Chemoenzymatic synthesis of “click” xylosides and xylobiosides from lignocellulosic biomass

Abstract

Synthesis of bio-based molecules from plant biomass with chemoenzymatic pathways represents a challenging task for the development of green chemistry. In this context, an efficient two-step chemoenzymatic sequence has been achieved for the preparation of triazole-linked xylosides and xylobiosides from biomass-derived xylans. The synthesis of propargyl xyloside and xylobioside catalysed by a xylanase in an aqueous medium was first studied and improved according to different reaction parameters. Cycloaddition reactions between the terminal alkyne moiety of these xylosides or xylobiosides and various aliphatic, aromatic or functionalized azides (“click chemistry”) afforded various triazole-linked O-xylosides and O-xylobiosides in high yields. These molecules are of interest for different biological applications.

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Introduction

Lignocellulosic biomass is an abundant and low-cost source for renewable carbon. Sustainable refineries from lignocellulose represent an alternative for the petrochemical routes with the development of the production of bioethanol, biomaterials or bio-based added-value molecules.^1–4 Hemicelluloses, notably xylans, are the second most important polysaccharide in the biomass after cellulose. They are mainly constituted of pentoses and especially D-xylose that corresponds to the backbone unit of xylans.⁵ Natural conversion of xylans is achieved by enzymatic hydrolysis with glycoside hydrolases which can cleave glycosidic bonds by acid–base catalysis. Xylanases (EC 3.2.1.8) as endo-enzymes convert xylans into oligoxylosides whereas β-xylosidases (EC 3.2.1.37), exo-enzymes, hydrolyze oligoxylosides into D-xylose units. Besides these enzymes can also catalyze the production of various xylosides through reverse hydrolysis or transglycosylation reactions of glycosyl residues to alcohols.^6–8

Xylose is an unusual carbohydrate in mammalian cells, only present in the structure of proteoglycans. Proteoglycans are natural macromolecules composed of a core protein and glycosaminoglycan (GAG) side chains constituted of a repeating disaccharide of a hexosamine (GlcNac or GalNAc) and a hexuronic acid residue (GlcA or IdoA). This polysaccharide is covalently attached to the core protein through a specific tetrasaccharidic linkage, containing a β-D-xylopyranose directly bound to a serine residue.⁹ These GAG side chains, mainly sulfated, display many interactions with various proteins and thus play important roles in the regulation of numerous biological functions.¹⁰

Biosynthesis of GAG chains can also take place in cells via an exogenous pathway, independently of a core protein, by using simple xylosides as primers. Xylosides with hydrophobic aglycone groups have been shown to induce free GAG chains synthesis in different cellular systems by serving as acceptors for a galactosyl transferase in the first galactosylation step.¹¹ A range of β-D-xylopyranosides have been synthesized and studied for many years and some of them have exhibited a broad spectrum of biological applications as antithrombotic agents (Odiparcil),¹² antiproliferative agents¹³ or for skin anti-aging applications (Pro-Xylane™)¹⁴ (Fig. 1).

Fig. 1 Some representative reported xylosides with biological activities.

Recently, a wide library of triazole-linked N-xylosides containing a variety of hydrophobic groups, prepared by the “click chemistry” methodology from an azido xyloside, has been synthesized (Fig. 1). These N-xylosides have been shown to prime efficiently a diverse array of GAG chains and have exhibited potential anti-cancer activities¹⁵ or glycogen phosphorylase inhibition.¹⁶ Few examples of triazole-linked O-xylosides were described in the literature. These compounds have been obtained from a fully acetylated β-propargyl xyloside, synthesized in four chemical steps from D-xylose,¹⁷ and used for the synthesis of surfactants,¹⁸ water-soluble silicon-based dendrimers,¹⁹ or nucleotide analogues.²⁰

Preparation of glycosides using an enzymatic pathway via reverse hydrolysis or transglycosylation reactions represents an interesting and promising approach due to the use of unprotected carbohydrates, the high selectivity of the anomeric linkage and the mild reaction conditions. Previously, we have reported the synthesis of aliphatic β-xylosides by transglycosylation reactions from activated xyloside donors using a β-xylosidase.²¹ More recently, the use of a xylanase has led to the preparation of mixtures of aliphatic xylosides and oligoxylosides from xylans or directly from destarched wheat bran.²² One recent example in the literature described glycosidase-catalyzed synthesis of propargyl β-D-glucoside or galactoside and their use as substrates for a “click chemistry” step.²³ To our knowledge, no example of enzymatic synthesis of propargyl β-D-xyloside or xylobioside was previously described.

In this study, we developed a chemoenzymatic synthesis of a series of xylosides and xylobiosides featuring a triazole heterocycle using a xylanase-catalyzed synthesis of propargyl β-D-xyloside 1 and xylobioside 2 from xylans in a first step and the copper(I) catalyzed azide–alkyne cycloaddition (CuAAC) or click chemistry in a second step (Scheme 1). Both original xylosides 3 and xylobiosides 4 should exhibit potential biological activities for the priming of the biosynthesis of GAG chains, with different physico-chemical properties such as solubility according to the degree of polymerization (DP) of the xylose moiety.

Scheme 1 General strategy for the chemoenzymatic synthesis of triazole-linked O-xylosides.

Results and discussion

Enzymatic synthesis of propargyl xyloside and xylobioside from xylans

Transglycosylation reactions were performed with a commercial xylanase (NS 22083, Novozymes) using beechwood xylans and propargyl alcohol in water (pH = 6) at 50 °C. Propargyl xyloside and oligoxylosides were produced with DP from 1 to 6 according to mass spectra carried out on the crude reaction mixtures (Scheme 2).

Scheme 2 Enzymatic synthesis of propargyl xylosides using a xylanase.

In all reactions, propargyl xyloside 1 (n = 0, DP1) and propargyl xylobioside 2 (n = 1, DP2) were the most abundant compounds. Propargyl xylotrioside (n = 2, DP3) was also detected in the reaction mixture and purified in small amount but its purification was really tedious due to its polarity and to a mixture with xylose and oligoxylosides resulting from hydrolysis competing reaction. Propargyl xylosides with higher DP (DP 4 to DP 6) were produced in very small amounts and were only detected by mass spectrometry. Various experiments were focused on the different reaction parameters in order to optimize the enzymatic synthesis of propargyl xyloside and xylobioside. Enzymatic reactions were monitored by TLC (EtOAc/HOAc/H₂O 7 : 2 : 2), quantification of propargyl xyloside 1 and xylobioside 2 was achieved by HPLC and both compounds were isolated by flash chromatography.

First experiments were carried out in a water solution with 10% v/v of propargyl alcohol at 50 °C and 50 IU mL⁻¹ of xylanase with different xylan concentration (0.5 to 20% w/v, Fig. 2a and b).

