Cu–Al mixed oxide-catalysed multi-component synthesis of gluco- and allofuranose-linked 1,2,3-triazole derivatives

A series of carbohydrate-linked 1,2,3-triazole derivatives were synthesized in good yields from glucofuranose and allofuranose diacetonides using as key step a three-component 1,3-dipolar azide–alkyne cycloaddition catalysed by a Cu–Al mixed oxide. In this multi-component reaction, Cu–Al mixed oxide/sodium ascorbate system serves as a highly reactive, recyclable and efficient heterogeneous catalyst for the regioselective synthesis of 1,4-disubstituted 1,2,3-triazoles. The reported protocol has significant advantages over classical CuI/N,N-diisopropylethylamine (DIPEA) or CuSO4/sodium ascorbate conditions in terms of efficiency and reduced synthetic complexity. In addition, the selective deprotection of synthesized di-O-isopropylidene derivatives was also carried out leading to the corresponding mono-O-isopropylidene products in moderate yields. Some of the synthesized triazole glycoconjugates were tested for their in vitro antimicrobial activity using the disc diffusion method against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), as well as fungus (Aspergillus niger) and yeast (Candida utilis). The results revealed that these compounds exhibit moderate to good antimicrobial activity mainly against Gram-negative bacteria.

Carbohydrates are an invaluable source of stereochemically pure molecules, and they are relevant as signalling molecules and important for cellular recognition events. As a consequence, the construction of carbohydrate-based bioactive compounds has become an active research area [15]. Compounds containing the 1,2,3-triazole ring have gained increased attention in the drug-discovery field since the introduction of the 'click' chemistry concept which was reported simultaneously and independently by the groups of Meldal in Denmark [16] and Fokin and Sharpless in the United States [17,18]. Particularly, the copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) to give a 1,4-disubstituted 1,2,3-triazole derivative is a powerful method for glycoconjugation due to its reliability, specificity and biocompatibility [19]. Click chemistry has great potential for use in binding between nucleic acids, lipids, proteins and carbohydrates. In this context, metabolic glycoengineering allows direct modification of living cells with substrates for click chemistry either in vitro or in vivo, becoming a powerful tool for cell transplantation and drug delivery [20]. In this sense, carbohydrates represent versatile starting substrates for the CuAAC reaction because it is possible to synthesize from them both from the azide-or the terminal alkyne-containing moieties needed for this cycloaddition reaction.
Considerable efforts have been made in order to increase the general efficiency of the process for obtaining hybrid 1,2,3-triazoles. In this aspect, heterogeneous catalysts offer several advantages over their homogeneous counterparts, such as enhanced stability, easy recovery and recycling [21,22]. For CuAAC process, almost any Cu catalyst able to furnish Cu(I) species in the reaction medium can be successfully used. In this context, the two possible approaches are either to use a Cu(I) source directly into reaction with the presence of stabilizing ligands, or to generate it either by reduction of Cu(II) salts or by oxidation of elemental Cu. In situ generation of Cu(I) species using Cu(II) salts such as CuSO 4 or copper acetate is a more effective way, since these conditions allow reactions to be carried out without an inert atmosphere and it avoids the use of anhydrous solvents, endures aqueous conditions, and at the same time maintains a high Cu(I) concentration during the course of the reaction [23].
On the other hand, hydrotalcite-like compounds constitute a class of two-dimensional materials, which are represented by a general chemical formula [M II 1−x M III x (OH) 2 ] x+ (A n− ) x/n · mH 2 O, where M II , M III represent divalent and trivalent metal cations, and A n− is an anion (usually CO 3 2− or NO 3 − ).
