Chemoenzymatic synthesis of click xylosides and xylobiosides from lignocellulosic biomass

Article abstract of DOI:10.1039/c3ra46173d

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.

Full text of DOI:10.1039/c3ra46173d

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Chemoenzymatic synthesis of “click” xylosides
and xylobiosides from lignocellulosic biomass
Cite this: RSC Adv., 2014, 4, 9330
a
Charlotte Brusa,abc Marjorie Ochs,†abc Caroline Remond,bc Murielle Muzard
*
´
a
*
and Richard Plantier-Royon
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.
Received 28th October 2013
Accepted 23rd January 2014
DOI: 10.1039/c3ra46173d
www.rsc.org/advances
glycosaminoglycan (GAG) side chains constituted of a repeating
disaccharide of a hexosamine (GlcNac or GalNAc) and a hex-
Introduction
uronic acid residue (GlcA or IdoA). This polysaccharide is
covalently attached to the core protein through a specic tet-
rasaccharidic linkage, containing a b-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 bio-
logical functions.10
Lignocellulosic biomass is an abundant and low-cost source for
renewable carbon. Sustainable reneries 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 aer 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 b-xylosi-
dases (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
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 agly-
cone groups have been shown to induce free GAG chains
synthesis in different cellular systems by serving as acceptors
for a galactosyl transferase in the rst galactosylation step.11
A
range of b-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
a
´
´
Universite de Reims Champagne-Ardenne, Institut de Chimie Moleculaire de Reims
ˆ
(ICMR), CNRS UMR 7312, UFR Sciences Exactes et Naturelles, Batiment 18
Europol'Agro, BP 1039, F-51687 Reims Cedex 2, France. E-mail: richard.
plantier-royon@univ-reims.fr; murielle.muzard@univ-reims.fr; Fax: +33 326913166;
Tel: +33 326913308
b
´
Universite de Reims Champagne-Ardenne, UMR 614 Fractionnement des
AgroRessources et Environnement, F-51100, France
cINRA, UMR 614, Fractionnement des AgroRessources et Environnement, F-51100,
France
† Present address: LISBP, INSA de Toulouse, 135 Avenue de Rangueil, 31077
Toulouse Cedex 04, France.
Fig.
activities.
1 Some representative reported xylosides with biological
9330 | RSC Adv., 2014, 4, 9330–9338
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Scheme 1 General strategy for the chemoenzymatic synthesis of triazole-linked O-xylosides.
(Odiparcil),12 antiproliferative agents13 or for skin anti-aging and oligoxylosides were produced with DP from 1 to 6 according
applications (Pro-Xylane™)14 (Fig. 1).
to mass spectra carried out on the crude reaction mixtures
Recently, a wide library of triazole-linked N-xylosides con- (Scheme 2).
taining a variety of hydrophobic groups, prepared by the “click
In all reactions, propargyl xyloside 1 (n ¼ 0, DP1) and prop-
chemistry” methodology from an azido xyloside, has been argyl xylobioside 2 (n ¼ 1, DP2) were the most abundant
synthesized (Fig. 1). These N-xylosides have been shown to compounds. Propargyl xylotrioside (n ¼ 2, DP3) was also
prime efficiently a diverse array of GAG chains and have detected in the reaction mixture and puried in small amount
exhibited potential anti-cancer activities15 or glycogen phos- but its purication was really tedious due to its polarity and to a
phorylase inhibition.16 Few examples of triazole-linked O-xylo- mixture with xylose and oligoxylosides resulting from hydrolysis
sides were described in the literature. These compounds have competing reaction. Propargyl xylosides with higher DP (DP 4 to
been obtained from a fully acetylated b-propargyl xyloside, DP 6) were produced in very small amounts and were only
synthesized in four chemical steps from D-xylose,17 and used for detected by mass spectrometry. Various experiments were
the synthesis of surfactants,18 water-soluble silicon-based den- focused on the different reaction parameters in order to opti-
drimers,19 or nucleotide analogues.20
mize the enzymatic synthesis of propargyl xyloside and xylo-
Preparation of glycosides using an enzymatic pathway via bioside. Enzymatic reactions were monitored by TLC (EtOAc/
reverse hydrolysis or transglycosylation reactions represents an HOAc/H2O 7 : 2 : 2), quantication of propargyl xyloside 1 and
interesting and promising approach due to the use of unpro- xylobioside 2 was achieved by HPLC and both compounds were
tected carbohydrates, the high selectivity of the anomeric isolated by ash chromatography.
linkage and the mild reaction conditions. Previously, we have
First experiments were carried out in a water solution with
reported the synthesis of aliphatic b-xylosides by trans- 10% v/v of propargyl alcohol at 50 ꢀC and 50 IU mLꢁ1 of xylanase
glycosylation reactions from activated xyloside donors using a with different xylan concentration (0.5 to 20% w/v, Fig. 2a and b).
b-xylosidase.21 More recently, the use of a xylanase has led to the
Results clearly show that the best yields in propargyl xylo-
preparation of mixtures of aliphatic xylosides and oligoxylo- sides were reached with the lower xylan concentrations (0.5%
sides from xylans or directly from destarched wheat bran.22 One and 1% w/v, Fig. 2a). In the above mentioned conditions,
recent example in the literature described glycosidase-catalyzed propargyl xyloside 1 and propargyl xylobioside 2 were produced
synthesis of propargyl b-D-glucoside or galactoside and their use in the same amounts (290 mg gꢁ1 xylan for 0.5% xylan solution
as substrates for a “click chemistry” step.23 To our knowledge, and 280 mg gꢁ1 xylan for 1% xylan solution), with a 16% overall
no example of enzymatic synthesis of propargyl b-D-xyloside or yield calculated on the basis of xylan exclusively constituted of
xylobioside was previously described.