Fig. 2 Influence of reaction parameters on propargyl xyloside and xylobioside synthesis. The reactions were performed at 50 °C, 1 h incubation time (a–d): 10% (v/v) of propargyl alcohol and 50 IU mL⁻¹ of xylanase (a and b); 10% (w/v) xylans and 10% (v/v) of propargyl alcohol (c); 10% (w/v) xylans and 50 IU mL⁻¹ of xylanase (d); 10% (v/v) of propargyl alcohol, 50 IU mL⁻¹ of xylanase and 10% (w/v) xylans (e).

Results clearly show that the best yields in propargyl xylosides were reached with the lower xylan concentrations (0.5% and 1% w/v, Fig. 2a). In the above mentioned conditions, propargyl xyloside 1 and propargyl xylobioside 2 were produced in the same amounts (290 mg g⁻¹ xylan for 0.5% xylan solution and 280 mg g⁻¹ xylan for 1% xylan solution), with a 16% overall yield calculated on the basis of xylan exclusively constituted of D-xylose units as the starting material (6% for 1 and 10% for 2). These calculated yields are underestimated since commercially available beechwood xylan is constituted of about 60% of xylose units as determined by classical sugar analysis (see Experimental section).^22a As our aim in this work was to obtain the largest amount of propargyl xylosides 1 and 2 in a single step, and not to optimize the conversion of xylans, yields were also expressed in mg of propargyl xylosides per mL of reaction (Fig. 2b). Higher xylan concentrations 10% w/v (19 mg mL⁻¹, overall yield 11%) and 20% w/v (22 mg mL⁻¹, overall yield 6%) led to the largest quantities of propargyl xylosides. The best compromise to maintain an acceptable yield and to insure the homogeneity of the reaction medium and an easy purification of the xylosides was to keep the xylan concentration at 10% w/v.

The effect of xylanase loading is depicted in Fig. 2c. During the reaction, the enzyme catalyzed transglycosylation (synthesis of propargyl xylosides) in competition with primary hydrolysis (hydrolysis of xylans into xylo-oligosaccharides) and secondary hydrolysis (hydrolysis of propargyl xylosides). Increasing the enzyme concentration from 2 to 100 IU mL⁻¹ led to higher amounts of transglycosylation reaction products. A slight decrease in the yield of propargyl xylosides was observed at 100 IU mL⁻¹ concentration, probably due to a higher secondary hydrolysis of both propargyl xyloside 1 and xylobioside 2. A more important effect on 2 was observed, leading to an increase of the ratio DP1/DP2.

The effect of concentration in propargyl alcohol in the reaction medium (1 to 20% v/v) was also studied (Fig. 2d). The best result was obtained with 20% v/v of propargyl alcohol (215 mg g⁻¹ xylan, 12% overall yield) but a modulation in the DP1/DP2 ratio from 1 : 6 to 1 : 3 was observed compared to the reaction with 10% v/v of propargyl alcohol. The lower amount of compound 1 could be attributed to the limitation of the secondary hydrolysis of propargyl oligoxylosides leading to the formation of propargyl xyloside 1. This secondary hydrolysis cannot be quantified since the propargyl xyloside is produced both by tranglycosylation and secondary hydrolysis.

The effect of the duration of incubation was investigated (Fig. 2e) from short times (5 min) to longer reaction times (24 h). Maximal transglycosylation yields were obtained from 1 h to 4 h (180–190 mg g⁻¹ xylan, overall yield 11%) at 50 °C. Interestingly, a modulation of the ratio 1/2 was observed according to the reaction time, from 1 : 6 (30 min) to 4.5 : 1 (24 h). In the latter case, the increase of the concentration in propargyl xyloside 1 is probably due a secondary hydrolysis of propargyl xylobioside 2. The decrease of the total amount of products for long reaction times was also observed and can be attributed to a secondary hydrolysis of propargyl xyloside 1.

Finally, an enzymatic reaction performed in the optimized reaction conditions: 10% w/v xylan concentration, 50 IU mL⁻¹ of commercially available xylanase, 10% v/v propargyl alcohol, for 1 h led to the production of DP1 propargyl xyloside 1 (45 mg g⁻¹ xylan) and DP2 propargyl xylobioside 2 (145 mg g⁻¹ xylan). A putative and acceptable 18% overall yield, based on the sugar composition of the starting material (60% of D-xylose), could be attributed for the transglycosylation in these reaction conditions. A scale-up reaction (500 mL solution) was then carried out in the same reaction conditions. The amounts of compounds 1 and 2 produced were not significantly modified as determined by HPLC quantification. After centrifugation of the reaction mixture, filtration through reverse phase C18 silica gel and a final purification by flash chromatography, isolated compounds 1 (2.3 g) and 2 (7.1 g) were obtained, so about 10 g for a 500 mL reaction medium.

“Click” reactions of propargyl xylosides with azides

Preparation of 1,2,3-triazoles rings by the Huisgen's 1,3-dipolar cycloaddition²⁴ catalyzed by Cu(I) species (CuAAC) has been applied to a wide variety of chemical research topics in the last decade²⁵ since its introduction by Sharpless²⁶ and Meldal.²⁷ Introduction of 1,2,3-triazole moieties in organic molecules can bring interesting favorable physico-chemical properties for drug discovery such as a greater stability under physiological conditions, π–π interactions with aromatic rings, hydrogen-bond accepting ability, a strong dipole moment or coordination of the nitrogen atoms with metal ions.²⁸

Click reactions were carried out in the previously described reaction conditions²³ with both purified propargyl xyloside 1 and propargyl xylobioside 2 and various azides (Scheme 3).

Scheme 3 Synthesis of triazole-linked O-xylosides 3 and triazole-linked O-xylobiosides 4.

All the reactions afforded the corresponding triazole-linked O-xylosides 3 and triazole-linked O-xylobiosides 4 in good yields (58% to 93%, Table 1).

Table 1 Yields obtained for triazole-linked O-xylosides 3 and triazole-linked O-xylobiosides 4

Entry	Compound	R	Yields (%)
1	3a		75
	4a		93
2	3b		88
	4b		73
3	3c		70
	4c		70
4	3d		80
	4d		80
5	3e		87
	4e		74
6	3f		58
	4f		61
7	3g		91
	4g		90

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Aliphatic (entry 1) and aromatic (entries 2–3) R groups were first introduced as substituents of the triazole ring to obtain xylosides with hydrophobic aglycone moieties 3a–c (75% to 88%) and 4a–c (70–93%), known as good candidates for an efficient priming of GAGs biosynthesis.

