to dryness. The crude transglucosylation reaction mixture was purified by Sephadex LH-20 column chromatography in 100% water and fractions monitored by TLC. When the product-containing fractions were frozen and lyophilized to dryness (overnight), the amount of white solid was more than expected (>1 g) due to the presence of sodium azide that eluted together with α-d-glucosylazide. The white solid was then dissolved with MeOH (40 mL) and then passed through a celite pad. The filtrate was concentrated under vacuum to afford a-d-glucosylazide (αGA1, 280 mg) in 68% yield as a white solid. Rf = 0.25 (silica gel TLC with EtOAc/MeOH 9:1 v/v as the solvent); 1H NMR (D2O, 500 MHz): δ 5.51 (H-1, d, J = 4 Hz, 1H), 3.86 (dd, J = 2, 12 Hz, 1H), 3.81–3.77 (m, 1H), 3.74 (dd, J = 5, 12 Hz, 1H), 3.63 (dd, J = 4.5, 10 Hz, 1H), 3.58 (appt, J = 9 Hz, 1H), and 3.39 (appt, J = 9 Hz, 1H); 13C NMR (D2O, 125 MHz) δ 89.1, 73.7, 72.6, 70.6, 69.1, and 60.4; LC-ToF-MS m/z [M + Na]+ calcd for C6H11N3 NaO5+ 228.0596, found 228.0594.

Preparation of Alkynes (1b–1m) Using the Reported Reaction Conditions

The amide and ester functional groups containing alkynes 1e, 1f, 1g, and 1k (Figure 2) were freshly prepared by coupling the propargylamine or propargyl alcohol with carboxylic acids through the well-known standard EDC/DMAP coupling reaction.60 Both O and N-propargyl alkynes 1c, 1h, 1i, 1m, and 1l were prepared by alkylating the phenolic hydroxyl group or amide −NH by propargyl bromide using sodium hydride.61 Phenyl acetylenes 1b and 1d (Figure 2) were freshly prepared from their respective aldehydes by reacting with the Ohira–Bestmann reagent via the Seyferth–Gilbert homologation reaction.62

General Procedures Followed for the Synthesis of 1,4-Substituted 1,2,3,-Triazole-a-d-glucoside Derivatives (AGT2-14)

Method A (Applied for Synthesis of AGT2, AGT6, AGT7, AGT8, AGT10, AGT11, AGT12, and AGT13)

To a solution of a-d-glucosylazide (αGA1) and alkyne (1a, 1e, 1f, 1g, 1i, 1j, 1k, and 1l) in 1.5 mL of CH3CN/H2O (2:1) were added CuI (2 equiv) and DIPEA (2 equiv) and the resulting yellow precipitated reaction mixture was stirred at room temperature overnight until the formation of major amounts of products. Reaction mixtures were diluted with methanol (5 mL) and then passed through a celite pad by washing with methanol (20–30 mL) for the removal of copper salt. The filtrate was concentrated and adsorbed on silica gel and then loaded on a silica gel column. The yellow nonpolar impurities and unreacted glucosylazide were eluted in MeOH/EtOAc (3–5/97–95%), and then, the desired 1,2,3,-triazole-a-d-glucosides were eluted in MeOH/EtOAc (5–10/95–90%).

Method B (Applied for Synthesis of AGT3, AGT4, AGT5, AGT9, and AGT14)

A solution of αGA1 and alkynes (1b, 1c, 1d, 1h, and 1m) in 1.5–2 mL of t-BuOH/H2O (5:1) was heated in a water bath at 50 °C until the reaction mixture became homogeneous, and then, CuSO4·5H2O (0.2–0.3 equiv) and sodium ascorbate (0.4–0.6 equiv) were added in that order. Reaction mixtures were stirred at room temperature for more than 1 week until the products were formed. Reaction mixtures were diluted with methanol (5 mL) and then passed through celite pads by washing with methanol (20–30 mL) to remove the copper salt. The filtrate was concentrated and adsorbed on silica gel and then loaded on a silica gel column. Unreacted αGA1, AGT3, AGT5, AGT5, AGT9, and AGT14 were eluted with MeOH/EtOAc (5–10/95–90%).