D-xylose units as the starting material (6% for 1 and 10% for 2).
In this study, we developed a chemoenzymatic synthesis of These calculated yields are underestimated since commercially
a series of xylosides and xylobiosides featuring a triazole available beechwood xylan is constituted of about 60% of xylose
heterocycle using a xylanase-catalyzed synthesis of propargyl b-D- units as determined by classical sugar analysis (see Experi-
xyloside 1 and xylobioside 2 from xylans in a rst step and the mental section).22a As our aim in this work was to obtain the
copper(I) catalyzed azide–alkyne cycloaddition (CuAAC) or click largest amount of propargyl xylosides 1 and 2 in a single step,
chemistry in a second step (Scheme 1). Both original xylosides 3 and not to optimize the conversion of xylans, yields were also
and xylobiosides 4 should exhibit potential biological activities expressed in mg of propargyl xylosides per mL of reaction
for the priming of the biosynthesis of GAG chains, with different (Fig. 2b). Higher xylan concentrations 10% w/v (19 mg mLꢁ1
,
physico-chemical properties such as solubility according to the overall yield 11%) and 20% w/v (22 mg mLꢁ1, overall yield 6%)
degree of polymerization (DP) of the xylose moiety.
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
Scheme
2 Enzymatic synthesis of propargyl xylosides using a
propargyl alcohol in water (pH ¼ 6) at 50 ꢀC. Propargyl xyloside xylanase.
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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 mLꢁ1 of xylanase (a and b); 10% (w/v) xylans and 10% (v/v) of propargyl alcohol (c); 10% (w/v)
xylans and 50 IU mLꢁ1 of xylanase (d); 10% (v/v) of propargyl alcohol, 50 IU mLꢁ1 of xylanase and 10% (w/v) xylans (e).
led to the largest quantities of propargyl xylosides. The best best result was obtained with 20% v/v of propargyl alcohol
compromise to maintain an acceptable yield and to insure the (215 mg gꢁ1 xylan, 12% overall yield) but a modulation in the
homogeneity of the reaction medium and an easy purication DP1/DP2 ratio from 1 : 6 to 1 : 3 was observed compared to the
of the xylosides was to keep the xylan concentration at 10% w/v. reaction with 10% v/v of propargyl alcohol. The lower amount of
The effect of xylanase loading is depicted in Fig. 2c. During compound 1 could be attributed to the limitation of the
the reaction, the enzyme catalyzed transglycosylation (synthesis secondary hydrolysis of propargyl oligoxylosides leading to the
of propargyl xylosides) in competition with primary hydrolysis formation of propargyl xyloside 1. This secondary hydrolysis
(hydrolysis of xylans into xylo-oligosaccharides) and secondary cannot be quantied since the propargyl xyloside is produced
hydrolysis (hydrolysis of propargyl xylosides). Increasing the both by tranglycosylation and secondary hydrolysis.
enzyme concentration from 2 to 100 IU mLꢁ1 led to higher
The effect of the duration of incubation was investigated
amounts of transglycosylation reaction products. A slight (Fig. 2e) from short times (5 min) to longer reaction times (24 h).
decrease in the yield of propargyl xylosides was observed at Maximal transglycosylation yields were obtained from 1 h to 4 h
100 IU mLꢁ1 concentration, probably due to a higher secondary (180–190 mg gꢁ1 xylan, overall yield 11%) at 50 ꢀC. Interestingly,
hydrolysis of both propargyl xyloside 1 and xylobioside 2. A a modulation of the ratio 1/2 was observed according to the
more important effect on 2 was observed, leading to an increase reaction time, from 1 : 6 (30 min) to 4.5 : 1 (24 h). In the latter
of the ratio DP1/DP2.
case, the increase of the concentration in propargyl xyloside 1 is
The effect of concentration in propargyl alcohol in the probably due a secondary hydrolysis of propargyl xylobioside 2.
reaction medium (1 to 20% v/v) was also studied (Fig. 2d). The The decrease of the total amount of products for long reaction
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Table 1 Yields obtained for triazole-linked O-xylosides 3 and triazole-
linked O-xylobiosides 4
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 mLꢁ1
of commercially available xylanase, 10% v/v propargyl alcohol,
for 1 h led to the production of DP1 propargyl xyloside 1 (45 mg
gꢁ1 xylan) and DP2 propargyl xylobioside 2 (145 mg gꢁ1 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 condi-
tions. 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 signicantly modied as determined
by HPLC quantication. Aer centrifugation of the reaction
mixture, ltration through reverse phase C18 silica gel and a
nal purication by ash chromatography, isolated compounds
1 (2.3 g) and 2 (7.1 g) were obtained, so about 10 g for a 500 mL
reaction medium.
Entry
1
Compound
R
Yields (%)
3a
4a
75
93
3b
4b
88
73
2
3
3c
4c
70
70
3d
4d
80
80
4
5
3e
4e
87
74
3f
4f
58
61
6
7
“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, p–p 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 puried propargyl xyloside 1
and propargyl xylobioside 2 and various azides (Scheme 3).
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).
Aliphatic (entry 1) and aromatic (entries 2–3) R groups were
rst 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
3g
4g
91
90
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 corre-
sponding 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 b-
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
Scheme 3 Synthesis of triazole-linked O-xylosides 3 and triazole-linked O-xylobiosides 4.