Introduction of functional groups such as hydroxyl group from azidoethanol (entry 4, 3d–4d) or an oligoethylene glycol (OEG) part (entry 5, 3e–4e) was achieved in order to modulate the water-solubility and the polarity of these molecules which seem to be important factors for the nature of the GAGs produced and the antiproliferative activity.^13c Additionally, it is worth to note that N-xylosides with OEG linkers were recently prepared to study the effect of the length of the OEG linker on the GAG priming ability and antiproliferative activity.²⁹ A N-protected serine-analogue was also used to give the corresponding triazole-linked xyloside 3f (58%) and xylobioside 4f (61%) attached to an aminoacid moiety (entry 6).³⁰ Finally, click reaction of 1 and 2 with commercially available peracetylated β-D-xylopyranosyl azide afforded the original compounds 3g (91%) and 4g (90%). Similar disaccharidic compounds with a hydroxynaphtyl aromatic part were previously synthesized.³¹ More recently, a library of cluster xylosides with two or more xylose residues attached via triazole linkers was synthesized and some compounds showed an interesting priming of multiple GAG chains.³²

Experimental section

Materials

Propargyl alcohol was purchased from Sigma-Aldrich Corp. (St. Louis, USA). Benzyl azide solution, 2-[2-(2-azidoethoxy)ethoxy]ethanol solution, Fmoc-β-azido-Ala-OH and 2,3,4-tri-O-acetyl-β-D-xylopyranosyl azide, were purchased from Sigma-Aldrich. Butyl azide,³³ 2-azidoethanol²³ and azidobenzene³⁴ were synthesized as previously described. Beechwood xylans were obtained from Carl Roth (Karlsruhe, Germany). The sugar composition of commercial beechwood xylan was determined after acid hydrolysis and chromatographic analysis.^22a For the starting material used in our study, the total ratio in monosaccharides was determined at 65% with an overall composition of D-xylose units of about 60%. The purified thermophilic endo-xylanase was purchased from Novozymes (NS-22083). Specific activity of xylanase NS 22083 (batch VHN00002) on beechwood xylan was 38 IU mg⁻¹.

Enzymatic synthesis of propargyl β-D-oligoxylosides

Enzymatic reactions (1 mL) were carried out in closed glass vessels with a magnetic stirring and were incubated in a thermostated oil bath for 1 h at 50 °C. The reactions were stopped by incubating the reaction mixtures during 10 min at 100 °C and centrifugating them for 10 min (2000g) in order to pellet the residual xylans. For the scale-up, the 500 mL reaction was incubated for 3 h at 50 °C in a reactor IKA®-WERKE (Eurostar).

Purification, characterization and quantification of the transglycosylation products

The qualitative analysis was performed by TLC, using Kieselgel 60 F₂₅₄ aluminium-backed sheets (E. Merck) and EtOAc/HOAc/water (7 : 2 : 2) as the mobile phase. Products were detected at 130 °C using 0.2% (v/v) orcinol in H₂SO₄ (20%, v/v). NMR spectra were recorded on Bruker spectrometer (500 MHz for ¹H and 125 MHz for ¹³C or 600 MHz for ¹H and 150 MHz for ¹³C). Chemical shifts are expressed in parts per million (ppm) using TMS as an internal standard. Coupling constants J are given in Hertz (Hz) and pattern abbreviations are as follows: s singlet, d doublet, t triplet, qu quintet, sex sextuplet, m multiplet. High resolution mass spectra (HRMS) were performed on Q-TOF Micro micromass positive ESI (CV = 30 V). After centrifugation to eliminate the residual xylan, the crude reaction was concentrated to half volume under reduced pressure. The purification of transglycosylation products DP1 and DP2 was achieved by silica gel chromatography (Polygoprep® 60–50 C18, Macherey-Nagel) with H₂O as the mobile phase. Fractions containing DP1 and DP2 were lyophilized and purified by silica gel chromatography (9385 Merck Kieselgel 60, 230–400 mesh, 40–63 μm) with EtOAc : MeOH (90 : 10) as mobile phase. These compounds were identified by ¹H and ¹³C-NMR, COSY, ¹H–¹³C correlation experiments and ESI-HRMS.

The quantification of DP1 and DP2 was performed by HPLC using a RP-C18 column (Kinetex 5 μm C18 100A, 150 × 4.6 mm, Phenomenex). Standards propargyl xylosides were purified as described above. Products were eluted at 0.8 mL min⁻¹ with a mobile phase composed of a methanol : water mixture (3 : 97). The detection of eluates was performed with a dynamic light scattering detector (ELSD-LTII, Shimadzu).

Propargyl β-D-xylopyranoside 1. White solid, m.p. 132 °C. [α]²⁰_D −85.9 (c 0.21 in H₂O). δ_H (500 MHz; D₂O; Me₄Si) 2.92 (1H, br s, H alkyne), 3.28 (1H, dd, J_2,1 7.8, J_2,3 9.2, 2-H), 3.33 (1H, dd, J_5,4 10.5, J_5,5 11.6, 5-H), 3.45 (1H, t, J_3,2 = J_3,4 9.2, 3-H), 3.62 (1H, ddd, J_4,5 5.4, J_4,3 9.2, J_4,5 10.5, 4-H), 3.97 (1H, dd, J_5,4 5.4, J_5,5 11.6, 5-H), 4.44 (2H, br s, OCH₂), 4.58 (1H, d, J_1,2 7.8, 1-H); δ_C (125 MHz; D₂O; Me₄Si) 56.7 (OCH₂), 65.2 (C-5), 69.1 (C-4), 72.8 (C-2), 75.6 (C-3), 76.3 (C–H alkyne), 78.8 (C-alkyne), 101.5 (C-1); ESI-HRMS: m/z calcd for C₈H₁₂O₅Na: [M + Na]⁺: 211.0582, found: 211.0585.

Propargyl β-D-xylobioside 2. White solid, m.p. 95 °C. [α]²⁰_D −83.3 (c 0.38 in H₂O). δ_H (500 MHz; D₂O; Me₄Si) 2.83 (1H, br s, H alkyne), 3.18 (1H, dd, J_2′,1′ 7.9, J_2′,3′ 9.2, 2′-H), 3.22–3.24 (1H, m, 5′-H), 3.24 (1H, dd, J_2,1 7.7, J_2,3 8.2, 2-H), 3.33 (1H, dd, J_5,4 9.2, J_5,5 11.8, 5-H), 3.35 (1H, dd, J_3′,4′ 5.9, J_3′,2′ 9.2, 3′-H), 3.50 (1H, dd, J_3,2 8.2, J_3,4 9.8, 3-H), 3.54–3.56 (1H, m, 4′-H), 3.71 (1H, ddd, J_4,5 5.2, J_4,5 9.2, J_4,3 9.8, 4-H), 3.89 (1H, dd, J_5′,4′ 5.5, J_5′,5′ 11.6, 5′-H), 4.03 (1H, dd, J_5,4 5.2, J_5,5 11.8, 5-H), 4.36 (2H, br s, OCH₂), 4.38 (1H, d, J_1′,2′ 7.9, 1′-H), 4.53 (1H, d, J_1,2 7.7, 1-H); δ_C (125 MHz; D₂O; Me₄Si) 56.6 (OCH₂), 62.8 (C-5), 65.1 (C-5′), 69.0 (C-4′), 72.5 (C-2), 72.6 (C-2′), 73.5 (C-3), 75.4 (C-3′), 76.1 (C-4), 76.2 (C–H alkyne), 78.5 (C-alkyne), 101.1 (C-1), 101.7 (C-1′); ESI-HRMS: m/z calcd for C₁₃H₂₀O₉Na: [M + Na]⁺: 343.1005, found: 343.0996.