Method C (Applied for Synthesis of AGT3, AGT4, AGT5, AGT8, AGT12, and AGT14)

This method improved the yields of AGT3, AGT4, AGT5, ATG8, ATG12, and ATG14. To a solution of αGA1 and alkyne (1b, 1c, 1d, 1g, 1k, or 1m) dissolved in 1.5 mL of CH3CN were added CuI (3 equiv), DIPEA (3 equiv), and TBTA (0.5 equiv). TCEP (0.4 equiv) was used instead of DIPEA for the synthesis of AGT8. The reaction mixture was stirred at room temperature overnight. Reaction mixtures were diluted with methanol (5 mL) and then passed through a celite pad by washing with methanol (20–30 mL) for the removal of copper salt. The filtrate was concentrated and adsorbed on silica gel and then loaded on a silica column. Unreacted αGA1, AGT3, AGT4, AGT5, ATG8, and ATG12 eluted with MeOH/EtOAc (5–10/95–90%), and AGT14 was purified with MeOH/DCM (5/95%, 8/92%).

1-(α-d-Glucopyranosyl)-4-hydroxymethyl-[1,2,3]-triazole (AGT2)

αGA1 (20 mg, 0.097 mmol) and propargyl alcohol 1a (30 μL, 0.485 mmol) reacted following method A to produce AGT2 as a white solid (20 mg, 78%). The product had silica gel TLC Rf = 0.13 (with EtOAc/MeOH 9:1 v/v as the solvent); 1H NMR (D2O, 500 MHz): δ 8.13 (s, 1H), 6.31 (d, J = 6 Hz, 1H), 4.75 (s, 2H), 4.43 (appt, J = 9 Hz, 1H), 4.13 (dd, J = 4, 10 Hz, 1H), 3.77–3.61 (m, 3H), and 3.59 (appt, J = 9 Hz, 1H); 13C NMR (D2O, 125 MHz) δ 146.2, 126.0, 84.9, 75.1, 73.1, 70.2, 69.3, 60.2, and 54.4; LC-ToF-MS m/z [M + H]+ calcd for C9H16N3O6+ 262.1039, found 262.1031.

1-(α-d-Glucopyranosyl)-4-cyano-phenyl-[1,2,3]-triazole (AGT3)

αGA1 (22 mg, 0.107 mmol) and 4-cyano-phenylacetylene 1b (20 mg, 0.160 mmol) were reacted following method B to produce AGT3 as a white solid (17 mg, 48%).

αGA1 (13 mg, 0.063 mmol) and 4-cyano-phenylacetylene 1b (16 mg, 0.126 mmol) were reacted following method C to produce AGT3 as a white solid (14 mg, 66%).

AGT3 had silica gel TLC Rf = 0.19 (with EtOAc/MeOH 9:1 v/v as the solvent); 1H NMR (DMSO-d6, 500 MHz): δ 8.96 (s, 1H), 8.16 (d, J = 8.5 Hz, 2H), 7.94 (d, J = 8.5 Hz, 2H), 6.42 (d, J = 6 Hz, 1H), 5.57 (d, J = 5.0 Hz, 1H), 5.21 (dd, J = 5, 7 Hz, 2H), 4.57 (appt, J = 6 Hz, 1H), 4.21–4.17 (m, 1H), 3.90–3.86 (m, 1H), 3.79–3.76 (m, 1H), 3.71–3.67 (m, 1H), and 3.36–3.34 (m, 1H); 13C NMR (DMSO-d6, 125 MHz): δ 144.2, 135.5, 133.5, 126.2, 125.6, 119.3, 110.6, 86.2, 77.2, 73.3, 70.8, 70.3, and 61.2; LC-ToF-MS m/z [M + H]+ calcd for C15H17N4O5+ 333.1199, found 333.1206.