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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).
Experimental section
Materials
Propargyl b-D-xylopyranoside 1. White solid, m.p. 132 ꢀC.
[a]2D0 ꢁ85.9 (c 0.21 in H2O). dH (500 MHz; D2O; Me4Si) 2.92 (1H,
br s, H alkyne), 3.28 (1H, dd, J2,1 7.8, J2,3 9.2, 2-H), 3.33 (1H, dd,
J5,4 10.5, J5,5 11.6, 5-H), 3.45 (1H, t, J3,2 ¼ J3,4 9.2, 3-H), 3.62 (1H,
ddd, J4,5 5.4, J4,3 9.2, J4,5 10.5, 4-H), 3.97 (1H, dd, J5,4 5.4, J5,5 11.6,
5-H), 4.44 (2H, br s, OCH2), 4.58 (1H, d, J1,2 7.8, 1-H); dC (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 alcohol was purchased from Sigma-Aldrich Corp. (St.
Louis, USA). Benzyl azide solution, 2-[2-(2-azidoethoxy)ethoxy]-
ethanol solution, Fmoc-b-azido-Ala-OH and 2,3,4-tri-O-acetyl-b-
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
aer acid hydrolysis and chromatographic analysis.22a For the
starting material used in our study, the total ratio in mono-
saccharides was determined at 65% with an overall composition
of D-xylose units of about 60%. The puried thermophilic endo-
xylanase was purchased from Novozymes (NS-22083). Specic
activity of xylanase NS 22083 (batch VHN00002) on beechwood
Propargyl b-D-xylobioside 2. White solid, m.p. 95 ꢀC.
[a]2D0 ꢁ83.3 (c 0.38 in H2O). dH (500 MHz; D2O; Me4Si) 2.83 (1H,
br s, H alkyne), 3.18 (1H, dd, J2 ,1 7.9, J2 ,3 9.2, 20-H), 3.22–3.24
0
0
0
0
(1H, m, 50-H), 3.24 (1H, dd, J2,1 7.7, J2,3 8.2, 2-H), 3.33 (1H, dd, J5,4
xylan was 38 IU mgꢁ1
.
0
0
0
0
0
9.2, J5,5 11.8, 5-H), 3.35 (1H, dd, J3 ,4 5.9, J3 ,2 9.2, 3 -H), 3.50 (1H,
dd, J3,2 8.2, J3,4 9.8, 3-H), 3.54–3.56 (1H, m, 40-H), 3.71 (1H, ddd,
J4,5 5.2, J4,5 9.2, J4,3 9.8, 4-H), 3.89 (1H, dd, J5 ,4 5.5, J5 ,5 11.6, 50-
0
0
0
0
Enzymatic synthesis of propargyl b-D-oligoxylosides
H), 4.03 (1H, dd, J5,4 5.2, J5,5 11.8, 5-H), 4.36 (2H, br s, OCH2),
Enzymatic reactions (1 mL) were carried out in closed glass
vessels with a magnetic stirring and were incubated in a ther-
mostated oil bath for 1 h at 50 ꢀC. The reactions were stopped by
0
0
0
4.38 (1H, d, J1 ,2 7.9, 1 -H), 4.53 (1H, d, J1,2 7.7, 1-H); dC (125 MHz;
D2O; Me4Si) 56.6 (OCH2), 62.8 (C-5), 65.1 (C-50), 69.0 (C-40), 72.5
(C-2), 72.6 (C-20), 73.5 (C-3), 75.4 (C-30), 76.1 (C-4), 76.2 (C–H
alkyne), 78.5 (C-alkyne), 101.1 (C-1), 101.7 (C-10); ESI-HRMS: m/z
calcd for C13H20O9Na: [M + Na]+: 343.1005, found: 343.0996.
Propargyl b-D-xylotrioside. Amorphous solid. [a]2D0 ꢁ68.6
(c 0.38 in H2O). dH (500 MHz; D2O; Me4Si) 2.91 (1H, br s, H
ꢀ
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).
alkyne), 3.27 (1H, dd, J2 ,1 7.9, J2 ,3 9.2, 200-H), 3.29–3.37 (3H,
m, 2-H and 20-H and 500-H), 3.38–3.44 (2H, m, 50-H and 5-H), 3.45
00 00
00 00
Purication, characterization and quantication of the
transglycosylation products
(1H, t, J3 ,2 ¼ J3 ,4 9.2, 300-H), 3.57 (1H, t, J3 ,2 ¼ J3 ,4 9.2, 30-H),
00 00
00 00
0
0
0
0
00 00
00 00
The qualitative analysis was performed by TLC, using Kieselgel 3.60 (1H, t, J3,2 ¼ J3,4 9.2, 3-H), 3.63 (1H, ddd, J4 ,5 5.5, J4 ,3 9.2,
60 F254 aluminium-backed sheets (E. Merck) and EtOAc/HOAc/ J4 ,5 10.1, 400-H), 3.77–3.82 (2H, m, 40-H and 4-H), 3.98 (1H, dd,
00 00
00
0
00 00
00 00
water (7 : 2 : 2) as the mobile phase. Products were detected at J5 ,4 5.5, J5 ,5 11.6, 5 -H), 4.09–4.13 (2H, m, 5 -H and 5-H), 4.44–
130 ꢀC using 0.2% (v/v) orcinol in H2SO4 (20%, v/v). NMR 4.45 (2H, m, OCH2), 4.47 (1H, d, J1 ,2 7.9, 1 -H), 4.49 (1H, d, J1 ,2
00
00 00
0
0
spectra were recorded on Bruker spectrometer (500 MHz for 1H 7.8, 10-H), 4.61 (d, J1,2 7.7, 1-H); dC (125 MHz; D2O; Me4Si) 62.8
and 125 MHz for 13C or 600 MHz for 1H and 150 MHz for 13C). (C-5), 62.9 (C-50), 65.2 (C-500), 69.1 (C-400), 72.6 (C-2 and C-20), 72.7
Chemical shis are expressed in parts per million (ppm) using (C-200), 79.6 (C-3 and C-30), 75. 6 (C-300), 76.2 (C-40), 76.3 (C-4), 76.8
TMS as an internal standard. Coupling constants J are given in (C–H alkyne), 78.5 (C-alkyne), 101.3 (C-100), 101.6 (C-10), 101.8
Hertz (Hz) and pattern abbreviations are as follows: s singlet, d (C-1); ESI-HRMS: m/z calcd for C18H28O13Na: [M + Na]+:
doublet, t triplet, qu quintet, sex sextuplet, m multiplet. High 475.1428, found: 475.1436.