Propargyl β-D-xylotrioside. Amorphous solid. [α]²⁰_D −68.6 (c 0.38 in H₂O). δ_H (500 MHz; D₂O; Me₄Si) 2.91 (1H, br s, H alkyne), 3.27 (1H, dd, J_{2′′,1′′} 7.9, J_{2′′,3′′} 9.2, 2′′-H), 3.29–3.37 (3H, m, 2-H and 2′-H and 5′′-H), 3.38–3.44 (2H, m, 5′-H and 5-H), 3.45 (1H, t, J_{3′′,2′′} = J_{3′′,4′′} 9.2, 3′′-H), 3.57 (1H, t, J_3′,2′ = J_3′,4′ 9.2, 3′-H), 3.60 (1H, t, J_3,2 = J_3,4 9.2, 3-H), 3.63 (1H, ddd, J_{4′′,5′′} 5.5, J_{4′′,3′′} 9.2, J_{4′′,5′′} 10.1, 4′′-H), 3.77–3.82 (2H, m, 4′-H and 4-H), 3.98 (1H, dd, J_{5′′,4′′} 5.5, J_{5′′,5′′} 11.6, 5′′-H), 4.09–4.13 (2H, m, 5′-H and 5-H), 4.44–4.45 (2H, m, OCH₂), 4.47 (1H, d, J_{1′′,2′′} 7.9, 1′′-H), 4.49 (1H, d, J_1′,2′ 7.8, 1′-H), 4.61 (d, J_1,2 7.7, 1-H); δ_C (125 MHz; D₂O; Me₄Si) 62.8 (C-5), 62.9 (C-5′), 65.2 (C-5′′), 69.1 (C-4′′), 72.6 (C-2 and C-2′), 72.7 (C-2′′), 79.6 (C-3 and C-3′), 75. 6 (C-3′′), 76.2 (C-4′), 76.3 (C-4), 76.8 (C–H alkyne), 78.5 (C-alkyne), 101.3 (C-1′′), 101.6 (C-1′), 101.8 (C-1); ESI-HRMS: m/z calcd for C₁₈H₂₈O₁₃Na: [M + Na]⁺: 475.1428, found: 475.1436.

General procedure for the click reactions

Propargyl xyloside 1 or propargyl xylobioside 2 (0.5 mmol), azide (0.6 mmol), copper(II) sulfate (9 mg, 0.05 mmol), and sodium ascorbate (20 mg, 0.1 mmol) were solubilized in a mixture tert-butyl alcohol : water (1 : 1; 4 mL) and stirred at room temperature overnight. The solution was directly subjected to flash column chromatography with EtOAc : MeOH (from 90 : 10 to 50 : 50) as the mobile phase to afford the corresponding triazole-linked O-xylosides 3 or xylobiosides 4.

Xyloside 3a. Yellow oil. [α]²⁰_D −57.9 (c 0.21 in MeOH). δ_H (600 MHz; CD₃OD; Me₄Si) 0.97 (3H, t, J 7.4, triazolyl-CH₂-CH₂-CH₂-CH₃), 1.34 (2H, sex, J 7.4, triazolyl-CH₂-CH₂-CH₂), 1.89 (2H, qu, J 7.4, triazolyl-CH₂-CH₂), 3.20 (1H, dd, J_2,1 7.5, J_2,3 9.0, 2-H), 3.22 (1H, dd, J_5,4 10.3, J_5,5 11.5, 5-H), 3.32–3.35 (1H, m, 3-H), 3.49 (1H, ddd, J_4,5 5.4, J_4,3 9.0, J_4,5 10.3, 4-H), 3.89 (1H, dd, J_5,4 5.4, J_5,5 11.5, 5-H), 4.33 (1H, d, J_1,2 7.5, 1-H), 4.41 (2H, t, J 7.4, triazolyl-CH₂), 4.73 (1H, d, J 12.4, 1H OCH₂), 4.91 (1H, d, J 12.4, 1H OCH₂), 7.90 (1H, s, H triazolyl); δ_C (150 MHz; CD₃OD; Me₄Si) 13.7 (triazolyl-CH₂-CH₂-CH₂-CH₃), 20.6 (triazolyl-CH₂-CH₂-CH₂), 33.3 (triazolyl-CH₂-CH₂), 51.1 (triazolyl-CH₂), 63.0 (OCH₂), 67.0 (C-5), 71.2 (C-4), 74.9 (C-2), 77.7 (C-3), 104.3 (C-1), 125.1 (C–H triazolyl), 145.6 (C-triazolyl); ESI-HMRS: m/z calcd for C₁₂H₂₁N₃O₅: [MH]⁺: 310.1379, found: 310.1387.

Xyloside 4a. Yellow oil. [α]²⁰_D −63.9 (c 1.55 in MeOH). δ_H (500 MHz; CD₃OD; Me₄Si) 0.92 (3H, t, J 7.5, triazolyl-CH₂-CH₂-CH₂-CH₃), 1.30 (2H, sex, J 7.5, triazolyl-CH₂-CH₂-CH₂), 1.85 (2H, qu, J 7.5, triazolyl-CH₂-CH₂), 3.16–3.23 (3H, m, 2-H and 2′-H and 5′-H), 3.26–3.30 (2H, m, 5-H and 3′-H), 3.41 (1H, t, J_3,2 = J_3,4 8.8, 3-H), 3.47 (1H, ddd, J_4′,5′ 5.4, J_4′,3′ 8.8, J_4′,5′ 10.3, 4′-H), 3.62 (1H, ddd, J_4,5 5.3, J_4,3 8.8, J_4,5 10, 4-H), 3.85 (1H, dd, J_5′,4′ 5.4, J_5′,5′ 11.5, 5′-H), 4.00 (1H, dd, J_5,4 5.3, J_5,5 11.7, 5-H), 4.29 (1H, d, J_1′,2′ 7.7, 1′-H), 4.30 (d, J_1,2 7.4, 1-H), 4.35 (2H, t, J 7, triazolyl-CH₂), 4.69 (1H, d, J 12.4, 1H OCH₂), 4.87 (1H, d, J 12.4, 1H OCH₂), 7.94 (s, H triazolyl); δ_C (125 MHz; CD₃OD; Me₄Si) 13.7 (triazolyl-CH₂-CH₂-CH₂-CH₃), 20.6 (triazolyl-CH₂-CH₂-CH₂), 33.3 (triazolyl-CH₂-CH₂), 51.1 (triazolyl-CH₂), 63.0 (OCH₂), 64.6 (C-5), 67.1 (C-5′), 71.1 (C-4′), 74.3 (C-2′), 74.6 (C-2), 75.8 (C-3), 77.6 (C-3′), 78.2 (C-4), 104.0 (C-1), 104.1 (C-1′), 124.1 (C-H triazolyl), 145.6 (C-triazolyl); ESI-HMRS: m/z calcd for C₁₇H₃₀N₃O₉Na: [MH]⁺: 420.1982, found: 420.1979.

