An alternative approach for the synthesis of fluorogenic substrates of endo-β-(1→4)-xylanases and some applications

Article abstract of DOI:10.1016/j.carres.2007.11.011

Fluorogenic substrates of endo-β-(1→4)-xylanases (EXs), 4-methylumbelliferyl β-glycosides of xylobiose and xylotriose were synthesized from fully acetylated oligosaccharides using the α-trichloroacetimidate procedure. A commercially available syrup containing xylose and xylo-oligosaccharides was used as the starting material. Both fluorogenic glycosides were found to be suitable substrates for EXs, particularly for sensitive detection of the enzymes in electrophoretic gels and their in situ localization on sections of fruiting bodies of some plants, such as tomato, potato and eggplant, all of the family Solanaceae.

Full text of DOI:10.1016/j.carres.2007.11.011

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 541–548
Note
An alternative approach for the synthesis of fluorogenic
substrates of endo-b-(1!4)-xylanases and some applications
a
b
b
a,
*
´
´
Maria Vrsˇanska, Wim Nerinckx, Marc Claeyssens and Peter Biely
aInstitute of Chemistry, Slovak Academy of Sciences, Du´bravska´ cesta 9, SK-84538 Bratislava, Slovakia
bLaboratory of Glycobiology, Department of Biochemistry, Physiology and Microbiology, Ghent University,
K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
Received 24 August 2007; received in revised form 9 November 2007; accepted 9 November 2007
Available online 19 November 2007
Abstract—Fluorogenic substrates of endo-b-(1!4)-xylanases (EXs), 4-methylumbelliferyl b-glycosides of xylobiose and xylotriose
were synthesized from fully acetylated oligosaccharides using the a-trichloroacetimidate procedure. A commercially available syrup
containing xylose and xylo-oligosaccharides was used as the starting material. Both fluorogenic glycosides were found to be suitable
substrates for EXs, particularly for sensitive detection of the enzymes in electrophoretic gels and their in situ localization on sections
of fruiting bodies of some plants, such as tomato, potato and eggplant, all of the family Solanaceae.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: endo-b-(1!4)-Xylanase; Fluorogenic substrates; 4-Methylumbelliferyl b-xylobioside; 4-Methylumbelliferyl b-xylotrioside; Activity stain
Microbial endo-b-(1!4)-xylanases (EXs, EC 3.2.1.8)
represent important industrial enzymes.1 EXs also play
important physiological roles in plant tissues.2 They
seem to be involved in fruit softening, seed germination,
pollinization and possibly in cell wall extension.3–5
These enzymes have been reported in germinating
wheat,6,7 and have been characterized more extensively
in germinating barley.8,9 An EX was identified as the
predominant protein on the surface of maize pollen.10
Due to low levels of EXs in plants, their studies re-
quire sensitive substrates such as 4-methylumbelliferyl
b-glycosides of xylobiose (Umb-Xyl2) and xylotriose
(Umb-Xyl3). The first synthesis of these artificial sub-
strates, carried out in our group,11 was based on the
Koenigs–Knorr synthesis. The principal xylosyl accep-
tor for the synthesis was 4-methylumbelliferyl 2,3-di-
O-acetyl-b-D-xylopyranoside isolated from a mixture
of products obtained on partial deacetylation of the
per-O-acetylglycoside by hydrazine. The acceptor was
then condensed with 2,3,4-tri-O-acetyl-a-D-xylopyran-
osyl bromide or 2,3,20,30,40-penta-O-acetyl-a-xylobiosyl
bromide to give the acetylated forms of the desired sub-
strates. Umb-Xyl2 and Umb-Xyl3 were then obtained on
deacetylation.11 A bromosugar route towards Umb-
Xyl2 from xylobiose was also reported by Kaneko
et al.12 Similar strategies were used for preparation of
other types of aryl b-glycosides of xylobiose.13–16 4-
Methylumbelliferyl glycosides of higher xylo-oligosac-
charides, including Umb-Xyl3, were obtained from
Umb-Xyl2 in transglycosylation reactions catalyzed by
a b-xylosidase from Aspergillus sp.17 The glycosyl donor
of this reaction, Umb-Xyl2, was obtained by the conden-
sation of protected Umb-Xyl with ethyl 2,3,4-tri-O-
acetyl-1-thio-b-D-xylopyranoside.17 In this paper we
describe an alternative route leading with relatively high
yields to Umb-Xyl2 and Umb-Xyl3, starting from an
inexpensive commercially available syrupy mixture of
Xyl, Xyl2 and Xyl3 as a source of oligosaccharides.
The mixture was deprived of water and subsequently
acetylated. The per-O-acetylated xylo-oligosaccharides
Abbreviations: EX, endo-b-(1!4)-xylanase; Xyl, D-xylose; Xyl2–Xyl5,
xylobiose up to xylopentaose; Umb-Xyl, 4-methylumbelliferyl b-D-
xyloside; Umb-Xyl2, 4-methylumbelliferyl b-xylobioside; Umb-Xyl3,
4-methylumbelliferyl b-xylotrioside; Umb-Cel, 4-methylumbelliferyl
b-cellobioside
*
Corresponding author. Tel.: +421 259410275; fax: +421
259410222; e-mail: chempbsa@savba.sk
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.11.011