resolution mass spectra (HRMS) were performed on Q-TOF
Micro micromass positive ESI (CV ¼ 30 V). Aer centrifugation
General procedure for the click reactions
to eliminate the residual xylan, the crude reaction was
concentrated to half volume under reduced pressure. The Propargyl xyloside 1 or propargyl xylobioside 2 (0.5 mmol), azide
purication of transglycosylation products DP1 and DP2 was (0.6 mmol), copper(II) sulfate (9 mg, 0.05 mmol), and sodium
achieved by silica gel chromatography (Polygoprep® 60–50 C18, ascorbate (20 mg, 0.1 mmol) were solubilized in a mixture tert-
Macherey-Nagel) with H2O as the mobile phase. Fractions butyl alcohol : water (1 : 1; 4 mL) and stirred at room temper-
containing DP1 and DP2 were lyophilized and puried by silica ature overnight. The solution was directly subjected to ash
gel chromatography (9385 Merck Kieselgel 60, 230–400 mesh, column chromatography with EtOAc : MeOH (from 90 : 10 to
40–63 mm) with EtOAc : MeOH (90 : 10) as mobile phase. These 50 : 50) as the mobile phase to afford the corresponding tri-
compounds were identied by 1H and 13C-NMR, COSY, 1H–13C azole-linked O-xylosides 3 or xylobiosides 4.
correlation experiments and ESI-HRMS.
Xyloside 3a. Yellow oil. [a]2D0 ꢁ57.9 (c 0.21 in MeOH). dH (600
The quantication of DP1 and DP2 was performed by HPLC MHz; CD3OD; Me4Si) 0.97 (3H, t, J 7.4, triazolyl-CH2-CH2-CH2-
using a RP-C18 column (Kinetex 5 mm C18 100A, 150 ꢂ 4.6 mm, CH3), 1.34 (2H, sex, J 7.4, triazolyl-CH2-CH2-CH2), 1.89 (2H, qu,
Phenomenex). Standards propargyl xylosides were puried as J 7.4, triazolyl-CH2-CH2), 3.20 (1H, dd, J2,1 7.5, J2,3 9.0, 2-H), 3.22
described above. Products were eluted at 0.8 mL minꢁ1 with a (1H, dd, J5,4 10.3, J5,5 11.5, 5-H), 3.32–3.35 (1H, m, 3-H), 3.49 (1H,
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ddd, J4,5 5.4, J4,3 9.0, J4,5 10.3, 4-H), 3.89 (1H, dd, J5,4 5.4, J5,5 11.5, 7.31–7.43 (5H, m, Ph), 7.99 (1H, s, H triazolyl); dC (125 MHz;
5-H), 4.33 (1H, d, J1,2 7.5, 1-H), 4.41 (2H, t, J 7.4, triazolyl-CH2), CD3OD; Me4Si) 53.5 (CH2-Ph), 61.6 (OCH2), 65.6 (C-5), 69.7 (C-4),
4.73 (1H, d, J 12.4, 1H OCH2), 4.91 (1H, d, J 12.4, 1H OCH2), 7.90 73.4 (C-2), 76.3 (C-3), 102.9 (C-1), 123.8 (C-H triazolyl), 127.7 and
(1H, s, H triazolyl); dC (150 MHz; CD3OD; Me4Si) 13.7 (triazolyl- 128.2 and 128.6 (C-H Ph), 135.3 (C-Ph), 146.6 (C-triazolyl); ESI-
CH2-CH2-CH2-CH3), 20.6 (triazolyl-CH2-CH2-CH2), 33.3 (tri- HMRS: m/z calcd for C15H19N3O5Na: [M + Na]+: 344.1222, found:
azolyl-CH2-CH2), 51.1 (triazolyl-CH2), 63.0 (OCH2), 67.0 (C-5), 344.1227.
71.2 (C-4), 74.9 (C-2), 77.7 (C-3), 104.3 (C-1), 125.1 (C–H tri-
Xyloside 4c. White solid, decomposition at T > 225 ꢀC.
azolyl), 145.6 (C-triazolyl); ESI-HMRS: m/z calcd for C12H21N3O5: [a]2D0 ꢁ61.8 (c 0.18 in DMSO). dH (500 MHz; DMSO-d6; Me4Si)
[MH]+: 310.1379, found: 310.1387.