Xyloside 3b. Pale yellow solid, m.p. 128 °C. [α]²⁰_D −52.9 (c 0.2 in H₂O). δ_H (500 MHz; CD₃OD; Me₄Si) 3.23–3.24 (1H, m, 2-H), 3.24–3.26 (1H, m, 5-H), 3.34 (1H, t, J_3,2 = J_3,4 8.9, 3-H), 3.52 (1H, ddd, J_4,5 5.3, J_4,3 8.9, J_4,5 10.2, 4-H), 3.91 (1H, dd, J_5,4 5.3, J_5,5 11.4, 5-H), 4.40 (1H, d, J_1,2 7.3, 1-H), 4.82–4.84 (1H, m, 1H OCH₂), 5.00 (1H, d, J 12.5, 1H OCH₂), 7.50 and 7.58 (2H, t, J 7.8, Ph), 7.83 (1H, d, J 7.8, Ph), 8.53 (1H, s, H triazolyl); δ_C (125 MHz; CD₃OD; Me₄Si) 63.0 (OCH₂), 67.0 (C-5), 71.2 (C-4), 74.9 (C-2), 77.7 (C-3), 104.4 (C-1), 121.6 (C-H Ph), 123.4 (C–H triazolyl), 130.1 and 131.0 (C-H Ph), 138.4 (C-Ph), 146.7 (C-triazolyl); ESI-HRMS: m/z calcd for C₁₄H₁₈N₃O₅: [MH]⁺: 308.1246, found: 308.1239.

Xyloside 4b. Pale yellow solid, m.p. 168 °C. [α]²⁰_D −49.0 (c 0.5 in H₂O). δ_H (500 MHz; CD₃OD; Me₄Si) 3.23–3.24 (1H, m, 2′-H), 3.24–3.26 (1H, m, 5′-H), 3.31–3.33 (1H, m, 2-H), 3.37–3.39 (2H, m, 5-H and 3-H), 3.49–3.51 (1H, m, 3′-H), 3.52–3.54 (1H, m, 4′-H), 3.68–3.70 (1H, m, 4-H), 3.92 (1H, dd, J_5′,4′ 5.3, J_5′,5′ 11.7, 5′-H), 4.08 (1H, dd, J_5,4 5.3, J_5,5 11.7, 5-H), 4.36 (1H, d, J_1′,2′ 7.5, 1′-H), 4.43 (1H, d, J_1,2 7.5, 1-H), 4.86 (1H, d, J 12.5, 1H OCH₂), 5.02 (1H, d, J 12.5, 1H OCH₂), 7.52 and 7.61 (2H, t, J 7.8, Ph), 7.86 (1H, d, J 7.8, Ph), 8.56 (1H, s, H triazolyl); δ_C (125 MHz; CD₃OD; Me₄Si) 63.0 (OCH₂), 64.6 (C-5), 67.1 (C-5′), 71.1 (C-4′), 74.3 (C-2′), 74.7 (C-2), 75.8 (C-3′), 77.6 (C-3), 78.2 (C-4), 104.0 (C-1′), 104.1 (C-1), 121.6 (C-H Ph), 123.4 (C-H triazolyl), 130.1 and 131.0 (C-H Ph), 138.4 (C-Ph), 146.6 (C-triazolyl); ESI-HRMS: m/z calcd for C₁₉H₂₆N₃O₉: [MH]⁺: 440.1669, found: 440.1681.

Xyloside 3c. White solid, m.p. 135 °C. [α]²⁰_D −33.9 (c 0.22 in H₂O). δ_H (500 MHz; CD₃OD; Me₄Si) 3.16–3.23 (1H, dd, J_2,1 7.5, J_2,3 9.0, 2-H), 3.18–3.26 (1H, m, 5-H), 3.31 (1H, t, J_3,2 = J_3,4 7.5, 3-H), 3.50 (1H, ddd, J_4,5 5.3, J_4,3 9.0, J_4,5 10.1, 4-H), 3.88 (1H, dd, J_5,4 5.3, J_5,5 11.4, 5-H), 4.33 (1H, d, J_1,2 7.5, 1-H), 4.73 (1H, d, J 12.4, 1H OCH₂), 4.91 (1H, d, J 12.4, 1H OCH₂), 5.61 (2H, s, CH₂-Ph), 7.31–7.43 (5H, m, Ph), 7.99 (1H, s, H triazolyl); δ_C (125 MHz; CD₃OD; Me₄Si) 53.5 (CH₂-Ph), 61.6 (OCH₂), 65.6 (C-5), 69.7 (C-4), 73.4 (C-2), 76.3 (C-3), 102.9 (C-1), 123.8 (C-H triazolyl), 127.7 and 128.2 and 128.6 (C-H Ph), 135.3 (C-Ph), 146.6 (C-triazolyl); ESI-HMRS: m/z calcd for C₁₅H₁₉N₃O₅Na: [M + Na]⁺: 344.1222, found: 344.1227.