1-(α-d-Glucopyranosyl)-4-methoxy-fluorescein-[1,2,3]-triazole (AGT4)

αGA1 (15 mg, 0.073 mmol) and fluorescein-propargyl ether 1c (15 mg, 0.0365 mmol) were reacted following method B, and AGT4 was purified as a yellow solid (10 mg, 47%).

αGA1 (20 mg, 0.098 mmol) and fluorescein-propargyl ether 1c (40 mg, 0.098 mmol) were reacted by method C, and AGT4 was purified as a white solid (28 mg, 50%). The product had silica gel TLC Rf = 0.13 (with EtOAc/MeOH 9:1 v/v as the solvent); 1H NMR (DMSO-d6, 500 MHz): δ 10.23 (s, 1H), 8.44 (s, 1H), 8.07 (d, J = 7.5 Hz, 1H), 7.87 (appt, J = 7.5 Hz, 1H), 7.79 (appt, J = 7.2 Hz, 1H), 7.36 (d, J = 7.5 Hz, 1H), 7.20 (d, J = 2 Hz, 1H), 6.87 (dd, J = 2, 11 Hz, 1H), 6.78 (s, 1H), 6.73 (d, J = 9 Hz, 1H), 6.65 (s, 2H), 6.24 (d, J = 5.5 Hz, 1H), 5.52 (d, J = 5 Hz, 1H), 5.32 (s, 2H), 5.18 (dd, J = 5, 13 Hz, 2H), 4.56 (appt, J = 6 Hz, 1H), 4.17–4.12 (m, 1H), 3.85–3.80 (m, 1H), 3.74 (dd, J = 5, 9.5 Hz, 1H), 3.66 (dd, J = 6, 10.5 Hz, 1H), 3.54–3.50 (m, 1H), and 3.35–3.31 (m, 1H); 13C NMR (DMSO-d6, 125 MHz): δ 169.1, 160.2, 160.0, 152.9, 152.3, 152.2, 141.4, 136.1, 130.6, 129.5, 129.4, 127.5, 126.5, 125.1, 124.5, 124.4, 113.3, 112.9, 111.8, 109.9, 102.7, 102.0, 90.2, 85.7, 77.0, 73.4, 70.8, 69.9, 61.8, and 61.1; LC-ToF-MS m/z [M + H]+ calcd for C29H26N3O10+ 576.1618, found 576.1649.

1-(α-d-Glucopyranosyl)-4-nitro-phenyl-[1,2,3]-triazole (AGT5)

αGA1 (20 mg, 0.097 mmol) and 4-nitro-phenylacetylene 1d (36 mg, 0.243 mmol) were reacted following method B, and AGT5 was purified as a white solid (10 mg, 30%).

To improve the yield, αGA1 (15 mg, 0.073 mmol) and 4-nitro-phenylacetylene 1d (21 mg, 0.146 mmol) were reacted following method C and AGT5 was purified as a white solid (18 mg, 70%).

This compound had silica gel TLC Rf = 0.28 (TLC run twice with EtOAc/MeOH 9:1 v/v as the solvent); 1H NMR (DMSO-d6, 500 MHz): δ 9.09 (s, 1H), 8.41 (d, J = 9 Hz, 2H), 8.25 (d, J = 9 Hz, 2H), 6.28 (d, J = 6 Hz, 1H), 5.60 (d, J = 5 Hz, 1H), 5.22 (dd, J = 5.5, 6.5 Hz, 2H), 4.58 (appt, J = 5.5 Hz, 1H), 4.22–4.17 (m, 1H), 3.91–3.87 (m, 1H), 3.80–3.77 (m, 1H), 3.71–3.68 (m, 1H), 3.57–3.53 (m, 1H) and 3.37–3.35 (m, 1H); 13C NMR (DMSO-d6, 125 MHz): δ 147.1, 143.8, 137.4, 126.5, 126.0, 124.8, 86.2, 77.2, 73.3, 70.8, 70.3, and 61.2; LC-ToF-MS m/z [M + H]+ calcd for C14H17N4O7+ 353.1097, found 353.1095.