These materials have been used mainly as catalysts for the preparation of fine chemicals, intermediates and valuable molecules [24,25]. Recently, we discovered that Cu-Al mixed oxide, generated by calcination of corresponding hydrotalcite type layered double hydroxide (LDH), in the presence of sodium ascorbate (NaAsc) as a reducing agent, efficiently catalyses the 1,3-dipolar cycloaddition of in situ-generated benzyl azides and phenyl acetylene in short reaction times using EtOH/H 2 O as a solvent [26]. In continuation with our interest in the synthesis of functional triazole derivatives [27][28][29][30], and in order to introduce the use of mixed oxides in carbohydrate CuAAC click chemistry, herein we report a rapid and facile synthesis of a series of carbohydrate-linked 1,2,3-triazole derivatives using as key step a three-component 1,3-dipolar azide-alkyne cycloaddition catalysed by Cu-Al mixed oxide, and an insight into their antimicrobial properties.
Then, we focused on studying the Cu-Al mixed oxide-catalysed three-component 1,3-dipolar azidealkyne cycloaddition reaction to obtain 1,2,3-triazole-carbohydrate conjugates. Firstly, the catalyst was prepared using our previously reported method, from Cu(NO 3 ) 2 · 2.5H 2 O and AlCl 3 · 6H 2 O by coprecipitation with Na 2 CO 3 in water [26]. After heating at 60°C for 24 h, removal of water and drying in an oven (120°C, 18 h), a green solid was obtained, which was then calcinated in a N 2 atmosphere (550°C, 6 h) to obtain a fine black powder material corresponding to Cu-Al mixed oxide. The structure of mixed oxide was confirmed by X-ray diffraction. Figure 2 shows the diffractogram of calcinated layered double hydroxide (HLD), with the characteristic pattern of a periclase-like structure with (110), (111), (202), (022), (113), (311) and (220) plane reflections, typical of CuO, consistent with what was previously described.
With the raw materials and the catalyst in hand, in our initial experiment, the multi-component reaction between the glucose diacetonide 2, benzyl chloride and sodium azide was conducted in the presence of 10 mg of the Cu-Al mixed oxide and the same amount of sodium ascorbate at 80°C; with conventional heating for 18 h in ethanol-water as solvent (table 1, entry 1).
Under these conditions, the 1,2,3-triazole-carbohydrate conjugate 7a was obtained as a white crystalline solid in 69% yield after purification by crystallization with dichloromethane-hexane. Thus, we have demonstrated that Cu-Al mixed oxide can catalyse this three-component 1,3-dipolar azidealkyne cycloaddition reaction in a regioselective manner, giving exclusively the 1,4-disubstituted Ref. [40] Ref. [40] Ref. [39,45]   triazole. To determine the optimal conditions for the reaction, a series of experiments were performed varying parameters such as catalyst loading, solvent and heating method (table 1, entries 2-7). The effectiveness of the microwave irradiation and the conventional heating for this three-component click reaction was compared. In this case, a slightly lower yield than that obtained with the conventional heating was found (table 1, entry 2). However, although both methods are efficient, the microwaveassisted reaction was faster (10 min) in comparison with conventional thermal conditions, which require 18 h to complete the reaction. We also found that 30 mg of catalyst provides the best result, with optimum yields of the glycoconjugate product (83%), using an EtOH:H 2 O (3 : 1) mixture (   Yields of the isolated product after purification by crystallization. c The reaction was performed in the presence of 10 mg of catalyst and 10 mg of sodium ascorbate. d The reaction was performed in the presence of CuSO 4 · 5H 2 O (20 mol%) and sodium ascorbate (20 mol%). e The reaction was performed in the presence of CuI (20 mol%) and DIPEA (30 mol%). ND, not detected.
royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 7: 200290 diisopropylethylamine (DIPEA) systems were performed (table 1, entries 5 and 6). The Cu(I) salt and DIPEA were used in the reaction, under the same solvent and heating conditions, yielding 83% of triazole 7a after column chromatography. Surprisingly, the CuSO 4 · 5H 2 O/sodium ascorbate system using microwave irradiation with EtOH/H 2 O as solvent was not efficient for this transformation since 1,2,3-triazole compound could not be detected and only the formation of benzyl azide took place. This is probably due to the fact that Cu(I) species formed by the action of a reducing agent are not stable enough under the used conditions and an external stabilizing ligand needs to be added, which becomes a drawback for this method. Thus, we demonstrate that the described method is efficient and does not need any additional stabilizing ligands. Finally, although our ultimate goal was to obtain a heterogeneous and recyclable catalyst for the multi-component version of azide-alkyne cycloaddition applicable in carbohydrate CuAAC click chemistry, we also performed an experiment with the standard two-component reaction between phenylacetylene and benzyl azide catalysed by Cu-Al mixed oxide. It was expected that the reaction with the preformed organic azide was faster than with the multicomponent approach; however, after 30 min using microwave (MW) irradiation at 80°C in EtOH/H 2 O as solvent, only 43% yield of 1,4-disubstituted 1,2,3-triazole 7a was obtained, besides the recovery of part of the benzyl azide used as starting material (scheme 2). This could be attributed to the low solubility of benzyl azide in EtOH/H 2 O system and demonstrates that the three-component 1,3-dipolar azide-alkyne cycloaddition reaction catalysed by Cu-Al mixed oxide remains the best approach to perform this reaction.
It is worth noting that, under optimized conditions, the mixed oxide catalyst was successfully recovered, reactivated by calcination at 500°C, and re-used twice more in the same click reaction with similar efficiency. Subsequent to this reuse, the materials probably lose their initial structure and therefore their catalytic activity (figure 3).
Encouraged by these results, once the reaction was optimized and the catalyst recyclability was evaluated, we synthesized two series of 1,2,3-triazole-carbohydrate conjugates using the Opropargylated gluco-or allofuranose derivatives with a range of different p-halobenzyl chlorides in order to obtain 1,2,3-triazole-carbohydrate conjugates which could be used as valuable substrates for cross-coupling reactions (table 2).
Gratifyingly, in nearly all cases, very good yields of triazoles 7a-e and 8a-e were obtained and halogen substituents at the substrates were well tolerated. Both diastereomeric series of carbohydrate-triazole conjugates were fully characterized by FT-IR, HRMS, 1 H-and 13 C-NMR spectroscopy. The IR spectra of compounds 7a-e and 8a-e showed the characteristic absorption assigned to the N=N stretching for triazole compounds that appear nearly around 1490 cm − 1 and three or four intense absorption bands  Furthermore, the identity of the carbohydrate-triazole conjugates presented in this work was supported by high-resolution mass spectrometry (HRMS) which confirmed the expected mass for all compounds in accordance with their molecular formula. The analysis of the NMR spectroscopic data ( 1 H, 13 C, homonuclear correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments) reveals the diagnostic singlet in the resonance of the 1 H NMR due to triazolyl protons in the region of 7.50 ppm and around 122.0 ppm in 13 C NMR due to C-5 of the triazole ring, which confirms the formation of glycoconjugates. Representative 1 H NMR spectra of diastereomeric compounds 7c and 8c are shown in figure 4.
An analysis of spectroscopic data for both diastereomeric carbohydrate-triazole derivatives revealed a diagnostic pattern associated with the methylene protons (H-25). For glucofuranose derivative 7c, a simple signal at 5.48 ppm is assigned to H-25; while for analogue allofuranose derivative 8c, the same methylene group appearing as an AB quartet system in 5.49 ppm (J = 12.7 Hz). Besides, the double of doublet at 3.97 ppm (J = 8.7, 5.5 Hz) assigned to H-5 in 7c is slightly downshifted to 4.06 ppm (J = 8.7, 3.4 Hz) in the case of allofuranose derivative 8c. The regioselectivity of the cycloaddition reaction was elucidated by using 1 H-13 C long-range NMR experiments (HMBC). The selective formation of 1,4-disubstituted 1,2,3-triazoles was confirmed by a long-range correlation between the methylene protons H-19 and H-25 with the carbon-bearing H-21 in the triazole ring, since in 1,5-regioisomer the correlation between the benzyl protons and the carbon bearing the proton in the triazole ring would not be present ( figure 5).