2.98–3.04 (2H, m, 2-H and 20-H), 3.06–3.09 (1H, m, 50-H), 3.09–
Xyloside 4a. Yellow oil. [a]2D0 ꢁ63.9 (c 1.55 in MeOH). dH (500 3.13 (1H, m, 30-H), 3.16–3.18 (1H, m, 5-H), 3.21–3.25 (1H, m, 3-
MHz; CD3OD; Me4Si) 0.92 (3H, t, J 7.5, triazolyl-CH2-CH2-CH2- H), 3.26–3.31 (1H, m, 40-H), 3.50 (1H, dt, J4,5 5.5, J4,3 9.4, 4-H),
CH3), 1.30 (2H, sex, J 7.5, triazolyl-CH2-CH2-CH2), 1.85 (2H, qu, 3.70 (1H, dd, J5 ,4 5.5, J5 ,5 11.5, 50-H), 3.88 (1H, dd, J5,4 5.5, J5,5
0
0
0
0
J 7.5, triazolyl-CH2-CH2), 3.16–3.23 (3H, m, 2-H and 20-H and 50- 11.5, 5-H), 4.23 (1H, d, J1 ,2 7.5, 10-H), 4.26 (1H, d, J1,2 7.5, 1-H),
H), 3.26–3.30 (2H, m, 5-H and 30-H), 3.41 (1H, t, J3,2 ¼ J3,4 8.8, 4.59 (1H, d, J 12.1, 1H OCH2), 4.77 (1H, d, J 12.1, 1H OCH2), 5.00
0
0
3-H), 3.47 (1H, ddd, J4 ,5 5.4, J4 ,3 8.8, J4 ,5 10.3, 40-H), 3.62 (1H, (3 OH), 5.17 (2 OH), 5.59 (2H, s, CH2-Ph), 7.30–7.39 (10H, m, Ph),
0
0
0
0
0
0
0
0
0
0
ddd, J4,5 5.3, J4,3 8.8, J4,5 10, 4-H), 3.85 (1H, dd, J5 ,4 5.4, J5 ,5 11.5, 8.15 (1H, s, H triazolyl); dC (125 MHz; DMSO-d6; Me4Si) 52.8
50-H), 4.00 (1H, dd, J5,4 5.3, J5,5 11.7, 5-H), 4.29 (1H, d, J1 ,2 7.7, 1 - (CH2-Ph), 61.6 (OCH2), 63.2 (C-5), 65.8 (C-50), 69.5 (C-40), 72.6 (C-
H), 4.30 (d, J1,2 7.4, 1-H), 4.35 (2H, t, J 7, triazolyl-CH2), 4.69 (1H, 20), 73.1 (C-2), 74.2 (C-3), 75.4 (C-4), 76.3 (C-30), 102.0 (C-10),
d, J 12.4, 1H OCH2), 4.87 (1H, d, J 12.4, 1H OCH2), 7.94 (s, H 102.7 (C-1), 124.2 (C–H triazolyl), 127.9–128.7 (C-H Ph), 136.1 (C-
triazolyl); dC (125 MHz; CD3OD; Me4Si) 13.7 (triazolyl-CH2-CH2- Ph), 144.0 (C-triazolyl); ESI-HMRS: m/z calcd for C20H27N3O9Na:
CH2-CH3), 20.6 (triazolyl-CH2-CH2-CH2), 33.3 (triazolyl-CH2- [M + Na]+: 476.1645, found: 476.1653.
0
0
0
CH2), 51.1 (triazolyl-CH2), 63.0 (OCH2), 64.6 (C-5), 67.1 (C-50),
Xyloside 3d. Pale yellow solid, decomposition at T > 188 ꢀC.
71.1 (C-40), 74.3 (C-20), 74.6 (C-2), 75.8 (C-3), 77.6 (C-30), 78.2 [a]D20 ꢁ47.1 (c 0.28 in H2O). dH (500 MHz; D2O; Me4Si) 3.30 (1H,
(C-4), 104.0 (C-1), 104.1 (C-10), 124.1 (C-H triazolyl), 145.6 dd, J2,1 7.8, J2,3 9.2, 2-H), 3.36 (1H, dd, J5,4 10.5, J5,5 11.7, 5-H),
(C-triazolyl); ESI-HMRS: m/z calcd for C17H30N3O9Na: [MH]+: 3.43 (1H, t, J3,2 ¼ J3,4 9.2, 3-H), 3.62 (1H, ddd, J4,5 5.5, J4,3 9.2, J4,5
420.1982, found: 420.1979.
10.5, 4-H), 3.97 (1H, dd, J5,4 5.5, J5,5 11.7, 5-H), 4.01 (2H, t, J 5.0,
Xyloside 3b. Pale yellow solid, m.p. 128 ꢀC. [a]D20 ꢁ52.9 (c 0.2 triazolyl-CH2-CH2-OH), 4.51 (1H, d, J1,2 7.8, 1-H), 4.57 (2H, t,
in H2O). dH (500 MHz; CD3OD; Me4Si) 3.23–3.24 (1H, m, 2-H), J 5.0, triazolyl-CH2-CH2-OH), 4.87 (1H, d, J 12.6, 1H OCH2), 4.97
3.24–3.26 (1H, m, 5-H), 3.34 (1H, t, J3,2 ¼ J3,4 8.9, 3-H), 3.52 (1H, (1H, d, J 12.6, 1H OCH2), 8.10 (1H, s, H-triazolyl); dC (125 MHz;
ddd, J4,5 5.3, J4,3 8.9, J4,5 10.2, 4-H), 3.91 (1H, dd, J5,4 5.3, J5,5 11.4, D2O; Me4Si) 52.5 (triazolyl-CH2-CH2-OH), 60.1 (triazolyl-CH2-
5-H), 4.40 (1H, d, J1,2 7.3, 1-H), 4.82–4.84 (1H, m, 1H OCH2), 5.00 CH2-OH), 62.0 (OCH2), 65.1 (C-5), 69.1 (C-4), 72.9 (C-2), 75.6
(1H, d, J 12.5, 1H OCH2), 7.50 and 7.58 (2H, t, J 7.8, Ph), 7.83 (1H, (C-3), 102.1 (C-1), 125.7 (C-H triazolyl), 143.4 (C-triazolyl);
d, J 7.8, Ph), 8.53 (1H, s, H triazolyl); dC (125 MHz; CD3OD; ESI-HMRS: m/z calcd for C10H17N3O6Na: [M + Na]+: 298.1015,
Me4Si) 63.0 (OCH2), 67.0 (C-5), 71.2 (C-4), 74.9 (C-2), 77.7 (C-3), found 298.1021.