1-(α-d-Glucopyranosyl)-4-methylene-cinnamide-[1,2,3]-triazole (AGT6)

αGA1 (20 mg, 0.098 mmol) and propargyl-cinnamide 1e (36 mg, 0.195 mmol) were reacted following method A, and AGT6 was purified as a white solid (31 mg, 81%). The product had silica gel TLC Rf = 0.15 (with EtOAc/MeOH 9:1 v/v as the solvent); 1H NMR (D2O, 500 MHz): δ 8.12 (s, 1H), 7.63 (appt, J = 3.5 Hz, 2H), 7. 55 (d, J = 15.5 Hz, 1H), 7.46–7.45 (m, 3H), 6.65 (d, J =16 Hz, 1H), 6.30 (d, J = 6 Hz, 1H), 4.62 (s, 2H), 4.44 (appt, J = 9.5 Hz, 1H), 4.14 (dd, J = 5.5, 9.5 Hz, 1H), 3.78–3.72 (m, 3H), and 3.60 (appt, J = 9 Hz, 1H); 13C NMR (D2O, 125 MHz) δ 168.8, 141.6, 134.3, 130.3, 129.0, 128.0, 119.8, 85.0, 75.9, 73.1, 70.2, 69.3, 60.3, and 34.5; LC-ToF-MS m/z [M + H]+ calcd for C18H23N4O6+ 391.1618, found 391.1628.

1-(α-d-Glucopyranosyl)-4-methylene-benzamide-[1,2,3]-triazole (AGT7)

αGA1 (20 mg, 0.098 mmol) and propargyl-benzamide 1f (31 mg, 0.195 mmol) were reacted following method A, and AGT7 was purified as a white solid (26 mg, 73%). AGT7 had silica gel TLC Rf = 0.23 (with EtOAc/MeOH 8.5:1.5 v/v as the solvent); 1H NMR (D2O, 500 MHz): δ 8.13 (s, 1H), 7.79 (d, J = 7.5 Hz, 2H), 7.63 (appt, J = 7.0 Hz, 1H), 7.50 (appt, J = 8.0 Hz, 2H), 6.29 (d, J = 6 Hz, 1H), 4.71 (s, 2H), 4.44 (appt, J = 9 Hz, 1H), 4.14 (dd, J = 5.5, 9.5 Hz, 1H), 3.78–3.71 (m, 3H), and 3.60 (appt, J = 9.5 Hz, 1H); 13C NMR (D2O, 125 MHz): δ 170.9, 144.2, 133.2, 132.3, 128.8, 127.1, 125.8, 85.0, 75.2, 73.1, 70.3, 69.3, 60.3, and 34.8; LC-ToF-MS m/z [M + H]+ calcd for C16H21N4O6+ 365.1461, found 365.1466.

1-(α-d-Glucopyranosyl)-4-4-napthalene-1acetate-[1,2,3]-triazole (AGT8)

αGA1 (20 mg, 0.097 mmol) and propargyl-1-napthaleneacetate 1g (43 mg, 0.195 mmol) were reacted following method A, and AGT8 was purified as a white solid (11 mg, 26%).

To improve the yield, αGA1 (20 mg, 0.098 mmol) and propargyl-1-napthaleneacetate 1g (43 mg, 0.195 mmol) were reacted following method C and AGT8 was isolated as a white solid (21 mg, 50%).

The product had silica gel TLC Rf = 0.2 (TLC run twice with EtOAc/MeOH 9:1 v/v as the solvent); 1H NMR (DMSO-d6, 500 MHz): δ 8.32 (s, 1H), 8.02–7.98 (m, 2H), 7.94 (dd, J = 2, 7.0 Hz, 1H), 7.63–7.58 (m, 2H), 7.55–7.51 (m, 2H), 6.21 (d, J = 5.5 Hz, 1H), 5.51 (d, J = 5.0 Hz, 1H), 5.27 (s, 2H), 5.19 (dd, J = 5, 7.5 Hz, 2H), 4.58 (appt, J = 5.5 Hz, 1H), 4.26 (s, 2H), 4.16–4.12 (m, 1H), 3.85–3.81 (m, 1H), 3.75–3.73 (m, 1H), 3.69–3.66 (m, 1H), 3.56–3.51 (m, 1H), and 3.36–3.32 (m, 1H); 13C NMR (DMSO-d6, 125 MHz): δ 171.5, 140.9, 133.8, 132.2, 131.2, 128.9, 128.5, 12.1, 127.6, 126.7, 126.2, 125.9, 124.4, 85.6, 77.0, 73.3, 70.8, 70.3, 61.2, 57.9, and 38.2; LC-ToF-MS m/z [M + H]+ calcd for C21H24N3O7+ 430.1614, found 430.1612.