Xyloside 4c. White solid, decomposition at T > 225 °C. [α]²⁰_D −61.8 (c 0.18 in DMSO). δ_H (500 MHz; DMSO-d6; Me₄Si) 2.98–3.04 (2H, m, 2-H and 2′-H), 3.06–3.09 (1H, m, 5′-H), 3.09–3.13 (1H, m, 3′-H), 3.16–3.18 (1H, m, 5-H), 3.21–3.25 (1H, m, 3-H), 3.26–3.31 (1H, m, 4′-H), 3.50 (1H, dt, J_4,5 5.5, J_4,3 9.4, 4-H), 3.70 (1H, dd, J_5′,4′ 5.5, J_5′,5′ 11.5, 5′-H), 3.88 (1H, dd, J_5,4 5.5, J_5,5 11.5, 5-H), 4.23 (1H, d, J_1′,2′ 7.5, 1′-H), 4.26 (1H, d, J_1,2 7.5, 1-H), 4.59 (1H, d, J 12.1, 1H OCH₂), 4.77 (1H, d, J 12.1, 1H OCH₂), 5.00 (3 OH), 5.17 (2 OH), 5.59 (2H, s, CH₂-Ph), 7.30–7.39 (10H, m, Ph), 8.15 (1H, s, H triazolyl); δ_C (125 MHz; DMSO-d6; Me₄Si) 52.8 (CH₂-Ph), 61.6 (OCH₂), 63.2 (C-5), 65.8 (C-5′), 69.5 (C-4′), 72.6 (C-2′), 73.1 (C-2), 74.2 (C-3), 75.4 (C-4), 76.3 (C-3′), 102.0 (C-1′), 102.7 (C-1), 124.2 (C–H triazolyl), 127.9–128.7 (C-H Ph), 136.1 (C-Ph), 144.0 (C-triazolyl); ESI-HMRS: m/z calcd for C₂₀H₂₇N₃O₉Na: [M + Na]⁺: 476.1645, found: 476.1653.

Xyloside 3d. Pale yellow solid, decomposition at T > 188 °C. [α]²⁰_D −47.1 (c 0.28 in H₂O). δ_H (500 MHz; D₂O; Me₄Si) 3.30 (1H, dd, J_2,1 7.8, J_2,3 9.2, 2-H), 3.36 (1H, dd, J_5,4 10.5, J_5,5 11.7, 5-H), 3.43 (1H, t, J_3,2 = J_3,4 9.2, 3-H), 3.62 (1H, ddd, J_4,5 5.5, J_4,3 9.2, J_4,5 10.5, 4-H), 3.97 (1H, dd, J_5,4 5.5, J_5,5 11.7, 5-H), 4.01 (2H, t, J 5.0, triazolyl-CH₂-CH₂-OH), 4.51 (1H, d, J_1,2 7.8, 1-H), 4.57 (2H, t, J 5.0, triazolyl-CH₂-CH₂-OH), 4.87 (1H, d, J 12.6, 1H OCH₂), 4.97 (1H, d, J 12.6, 1H OCH₂), 8.10 (1H, s, H-triazolyl); δ_C (125 MHz; D₂O; Me₄Si) 52.5 (triazolyl-CH₂-CH₂-OH), 60.1 (triazolyl-CH₂-CH₂-OH), 62.0 (OCH₂), 65.1 (C-5), 69.1 (C-4), 72.9 (C-2), 75.6 (C-3), 102.1 (C-1), 125.7 (C-H triazolyl), 143.4 (C-triazolyl); ESI-HMRS: m/z calcd for C₁₀H₁₇N₃O₆Na: [M + Na]⁺: 298.1015, found 298.1021.

Xyloside 4d. Yellow solid, decomposition at T > 225 °C. [α]²⁰_D −65.7 (c 0.28 in H₂O). δ_H (500 MHz; D₂O; Me₄Si) 3.28 (1H, dd, J_2,1 7.8, J_2,3 9.1, 2-H), 3.31 (1H, dd, J_2′,1′ 7.9, J_2′,3′ 9.2, 2′-H), 3.32 (1H, dd, J_5′,4′ 10.6, J_5′,5′ 11.6, 5′-H), 3.41 (1H, dd, J_5,4 10.3, J_5,5 11.8, 5-H), 3.44 (1H, t, J_3′,2′ = J_3′,4′ 9.2, 3′-H), 3.56 (1H, t, J_3,2 = J_3,4 9.1, 3-H), 3.63 (1H, ddd, J_4′,5′ 5.5, J_4′,3′ 9.2, J_4′,5′ 10.6, 4′-H), 3.78 (1H, ddd, J_4,5 5.3, J_4,3 9.1, J_4,5 10.3, 4-H), 3.98 (1H, dd, J_5′,4′ 5.5, J_5′,5′ 11.6, 5′-H), 4.02 (2H, t, J 4.2, triazolyl-CH₂-CH₂-OH), 4.11 (1H, dd, J_5,4 5.3, J_5,5 11.8, 5-H), 4.48 (1H, d, J_1′,2′ 7.9, 1′-H), 4.54 (1H, d, J_1,2 7.8, 1-H), 4.58 (2H, t, J 5.1, triazolyl-CH₂-CH₂-OH), 4.88 (1H, d, J 12.7, 1H OCH₂), 4.97 (1H, d, J 12.7, 1H OCH₂), 8.10 (1H, s, H triazolyl); δ_C (125 MHz; D₂O; Me₄Si) 52.5 (triazolyl-CH₂-CH₂-OH), 60.1 (triazolyl-CH₂-CH₂-OH), 62.0 (OCH₂), 62.9 (C-5), 65.2 (C-5′), 69.1 (C-4′), 72.7 (C-2′), 72.8 (C-2), 73.7 (C-3), 75.7 (C-3′), 76.3 (C-4), 101.8 (C-1′), 102.0 (C-1), 125.7 (C-H triazolyl), 143.4 (C-triazolyl); ESI-HMRS: m/z calcd for C₁₅H₂₅N₃O₁₀Na: [M + Na]⁺: 430.1438, found 430.1434.

Xyloside 3e. Yellow oil. [α]²⁰_D −32.1 (c 0.12 in H₂O). δ_H (600 MHz; D₂O; Me₄Si) 3.27 (1H, dd, J_2,1 7.8, J_2,3 10, 2-H), 3.31–3.35 (1H, m, 5-H), 3.42 (1H, t, J_3,2 = J_3,4 10, 3-H), 3.54–3.56 (2H, m, e-CH₂), 3.59–3.61 (1H, m, 4-H), 3.61–3.63 (2H, m, c-CH₂), 3.62–3.64 (2H, m, d-CH₂), 3.64–3.69 (2H, m, f-CH₂), 3.96 (1H, t, J_5,4 = J_5,5 10.0, 5-H), 3.99 (2H, t, J 5, b-CH₂), 4.50 (1H, d, J_1,2 7.8, 1-H), 4.64 (2H, t, J 5, a-CH₂), 4.85 (1H, d, J 12.5, 1H OCH₂), 4.95 (1H, d, J 12.5, 1H OCH₂), 8.11 (1H, s, H triazolyl); δ_C (150 MHz; D₂O; Me₄Si) 50.0 (C-a), 60.3 (C-f), 62.0 (OCH₂), 65.1 (C-5), 68.7 (C-b), 69.1 (C-4), 69.3 (C-c), 69.6 (C-d), 71.8 (C-e), 72.9 (C-2), 75.6 (C-3), 102.2 (C-1), 125.7 (C-H triazolyl), 143.5 (C-triazolyl); ESI-HMRS: m/z calcd for C₁₄H₂₅N₃O₈Na: [M + Na]⁺: 386.1539, found: 386.1537.