104.4 (C-1), 121.6 (C-H Ph), 123.4 (C–H triazolyl), 130.1 and
Xyloside 4d. Yellow solid, decomposition at T > 225 ꢀC.
131.0 (C-H Ph), 138.4 (C-Ph), 146.7 (C-triazolyl); ESI-HRMS: m/z [a]2D0 ꢁ65.7 (c 0.28 in H2O). dH (500 MHz; D2O; Me4Si) 3.28 (1H,
calcd for C14H18N3O5: [MH]+: 308.1246, found: 308.1239.
dd, J2,1 7.8, J2,3 9.1, 2-H), 3.31 (1H, dd, J2 ,1 7.9, J2 ,3 9.2, 20-H),
0
0
0
0
20
Xyloside 4b. Pale yellow solid, m.p. 168 ꢀC. [a]D ꢁ49.0 (c 0.5 3.32 (1H, dd, J5 ,4 10.6, J5 ,5 11.6, 5 -H), 3.41 (1H, dd, J5,4 10.3, J5,5
0
0
0
0
0
in H2O). dH (500 MHz; CD3OD; Me4Si) 3.23–3.24 (1H, m, 20-H), 11.8, 5-H), 3.44 (1H, t, J3 ,2 ¼ J3 ,4 9.2, 3 -H), 3.56 (1H, t, J3,2 ¼ J3,4
0
0
0
0
0
3.24–3.26 (1H, m, 50-H), 3.31–3.33 (1H, m, 2-H), 3.37–3.39 (2H, 9.1, 3-H), 3.63 (1H, ddd, J4 ,5 5.5, J4 ,3 9.2, J4 ,5 10.6, 40-H), 3.78
0
0
0
0
0
0
m, 5-H and 3-H), 3.49–3.51 (1H, m, 30-H), 3.52–3.54 (1H, m, 40- (1H, ddd, J4,5 5.3, J4,3 9.1, J4,5 10.3, 4-H), 3.98 (1H, dd, J5 ,4 5.5,
0
0
0
H), 3.68–3.70 (1H, m, 4-H), 3.92 (1H, dd, J5 ,4 5.3, J5 ,5 11.7, 5 -H), J5 ,5 11.6, 50-H), 4.02 (2H, t, J 4.2, triazolyl-CH2-CH2-OH), 4.11
0
0
0
0
0
0
4.08 (1H, dd, J5,4 5.3, J5,5 11.7, 5-H), 4.36 (1H, d, J1 ,2 7.5, 10-H), (1H, dd, J5,4 5.3, J5,5 11.8, 5-H), 4.48 (1H, d, J1 ,2 7.9, 10-H), 4.54
4.43 (1H, d, J1,2 7.5, 1-H), 4.86 (1H, d, J 12.5, 1H OCH2), 5.02 (1H, (1H, d, J1,2 7.8, 1-H), 4.58 (2H, t, J 5.1, triazolyl-CH2-CH2-OH),
d, J 12.5, 1H OCH2), 7.52 and 7.61 (2H, t, J 7.8, Ph), 7.86 (1H, d, 4.88 (1H, d, J 12.7, 1H OCH2), 4.97 (1H, d, J 12.7, 1H OCH2), 8.10
J 7.8, Ph), 8.56 (1H, s, H triazolyl); dC (125 MHz; CD3OD; Me4Si) (1H, s, H triazolyl); dC (125 MHz; D2O; Me4Si) 52.5 (triazolyl-CH2-
63.0 (OCH2), 64.6 (C-5), 67.1 (C-50), 71.1 (C-40), 74.3 (C-20), 74.7 CH2-OH), 60.1 (triazolyl-CH2-CH2-OH), 62.0 (OCH2), 62.9 (C-5),
(C-2), 75.8 (C-30), 77.6 (C-3), 78.2 (C-4), 104.0 (C-10), 104.1 (C-1), 65.2 (C-50), 69.1 (C-40), 72.7 (C-20), 72.8 (C-2), 73.7 (C-3), 75.7 (C-
121.6 (C-H Ph), 123.4 (C-H triazolyl), 130.1 and 131.0 (C-H Ph), 30), 76.3 (C-4), 101.8 (C-10), 102.0 (C-1), 125.7 (C-H triazolyl),
138.4 (C-Ph), 146.6 (C-triazolyl); ESI-HRMS: m/z calcd for 143.4 (C-triazolyl); ESI-HMRS: m/z calcd for C15H25N3O10Na:
0
0
0
0
C
19H26N3O9: [MH]+: 440.1669, found: 440.1681.
Xyloside 3c. White solid, m.p. 135 C. [a]D ꢁ33.9 (c 0.22 in
[M + Na]+: 430.1438, found 430.1434.