1-(α-d-Glucopyranosyl)-4-hydroquinone-methyleneether-[1,2,3]-triazole (AGT9)

A solution of αGA1 (30 mg, 0.146 mmol) and dipropargylated-hydroquinone 1h (13 mg, 0.073 mmol) was reacted following method B, and AGT9 was purified as a white solid (24 mg, 55%). Silica gel TLC demonstrated an Rf = 0. 17 (with EtOAc/MeOH 6:4 v/v as the solvent); 1H NMR (D2O, 500 MHz): δ 8.21 (s, 2H), 7.02 (s, 4H), 6.30 (d, J = 6 Hz, 2H), 5.25 (s, 4H), 4.42 (appt, J = 9 Hz, 2H), 4.14 (dd, J = 6, 10 Hz, 2H), 3.74–3.73 (m, 4H), and 3.67–3.59 (m, 4H); 13C NMR (D2O, 125 MHz): 152.2, 142.8, 127.1, 117.1, 85.1, 75.1, 73.1, 70.2, 69.2, 62.0, and 60.2; LC-ToF-MS m/z [M + Na]+ calcd for C24H32N6NaO12+ 619.1975, found 619.1974.

1-(α-d-Glucopyranosyl)-4-methylumbellifery-methyleneether-[1,2,3]-triazole (AGT10)

αGA1 (20 mg, 0.098 mmol) and 1i (41 mg, 0.195 mmol) were reacted following method A, which generated AGT10 as a white solid (29 mg, 71%).

Silica gel TLC demonstrated an Rf = 0.14 (with EtOAc/MeOH 8:2 v/v as the solvent); 1H NMR (DMSO-d6, 500 MHz): δ 8.46 (s, 1H), 7.78 (d, J = 9 Hz, 1H), 7.25 (d, J = 2.5 Hz, 1H), 7.14 (dd, J = 2.5, 9 Hz, 1H), 6.30 (s, 1H), 6.24 (d, J = 6 Hz, 1H), 5.54 (d, J = 5 Hz, 1H), 5.37 (s, 2H), 5.19 (dd, J = 4.5, 9.5 Hz, 2H), 4.57 (appt, J = 5.5 Hz, 1H), 4.17–4.12 (m, 1H), 3.85–3.81 (m, 1H), 3.75–3.72 (m, 1H), 3.68–3.65 (m, 1H), 3.54–3.52 (m, 1H), 3.37–3.32 (m, 1H), and 2,48 (s, 3H); 13C NMR (DMSO-d6, 125 MHz): δ 161.5, 160.6, 155.1, 153.9, 141.2, 127.6, 127.0, 113.8, 113.0, 111.7, 102.0, 85.73, 77.1, 73.3, 70.8, 70.3, 62.0, 61.2, and 18.6; LC-ToF-MS m/z [M + Na]+ calcd for C19H21N3NaO8+ 442.1226, found 442.1224.