On the other hand, one of the advantages of di-O-isopropylidene gluco-and allofuranose derivatives is that these compounds could be selectively deprotected to generate diols that can be subsequently functionalized for their use in different fields of scientific research. Tiwari and co-workers [41] reported the synthesis of a series of glycosyl triazoles and their evaluation as efficient ligands in Cheteroatom coupling reactions by using a mono-O-isopropylidene glucofuranose derivative as starting material. Srinivas et al. [42,43] reported a 1,2,3-triazole-linked allofuranose compound that can be a key intermediary in the preparation of a series of heterocyclic-containing carbohydrate-triazole hybrids with potential antimicrobial activity. The synthesis of such compounds involves a selective acidic hydrolysis of the corresponding acetonide to yield a chiral diol. Besides, it is being observed that the presence of multiple free hydroxyl groups in this type of structural scaffolding (unprotected carbohydrate-triazole hybrids) increases not only the solubility in the biological systems favoured by hydrogen bonds but also enhances the affinity and selectivity towards recognition proteins of carbohydrates [44]. In order to obtain new deprotected carbohydrate-linked 1,2,3-triazole, and considering the potential application of these products, we proceeded the removal of the acetonide protecting groups of carbohydrate-triazole conjugates 7a-e and 8a-e under acidic conditions. We   royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 7: 200290 found that HCl-MeOH system at room temperature (RT) was a clean method to obtain the desired deprotected derivatives 9a-e and their diastereomeric series of derivatives 10a-e in moderate yields. These results are summarized in table 3. According to the 1 H NMR spectra, the absence of two methyl groups around 1.20-1.40 ppm confirms the deprotection in the 5,6 positions of carbohydratetriazole 7a-e and 8a-e. The hydrolysis products were easily purified by column chromatography, allowing access to two new families of carbohydrate-triazole derivatives 9a-e and 10a-e through a simple structural modification in green and low-cost conditions. Finally, in order to have an insight into the possible biological activity of synthesized compounds, preliminary studies about the antimicrobial activity of triazoles 7a-e against different Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), as well as fungus (Aspergillus niger) and yeast (Candida utilis), were determined by using the disc diffusion test (table 4). Yields of the isolated product after purification. The results showed that almost all tested compounds exhibit a moderate antimicrobial activity against the tested bacteria strains. In general, compounds 7a-e were more active against Gram-negative than for Grampositive bacteria, where compound 7a was the most active compound against P. aeruginosa with an inhibition similar to that of streptomycin used as reference. No evident correlation between the electronic nature of halogen substituent and antibacterial activity was found. In the case of the antifungal activity, the tested triazole exhibited low activity against C. utilis and no antifungal activity was observed against A. niger strain.

Conclusion
We developed an easy and efficient method for the synthesis of two diastereomeric series of 1,2,3-triazolecarbohydrate conjugates derived from gluco-and allofuranose diacetonides through a three-component 1,3-dipolar azide-alkyne cycloaddition catalysed by Cu-Al mixed oxide. In this multi-component reaction, the microwave irradiation dramatically reduces the reaction time and the catalyst could be recycled and re-used at least for two runs without notable reduction of its catalytic activity. Some advantages of this protocol are mild reaction conditions, good yields, short reaction times and easy work-up and isolation. Besides, both protected and deprotected triazole-carbohydrate conjugates that were synthesized could be suitable starting materials for preparing novel compounds with an interesting biological profile. Further research to extend the substrate scope is under way in our laboratory.