20
Xyloside 3e. Yellow oil. [a]2D0 ꢁ32.1 (c 0.12 in H2O). dH (600
ꢀ
H2O). dH (500 MHz; CD3OD; Me4Si) 3.16–3.23 (1H, dd, J2,1 7.5, MHz; D2O; Me4Si) 3.27 (1H, dd, J2,1 7.8, J2,3 10, 2-H), 3.31–3.35 (1H,
J2,3 9.0, 2-H), 3.18–3.26 (1H, m, 5-H), 3.31 (1H, t, J3,2 ¼ J3,4 7.5, 3- m, 5-H), 3.42 (1H, t, J3,2 ¼ J3,4 10, 3-H), 3.54–3.56 (2H, m, e-CH2),
H), 3.50 (1H, ddd, J4,5 5.3, J4,3 9.0, J4,5 10.1, 4-H), 3.88 (1H, dd, J5,4 3.59–3.61 (1H, m, 4-H), 3.61–3.63 (2H, m, c-CH2), 3.62–3.64 (2H,
5.3, J5,5 11.4, 5-H), 4.33 (1H, d, J1,2 7.5, 1-H), 4.73 (1H, d, J 12.4, m, d-CH2), 3.64–3.69 (2H, m, f-CH2), 3.96 (1H, t, J5,4 ¼ J5,5 10.0,
1H OCH2), 4.91 (1H, d, J 12.4, 1H OCH2), 5.61 (2H, s, CH2-Ph), 5-H), 3.99 (2H, t, J 5, b-CH2), 4.50 (1H, d, J1,2 7.8, 1-H), 4.64 (2H, t,
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ꢀ
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); dC (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 3g. Pale yellow solid, decomposition at T > 180 C.
[a]2D0 ꢁ76.5 (c 0.40 in DMSO). dH (500 MHz; DMSO; Me4Si) 1.77
(3H, s, COCH3), 1.96 (3H, s, COCH3), 1.98 (3H, s, COCH3), 2.97
(1H, dd, J2,1 7.6, J2,3 9.0, 2-H), 3.07–3.08 (1H, m, 5-H), 3.10 (1H, t,
J3,2 ¼ J3,4 9.0, 3-H), 3.28 (1H, ddd, J4,5 5.4, J4,3 9.0, J4,5 10.1, 4-H),
3.71 (1H, dd, J5,4 5.4, J5,5 11.4, 5-H), 3.77 (1H, t, J5a,4a ¼ J5a,5a 10.0,
5a-H), 4.08 (1H, dd, J5a,4a 5.6, J5a,5a 10.0, 5a-H), 4.13 (1H, d, J1,2
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, J4a,3a ¼ J4a,5a 10.0, J4a,5a 5.6, 4a-H), 5.42 (1H,
t, J3a,2a ¼ J3a,4a 10.0, 3a-H), 5.55 (1H, t, J2a,1a ¼ J2a,3a 10.0, 2a-H),
6.13 (d, J1a,2a 10.0, 1a-H), 8.32 (1H, s, H-triazolyl); dC (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 4e. Yellow oil. [a]2D0 ꢁ19.1 (c 0.49 in H2O). dH (500
MHz; D2O; Me4Si) 3.27 (1H, dd, J2,1 7.8, J2,3 9.0, 2-H), 3.29–3.33
(2H, m, 20-H and 50-H), 3.41 (1H, dd, J5,4 10.5, J5,5 12, 5-H), 3.44
(1H, t, J3 ,2 ¼ J3 ,4 9.3, 30-H), 3.54–3.64 (3H, m, 3-H and e-CH2),
3.64–3.66 (1H, m, 40-H), 3.65–3.69 (6H, m, c-CH2 and d-CH2 and
f-CH2), 3.79 (1H, ddd, J4,5 5.3, J4,3 9.0, J4,5 10.5, 4-H), 3.96–3.99
(1H, m, 50-H), 3.99–4.01 (2H, m, b-CH2), 4.11 (1H, dd, J5,4 5.3, J5,5
0
0
0
0
12, 5-H), 4.47 (1H, d, J1,2 7.8, 1-H), 4.53 (d, J1 ,2 7.7, 10-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); dC (125 MHz; D2O;
Me4Si) 50.0 (C-a), 60.3 (C-f), 62.0 (OCH2), 62.9 (C-5), 65.2 (C-50),
68.7 (C-b), 69.2 (C-40), 69.3 (C-c), 69.7 (C-d), 71.6 (C-e), 72.7 (C-20),
72.8 (C-2), 73.7 (C-3), 75.6 (C-30), 76.3 (C-4), 101.8 (C-10), 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, decomposi-
tion at T > 215 ꢀC. [a]D20 ꢁ91.8 (c 0.39 in H2O). dH (500 MHz; D2O;
Me4Si) 3.0 (1H, t, J2,1 ¼ J2,3 8.5, 2-H), 3.11–3.17 (1H, m, 5-H), 3.36
(1H, t, J3,2 ¼ J3,4 8.5, 3-H), 3.41–3.45 (1H, m, 4-H), 3.80 (1H, dd,
J5,4 5.3 Hz, J5,5 11.6, 5-H), 4.08 (1H, m, NHCOOCH2CH), 4.16–
4.19 (1H, m, 1H NHCOOCH2), 4.30 (1H, d, J1,2 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); dC (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.