1-(α-d-Glucopyranosyl)-4-N-Boc-methyleneamine-[1,2,3]-triazole (AGT11)

αGA1 (20 mg, 0.097 mmol) and N-Boc-propargylamine 1j (45 mg, 0.292 mmol) were reacted following method A, resulting in AGT11 as a white solid (23 mg, 66%). The product had the silica gel TLC Rf = 0.25 (with EtOAc/MeOH 8:2 v/v as the solvent); 1H NMR (D2O, 500 MHz): δ 8.05 (s, 1H), 6.29 (d, J = 6 Hz, 1H), 4.44 (appt, J = 8.5 Hz, 1H), 4.37 (s, 2H), 4.34 (dd, J = 6, 10 Hz, 1H), 3.78–3.72 (m, 3H), 3.61 (appt, J = 9 Hz, 1H), and 1.43 (s, 9H); 13C NMR (D2O, 125 MHz): δ 158.0, 125.4, 84.9, 75.1, 73.1, 70.2, 69.3, 60.2, and 27.5; LC-ToF-MS m/z [M + H]+ calcd for C14H25N4O7+ 361.1723, found 361.1739.

1-(α-d-Glucopyranosyl)-4-methylene-indometacinamide-[1,2,3]-triazole (AGT12)

αGA1 (23 mg, 0.112 mmol) and Indomatecine propargylamine 1k (62 mg, 0.168 mmol) were reacted following method A, resulting in AGT12 as a pale-yellow solid (14 mg, 21%).

αGA1 (20 mg, 0.098 mmol) and Indomatecine propargylamine 1k (77 mg, 0.195 mmol) underwent a reaction following method C, resulting in AGT12 as a pale-yellow solid (23 mg, 40%). The silica gel TLC had Rf = 0.25 (with EtOAc/MeOH 8:2 v/v as the solvent); 1H NMR (DMSO-d6, 500 MHz): δ 8.57 (s, 1H), 8.01 (s, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.13 (s, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.70 (d, J = 9.0 Hz, 1H), 6.10 (d, J = 5.5 Hz, 1H), 5.38 (d, J = 4.5 Hz, 1H), 5.08 (d, J = 4.0 Hz, 1H), 4.47 (appt, J = 5.0 Hz, 1H), 4.34 (d, J = 4.5 Hz, 1H), 4.06 (d, J = 9.0 Hz, 1H), 3.74 (s, 3H), 3.63–3.61 (m, 1H), 3.55 (s, 2H), 3.46–3.44 (m, 1H), 3.28 (s, 2H) and 2.23 (s, 3H); 13C NMR (DMSO-d6, 125 MHz): δ 169.8, 168.3, 156.1, 143.9, 138.0, 135.7, 134.7, 131.6, 131.39, 130.8, 129.5, 125.6, 114.9, 114.7, 111.8, 102.4, 85.5, 76.9, 73.4, 70.9, 70.3, 61.3, 55.9, 34.7, 31.5 and 13.9; LC-ToF-MS m/z [M + H]+ calcd for C28H31ClN5O8+ 600.1861, found 600.1884.

Bis[1-(α-d-glucopyranosyl)]-4-(5-fluorouracil)-N,N′-bis[methylene-[1,2,3]-triazole] (AGT13)

αGA1 (20 mg, 0.097 mmol) and 5-fluorouracil bis-propargyl 1l (10 mg, 0.049 mmol) were reacted following method A to yield AGT13 as a pale-yellow solid (17 mg, 56%). AGT13 had a silica gel TLC Rf = 0.18 (with EtOAc/MeOH 8:2 v/v as the solvent); 1H NMR (D2O, 500 MHz): δ 8.38 (s, 1H), 8.29 (s, 1H), 8.24 (d, J = 6.0 Hz, 1H), 6.41 (dd, J = 5.5, 6.0 Hz, 2H), 5.47 (s, 2H), 5.31 (s, 2H), 4.56–4.52 (m, 2H), 4.20–4.17 (m, 2H), 3.92–3.87 (m, 2H), 3.73–3.66 (m, 2H), and 3.58 (s, 2H); 13C NMR (D2O, 125 MHz): δ 170.9, 170.4, 159.0, 155.3, 150.5, 135.1, 129.4, 129.1, 85.2, 85.1, 75.3, 73.1, 70.3, 69.4, 60.4, 48.9, 44.3, 39.2, 36.7 and 33.8; LC-ToF-MS m/z [M + H]+ calcd for C22H30FN8O12+ 617.1967, found 617.1984.