Material and methods
All reagents were obtained from commercial suppliers and used without further purification. Merck silica gel (type 60, 0.063-0.200 mm) was used for column chromatography. All compounds were characterized by IR spectra, recorded on an FT-IR Bruker Tensor 27 spectrophotometer, by means of attenuated total reflection (ATR) technique, and all data is expressed as wavenumbers (cm −1 ). Melting points were obtained on a Fisher-Johns apparatus and are uncorrected. NMR spectra were recorded on a Bruker Ascend-400 (400 MHz) and Bruker Avance DMX-500 (500 MHz) spectrometers in CDCl 3 or acetone-d6 and chemical shifts are given in ppm with tetramethylsilane (TMS) as the reference. Direct analysis in real time mass spectrometry (DART-MS) were obtained on a Jeol AccuTOF; the values of the signals are expressed as mass/charge units (m/z). Microwave irradiation experiments were performed using a Discover System (CEM Corporation, Matthews, NC, USA) single-mode microwave with standard sealed microwave glass vials. The reaction temperature was monitored by an IR sensor on the outside wall of the reaction.

Synthesis of 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (1)
A solution of D-glucose (5 g, 27.78 mmol) in dry acetone (250 ml) was added concentrated sulfuric acid (1.2 ml) at room temperature. The reaction mixture was stirred vigorously at room temperature for 6 h. Anhydrous copper(II) sulfate (15 g) was added to the reaction mixture and stirring was continued at room temperature for 18 h. The reaction mixture was then neutralized with sodium bicarbonate and the inorganic materials were filtered off. The filtrate was evaporated under reduced pressure to leave a white solid, which was partitioned between CH 2 Cl 2 and H 2 O. The organic layer was dried over Na 2 SO 4 , filtered and evaporated under reduced pressure providing the title compound as a white solid. Recrystallization from hexane gave the pure diacetal.

Synthesis of 1,2:5,6-di-O-isopropylidene-α-D-allofuranose
The D-glucofuranose 1 (1 g, 3.84 mmol) was dissolved in a mixture of CH 2 Cl 2 /NaClO (4 : 4 ml) then was added TBAHS (0.260 g, 0.768 mmol) and TEMPO (0.090 g, 0.576 mmol). The reaction mixture was stirred vigorously at 35°C for 15 min. After this time, the reaction mixture was extracted three times with CH 2 Cl 2 (30 ml). The organic phase was dried with Na 2 SO 4 and evaporated under reduced pressure, and the resultant residue was dissolved in 10 ml of methanol. The reaction mixture was stirred for 10 min at 0°C before the addition of NaBH 4 (0.290 g, 7.68 mmol) and allowed to react for 40 min at 25°C. After this time, the reaction was quenched with 30 ml of water, and the aqueous layer was extracted three times with ethyl acetate, was dried over anhydrous Na 2 SO 4 , filtered, concentrated under vacuum and purified by column chromatography (ethyl acetate : hexane 2 : 1) afforded desired sugar 4 [45] in 90%,  A solution of 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose 2 or 1,2:5,6-di-O-isopropylidene-α-Dallofuranose 5 (1 g, 3.84 mmol) in anhydrous DMF (10 ml) was cooled to 0°C and sodium hydride 60% dispersion in mineral oil (320 mg, 9.0 mmol) was added. The reaction mixture was stirred at 0°C under nitrogen atmosphere for 30 min. Propargyl bromide 80 wt% in toluene (0.47 ml, 1.2 mmol) was added at 0°C and allowed to stir for 12 h at room temperature. Upon completion of the reaction, remaining sodium hydride quenched by water, the solvent was removed under reduced pressure and extracted with ethyl acetate (3 × 15 ml). The organic layer was dried over anhydrous Na 2 SO 4 , filtered, concentrated under vacuum and purified by column chromatography (ethyl acetate : hexane 85 : 15) afforded desired sugar-based alkyne.

General procedure for deprotection of carbohydrate-1,2,3-triazole conjugates
The corresponding glucosyl-triazol 7a-e or 8a-e (50 mg, 0.11 mmol) were dissolved in MeOH (4 ml) and then a solution of HCl (2 ml, 4 N) was added. The solution was stirred at room temperature for 5 h. After this time, the reaction mixture was treated with a saturated aqueous solution of NaHCO 3 until pH = 7 and extracted with ethyl acetate (3 × 30 ml). The organic phase was dried over Na 2 SO 4 and evaporated under reduced pressure. The resultant residue was purified by column chromatography on silica gel.