Xyloside 4e. Yellow oil. [α]²⁰_D −19.1 (c 0.49 in H₂O). δ_H (500 MHz; D₂O; Me₄Si) 3.27 (1H, dd, J_2,1 7.8, J_2,3 9.0, 2-H), 3.29–3.33 (2H, m, 2′-H and 5′-H), 3.41 (1H, dd, J_5,4 10.5, J_5,5 12, 5-H), 3.44 (1H, t, J_3′,2′ = J_3′,4′ 9.3, 3′-H), 3.54–3.64 (3H, m, 3-H and e-CH₂), 3.64–3.66 (1H, m, 4′-H), 3.65–3.69 (6H, m, c-CH₂ and d-CH₂ and f-CH₂), 3.79 (1H, ddd, J_4,5 5.3, J_4,3 9.0, J_4,5 10.5, 4-H), 3.96–3.99 (1H, m, 5′-H), 3.99–4.01 (2H, m, b-CH₂), 4.11 (1H, dd, J_5,4 5.3, J_5,5 12, 5-H), 4.47 (1H, d, J_1,2 7.8, 1-H), 4.53 (d, J_1′,2′ 7.7, 1′-H), 4.65 (2H, t, J 4.5, a-CH₂), 4.87 (1H, d, J 12.5, 1H OCH₂), 4.96 (1H, d, J 12.5, 1H OCH₂), 8.11 (1H, s, H triazolyl); δ_C (125 MHz; D₂O; Me₄Si) 50.0 (C-a), 60.3 (C-f), 62.0 (OCH₂), 62.9 (C-5), 65.2 (C-5′), 68.7 (C-b), 69.2 (C-4′), 69.3 (C-c), 69.7 (C-d), 71.6 (C-e), 72.7 (C-2′), 72.8 (C-2), 73.7 (C-3), 75.6 (C-3′), 76.3 (C-4), 101.8 (C-1′), 102.0 (C-1), 125.7 (C-H triazolyl), 143.5 (C-triazolyl); ESI-HMRS: m/z calcd for C₁₉H₃₃N₃O₁₂Na: [M + Na]⁺: 518.1962, found: 518.1968.

Xyloside 3f. (mixture of rotamers). White solid, decomposition at T > 215 °C. [α]²⁰_D −91.8 (c 0.39 in H₂O). δ_H (500 MHz; D₂O; Me₄Si) 3.0 (1H, t, J_2,1 = J_2,3 8.5, 2-H), 3.11–3.17 (1H, m, 5-H), 3.36 (1H, t, J_3,2 = J_3,4 8.5, 3-H), 3.41–3.45 (1H, m, 4-H), 3.80 (1H, dd, J_5,4 5.3 Hz, J_5,5 11.6, 5-H), 4.08 (1H, m, NHCOOCH₂CH), 4.16–4.19 (1H, m, 1H NHCOOCH₂), 4.30 (1H, d, J_1,2 8.5, 1-H), 4.33–4.41 (2H, m, 1H CH₂ NHCOOCH₂ and CH₂CHCOOH), 4.54–4.60 (2H, m, 1H CH₂CHCOOH and 1H OCH₂), 4.69 (1H, d, J 11.9, 1H OCH₂), 4.81–4.83 (1H, m, 1H CH₂CHCOOH); δ_C (125 MHz; D₂O; Me₄Si) 46.8 (NHCOOCH₂CH), 51.9 (CH₂CHCOOH), 56.0 (CHCOOH), 61.7 (OCH₂), 65.1 (C-5), 66.5 (NHCOOCH₂), 69.1 (C-4), 72.8 (C-2), 75.5 (C-3), 102.2 (C-1), 120.1 (C-H triazolyl), 125.1 (CH-Ph), 125.4 (CH-Ph), 127.4 (CH-Ph), 127.9 (CH-Ph), 140.9 (CH-Ph), 143.7 (C-Ph), 143.2 (C-triazolyl), 157.0 (OCONH), 174.4 (COOH); ESI-HMRS: m/z calcd for C₂₆H₂₈N₄O₉Na: [M + Na]⁺: 563.1754, found: 563.1747.

Xyloside 4f. (mixture of rotamers). White solid, decomposition at T > 190 °C. [α]²⁰_D −42.1 (c 0.19 in H₂O). δ_H (600 MHz; D₂O; Me₄Si) 3.01–3.02 (1H, t, J_2,1 = J_2,3 9.0, 2-H), 3.16–3.22 (2H, m, 2′-H and 5-H), 3.27–3.31 (1H, m, 5′-H), 3.37–3.42 (3H, m, 3-H and 3′-H and 4-H), 3.61 (1H, ddd, J_4′,5′ 5.4, J_4′,3′ 9.5, J_4′,5′ 10.5, 4′-H), 3.80 (1H, dd, J_5,4 5.0, J_5,5 12.6, 5-H), 3.96 (dd, J_5′,4′ 5.4, J_5′,5′ 11.3, 5′-H), 4.11–4.14 (2H, m, NHCOOCH₂CH and 1H NHCOOCH₂), 4.27 (1H, d, J_1′,2′ 7.8, 1′-H), 4.29 (1H, d, J_1,2 8.0, 1-H), 4.45–4.47 (1H, m, CHCOOH), 4.49–4.51 (1H, m, 1H NHCOOCH₂), 4.55–4.58 (2H, m, 1H CH₂CHCOOH and 1H OCH₂), 4.67 (1H, d, J 12.4, 1H OCH₂), 4.84–4.87 (1H, m, 1H CH₂CHCOOH); δ_C (150 MHz; D₂O; Me₄Si) 46.8 (NHCOOCH₂CH), 51.9 (CH₂CHCOOH), 56,0 (CHCOOH), 61.8 (OCH₂), 62.7 (C-5), 65.1 (C-5′), 66.5 (NHCOOCH₂), 69.1 (C-4′), 72.5 (C-2), 72.7 (C-2′), 73.4 (C-3), 75.5 (C-3′), 76.1 (C-4), 101.7 (C-1′), 102.0 (C-1), 120.1 (C-H triazolyl), 125.1 (CH-Ph), 125.2 (CH-Ph), 125.4 (CH-Ph), 125.5 (CH-Ph), 125.4 (CH-Ph), 127.4 (CH-Ph), 127.5 (CH-Ph), 128.0 (CH-Ph), 128.1 (CH-Ph), 140.9 (C-triazolyl), 143.2 (C-Ph), 143.6 (C-Ph), 143.9 (C-Ph), 157.1 OCONH, 174.4 (COOH); ESI-HMRS: m/z calcd for C₃₁H₃₆N₄O₁₃Na: [M + Na]⁺: 695.2177 found: 695.2183.