1-(α-d-Glucopyranosyl)-4-chrysinyl-methyleneether-[1,2,3]-triazole (AGT14)

αGA1 (20 mg, 0.098 mmol) and chrysin-propargyl ether 1m (42 mg, 0.146 mmol) were reacted following method A to yield AGT14 as a yellow solid (5 mg, 10%).

αGA1 (20 mg, 0.098 mmol) and chrysin-propargyl ether 1m (57 mg, 0.195 mmol) were reacted following method C to yield AGT14 as a yellow solid (17 mg, 47%). AGT14 had a silica gel TLC Rf = 0.25 (TLC run twice with EtOAc/MeOH 9:1 v/v as the solvent); 1H NMR (DMSO-d6, 500 MHz): 12.84 (bs, 1H), 8.43 (s, 1H), 8.13 (s, 1H), 8.11 (s, 1H), 7.65–7.60 (m, 4H), 7.07(s, 1H), 7.01 (s, 1H), 6.55 (s, 1H), 6.21 (d, J = 7.5 Hz, 1H), 5.55 (bs, 1H), 5.34 (s, 2H), 5.19 (bs, 1H), 4.55 (bs, 1H), 4.10 (appt, J = 11.5 Hz, 1H), 3.80–3.76 (m, 1H), 3.71–3.67 (m, 1H), 3.61b (d, J = 14.5 Hz, 1H) and 3.49–3.45 (m, 2H); 13C NMR (DMSO-d6, 125 MHz): δ 182.1, 164.1, 163.6, 161.2, 157.4, 140.6, 132.2, 130.6, 129.2, 127.3, 126.5, 105.5, 105.2, 98.7, 93.5, 85.3, 76.6, 72.8, 70.4, 69.8, 61.7 and 60.8; LC-ToF-MS m/z [M + Na]+ calcd for C24H23N3NaO9+ 520.1332, found 520.1311.

α-Glucosidase Inhibitory Activity Assay

The α-glucosidase inhibitory activity assay was based on a standard fluorometric assay as previously described63 using human lysosomal α-glucosidase-overexpressing preparations from transiently transfected COS-7 cells.64 Briefly, the inhibitor was premixed with the appropriate amount of α-glucosidase-overexpressing homogenates (10–30 μg of protein) and incubated for 10 min at 37 °C. Except in the cases of αGA1, AGT2, AGT9, and AGT11, which were dissolved in water, the reactions contained 3.5% dimethyl sulfoxide from the inhibitor solutions. Subsequently, 4-methyl-umbelliferyl α-d-glucopyranoside (Sigma-Aldrich, St. Louis, MO) (final concentration = 1.26 mM) in 0.1 M citrate-phosphate buffer, pH 4.0, was added in an incubation mixture of 70 μL. The reaction was incubated for 60 min at 37 °C and was terminated by adding 200 μL of 0.5 M sodium carbonate, pH 10.7, with 0.25 g/L Triton X-100. The release of the product 4-methylumbelliferone was measured at 365 nm excitation/450 nm emission. The control samples were prepared without the inhibitor. The percent inhibition of enzyme activity was calculated using the following formula

graphic file with name ao1c03928_m001.jpg

where control activity is the enzyme activity under the same conditions but without the inhibitor.

The IC50 value is defined as the concentration of compound inhibiting 50% of α-glucosidase activity under the stated assay conditions.

Acknowledgments

The authors are thankful to Dr. Yanling Hua for assistance with NMR and LC–MS analyses. Financial support from Suranaree University of Technology, Thailand Science Research and Innovation (TSRI), the Synchrotron Light Research Institute, and the Thailand Research Fund (TRF) grant RSA6280073 is gratefully acknowledged.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03928.

  • Figure S1, TLC profile of compound αGA1 formation; Figures S2–S29, 1H and 13C NMR spectra of αGA1 and AGT2-14; and Figure S30, LC–MS spectra of AGT9 (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao1c03928_si_001.pdf (2.7MB, pdf)

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