0
0
Xyloside 4g. Pale yellow solid, m.p. 67 ꢀC. [a]D20 ꢁ91.8 (c 0.39
in MeOH). dH (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 20-H and 5-H), 3.33–3.36 (1H, m, 3-H), 3.35–3.38 (1H, m, 50-
H), 3.47 (1H, t, J3 ,2 ¼ J3 ,4 9.0, 30-H), 3.52 (1H, ddd, J4,5 5.3, J4,3
0
0
0
0
8.7, J4,5 10.3, 4-H), 3.68 (1H, ddd, J4 ,5 5.1, J4 ,3 9.0, J4 ,5 10.1, 40-
H), 3.79 (1H, dd, J5a,4a 10.5, J5a,5a 11.5, 5a-H), 3.91 (1H, dd, J5,4
0
0
0
0
0
0
5.4, J5,5 11.5, 5-H), 4.06 (dd, J5 ,4 5.3, J5 ,5 11.5, 50-H), 4.27 (1H,
0
0
0
0
dd, J5a,4a 5.6, J5a,5a 11.5, 5a-H), 4.31 (1H, d, J1 ,2 7.5, 10-H), 4.35
(1H, d, J1,2 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, J4a,5a 5.6, J4a,3a 9.4, J4a,5a 10.5,
0
0
4a-H), 5.52 (1H, t, J3a,2a ¼ J3a,4a 9.4, 3a-H), 5.58 (1H, t, J2a,1a
¼
J2a,3a 9.4, 2a-H), 6.05 (1H, d, J1a,2a 9.4, 1a-H), 8.28 (1H, s, H tri-
azolyl); dC (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-50), 69.8 (C-4a), 71.1 (C-40), 72.1 (C-2a), 73.7 (C-3a), 74.3 (C-2),
74.6 (C-20), 75.7 (C-3), 77.6 (C-30), 78.2 (C-4), 87.3 (C-1a), 103.6 (C-
10), 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.
Xyloside 4f. (mixture of rotamers). White solid, decomposi-
tion at T > 190 ꢀC. [a]D20 ꢁ42.1 (c 0.19 in H2O). dH (600 MHz; D2O;
Me4Si) 3.01–3.02 (1H, t, J2,1 ¼ J2,3 9.0, 2-H), 3.16–3.22 (2H, m, 20-
Conclusions
H and 5-H), 3.27–3.31 (1H, m, 50-H), 3.37–3.42 (3H, m, 3-H and In conclusion, a range of original triazole-linked O-xylosides
30-H and 4-H), 3.61 (1H, ddd, J4 ,5 5.4, J4 ,3 9.5, J4 ,5 10.5, 40-H), and O-xylobiosides was prepared in a straightforward chemo-
0
0
0
0
0
0
0
0
0
0
3.80 (1H, dd, J5,4 5.0, J5,5 12.6, 5-H), 3.96 (dd, J5 ,4 5.4, J5 ,5 11.3, enzymatic two-step sequence from xylans. The synthesis of O-
50-H), 4.11–4.14 (2H, m, NHCOOCH2CH and 1H NHCOOCH2), xylobiosides by this methodology is of particular interest since
4.27 (1H, d, J1 ,2 7.8, 10-H), 4.29 (1H, d, J1,2 8.0, 1-H), 4.45–4.47 these compounds are not accessible by means of conventional
(1H, m, CHCOOH), 4.49–4.51 (1H, m, 1H NHCOOCH2), 4.55– carbohydrate synthesis. Preparation of propargyl xyloside and
4.58 (2H, m, 1H CH2CHCOOH and 1H OCH2), 4.67 (1H, d, J 12.4, xylobioside was rst investigated via an enzymatic trans-
1H OCH2), 4.84–4.87 (1H, m, 1H CH2CHCOOH); dC (150 MHz; glycosylation reaction from xylans and propargyl alcohol using a
D2O; Me4Si) 46.8 (NHCOOCH2CH), 51.9 (CH2CHCOOH), 56,0 xylanase. Different reaction parameters were studied (xylan
(CHCOOH), 61.8 (OCH2), 62.7 (C-5), 65.1 (C-50), 66.5 concentration, alcohol concentration, enzyme loading, reaction
(NHCOOCH2), 69.1 (C-40), 72.5 (C-2), 72.7 (C-20), 73.4 (C-3), 75.5 time) to optimize the production of xylosides. In the second
(C-30), 76.1 (C-4), 101.7 (C-10), 102.0 (C-1), 120.1 (C-H triazolyl), step, click reactions in an aqueous solution were carried out
125.1 (CH-Ph), 125.2 (CH-Ph), 125.4 (CH-Ph), 125.5 (CH-Ph), with various azides to give the corresponding triazoles with
125.4 (CH-Ph), 127.4 (CH-Ph), 127.5 (CH-Ph), 128.0 (CH-Ph), good yields (53% to 93%). Aliphatic, aromatic, hydroxyl, xylose-
128.1 (CH-Ph), 140.9 (C-triazolyl), 143.2 (C-Ph), 143.6 (C-Ph), derived or aminoacid-derived groups were introduced as the
143.9 (C-Ph), 157.1 OCONH, 174.4 (COOH); ESI-HMRS: m/z substituent of the triazole ring. Biological evaluation of these
calcd for C31H36N4O13Na: [M + Na]+: 695.2177 found: 695.2183. new triazole-linked xylosides will be undertaken.
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Acknowledgements
´
This work was supported by the “Region Champagne-Ardenne”
(Xylocos Emergence program). The authors are grateful to the
´
“Region Champagne-Ardenne” and FEDER for nancial
support and for doctoral fellowships (M. O. and C. B.). NS-22083
xylanase was kindly provided by Novozymes (France).
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