Xyloside 3g. Pale yellow solid, decomposition at T > 180 °C. [α]²⁰_D −76.5 (c 0.40 in DMSO). δ_H (500 MHz; DMSO; Me₄Si) 1.77 (3H, s, COCH₃), 1.96 (3H, s, COCH₃), 1.98 (3H, s, COCH₃), 2.97 (1H, dd, J_2,1 7.6, J_2,3 9.0, 2-H), 3.07–3.08 (1H, m, 5-H), 3.10 (1H, t, J_3,2 = J_3,4 9.0, 3-H), 3.28 (1H, ddd, J_4,5 5.4, J_4,3 9.0, J_4,5 10.1, 4-H), 3.71 (1H, dd, J_5,4 5.4, J_5,5 11.4, 5-H), 3.77 (1H, t, J_5a,4a = J_5a,5a 10.0, 5a-H), 4.08 (1H, dd, J_5a,4a 5.6, J_5a,5a 10.0, 5a-H), 4.13 (1H, d, J_1,2 7.6, 1-H), 4.61 (1H, d, J 12.6, 1H OCH₂), 4.74 (1H, d, J 12.6, 1H OCH₂), 5.09 (1H, td, J_4a,3a = J_4a,5a 10.0, J_4a,5a 5.6, 4a-H), 5.42 (1H, t, J_3a,2a = J_3a,4a 10.0, 3a-H), 5.55 (1H, t, J_2a,1a = J_2a,3a 10.0, 2a-H), 6.13 (d, J_1a,2a 10.0, 1a-H), 8.32 (1H, s, H-triazolyl); δ_C (125 MHz; DMSO; Me₄Si) 20.4 (COCH₃), 20.8 (COCH₃), 20.9 (COCH₃), 61.5 (OCH₂), 64.8 (C-5a), 66.1 (C-5), 68.5 (C-4a), 69.9 (C-4), 70.7 (C-2a), 72.6 (C-3a), 73.5 (C-2), 76.7 (C-3), 85.2 (C-1a), 102.8 (C-1), 124.0 (C-H triazolyl), 144.8 (C-triazolyl), 169.5 (COCH₃), 170.5 (COCH₃), 170.6 (COCH₃); ESI-HMRS: m/z calcd for C₁₉H₂₃N₃O₁₂: [MH]⁺: 490.1673, found: 490.1659.

Xyloside 4g. Pale yellow solid, m.p. 67 °C. [α]²⁰_D −91.8 (c 0.39 in MeOH). δ_H (500 MHz; CD₃OD; Me₄Si) 1.87 (3H, s, COCH₃), 2.04 (3H, s, COCH₃), 2.07 (3H, s, COCH₃), 3.19–3.29 (3H, m, 2-H and 2′-H and 5-H), 3.33–3.36 (1H, m, 3-H), 3.35–3.38 (1H, m, 5′-H), 3.47 (1H, t, J_3′,2′ = J_3′,4′ 9.0, 3′-H), 3.52 (1H, ddd, J_4,5 5.3, J_4,3 8.7, J_4,5 10.3, 4-H), 3.68 (1H, ddd, J_4′,5′ 5.1, J_4′,3′ 9.0, J_4′,5′ 10.1, 4′-H), 3.79 (1H, dd, J_5a,4a 10.5, J_5a,5a 11.5, 5a-H), 3.91 (1H, dd, J_5,4 5.4, J_5,5 11.5, 5-H), 4.06 (dd, J_5′,4′ 5.3, J_5′,5′ 11.5, 5′-H), 4.27 (1H, dd, J_5a,4a 5.6, J_5a,5a 11.5, 5a-H), 4.31 (1H, d, J_1′,2′ 7.5, 1′-H), 4.35 (1H, d, J_1,2 7.5, 1-H), 4.78 (1H, d, J 12.7, 1H OCH₂), 4.91 (1H, d, J 12.7, 1H OCH₂), 5.22 (1H, ddd, J_4a,5a 5.6, J_4a,3a 9.4, J_4a,5a 10.5, 4a-H), 5.52 (1H, t, J_3a,2a = J_3a,4a 9.4, 3a-H), 5.58 (1H, t, J_2a,1a = J_2a,3a 9.4, 2a-H), 6.05 (1H, d, J_1a,2a 9.4, 1a-H), 8.28 (1H, s, H triazolyl); δ_C (125 MHz; CD₃OD; Me₄Si) 20.2 (COCH₃), 20.4 (COCH₃), 20.5 (COCH₃), 62.5 (OCH₂), 64.6 (C-5), 66.3 (C-5a), 67.1 (C-5′), 69.8 (C-4a), 71.1 (C-4′), 72.1 (C-2a), 73.7 (C-3a), 74.3 (C-2), 74.6 (C-2′), 75.7 (C-3), 77.6 (C-3′), 78.2 (C-4), 87.3 (C-1a), 103.6 (C-1′), 104.0 (C-1), 124.4 (C-H triazolyl), 146.0 (C-triazolyl), 170.5 (COCH₃), 171.4 (COCH₃), 171.5 (COCH₃); ESI-HMRS: m/z calcd for C₂₄H₃₅N₃O₁₆Na: [M + Na]⁺: 644.1915, found: 644.1921.

Conclusions

In conclusion, a range of original triazole-linked O-xylosides and O-xylobiosides was prepared in a straightforward chemoenzymatic two-step sequence from xylans. The synthesis of O-xylobiosides by this methodology is of particular interest since these compounds are not accessible by means of conventional carbohydrate synthesis. Preparation of propargyl xyloside and xylobioside was first investigated via an enzymatic transglycosylation reaction from xylans and propargyl alcohol using a xylanase. Different reaction parameters were studied (xylan concentration, alcohol concentration, enzyme loading, reaction time) to optimize the production of xylosides. In the second step, click reactions in an aqueous solution were carried out with various azides to give the corresponding triazoles with good yields (53% to 93%). Aliphatic, aromatic, hydroxyl, xylose-derived or aminoacid-derived groups were introduced as the substituent of the triazole ring. Biological evaluation of these new triazole-linked xylosides will be undertaken.

Acknowledgements

This work was supported by the “Région Champagne-Ardenne” (Xylocos Emergence program). The authors are grateful to the “Région Champagne-Ardenne” and FEDER for financial support and for doctoral fellowships (M. O. and C. B.). NS-22083 xylanase was kindly provided by Novozymes (France).

Notes and references

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Footnote

† Present address: LISBP, INSA de Toulouse, 135 Avenue de Rangueil, 31077 Toulouse Cedex 04, France.

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