542
M. Vrsˇanska´ et al. / Carbohydrate Research 343 (2008) 541–548
were isolated by chromatography on silica gel. The syn-
theses of Umb-Xyl2 (1) and Umb-Xyl3 (2) from per-O-
acetylxylobiose (3) and per-O-acetylxylotriose (4) were
accomplished in four steps, involving selective anomeric
deprotection to 5 and 6 by treatment with benzylamine,
conversion to the a-trichloroacetimidates18 7 and 8 and
subsequent coupling with 4-methylumbelliferone (7-hy-
droxy-4-methylcoumarin) to give 9 and 10, and finally
They were obtained as yellowish hygroscopic pow-
ders, which proved to be stable during storage under
dry conditions at ꢀ20 ꢁC. Care should also be taken that
the final products are not contaminated with the highly
fluorescent aglycon, 4-methylumbelliferone.
The present route is straightforward and more suit-
able for a large-scale synthesis of Umb-Xyl2 and Umb-
Xyl3 than previously published procedures.11,12,17 The
compounds are obtained in five steps, starting from
the unprotected oligosaccharides and avoiding the prep-
aration of unstable acetobromoxylo-oligosaccharides.
Both Umb-Xyl2 and Umb-Xyl3 served as substrates of
EXs of families 10 and 11. Umb-Xyl2 was found partic-
ularly useful for fluorometric determination of activity
of microbial EXs during investigation of their inter-
action with plant proteinaceous inhibitors.19 The same
work emphasized the significance of low-molecular mass
EX substrates in such studies due to interference of the
inhibitors with polymeric xylan used as EX substrate.19
Umb-Xyl2 was also successfully used in a rapid semi-
quantitative test of EX inhibitors present in proteins
extracted from germinated maize.20 Our findings that
Umb-Xyl2 is hydrolyzed to give 4-methylumbelliferone
by members of EX families 10 and 11 is in contrast to
´
Zemplen deacetylation (Scheme 1). The overall yields
of compounds 1 and 2 were reproducible, at around
35% from the per-O-acetylated sugars. The structure
1
of the products was confirmed by H and 13C NMR
spectroscopy (Table 1). Earlier published 1H NMR data
12,17
of only the carbohydrate regions of Umb-Xyl2
and
17
Umb-Xyl3 were helpful for proton assignments.
The anomeric deprotection of hexa-O-acetyl-xylo-
biose (3) and octa-O-acetyl-xylotriose (4) with benzyl-
amine to give 5 and 6 is accompanied by formation of
N-benzyl-penta-O-acetyl-xylobiosylamine (11) and N-
benzyl-penta-O-acetyl-xylotriosylamine (12), respec-
tively, in yields up to 30%. Both benzyl derivatives 11
and 12 can be hydrolyzed to give compounds 5 and 6
and returned to the respective reaction pathways to
increase the overall yields of the final products.
Scheme 1. Synthesis of fluorogenic xylo-oligosaccharides. Reagents and conditions: (a) benzylamine, CH2Cl2, 88%/87%; (b) Et2O, HCl 1 N; (c)
˚
Cl3C–CN, DBU, CH2Cl2, 79%/80%; (d) 4-methylumbelliferone, BF3OEt2, CH2Cl2, 4 A molecular sieves, 57%/56%; (e) NaOMe, MeOH, 87%/97%.
Table 1. Chemical shifts (ppm) for D-xylopyranosyl residues of Umb-Xyl3
Residue
H-1
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5a
H-5b
C-5
XylpI
XylpII
XylpIII
5.18
4.58
4.53
101.1
102.8
102.9
3.69
3.35
3.35
73.70
73.76
73.76
3.78
3.63
3.49
74.41
74.79
76.66
3.94
3.88
3.69
77.20
77.44
70.13
4.24
4.19
4.04
3.65
3.46
3.38
64.04
64.00
66.21
XylpI, xylosyl residue at the reducing end; XylpII, xylosyl residue in the middle; XylpIII, xylosyl residue at the non-reducing end.

M. Vrsˇanska´ et al. / Carbohydrate Research 343 (2008) 541–548
543
the report of Kaneko et al.12 that EXs of families 11 do
not liberate the aromatic aglycon from the xylobioside.
We did not have in our hands an EX of family 11 that
would not be detectable with Umb-Xyl2. A more
detailed study is required to find out whether the two
substrates could be used for a simple differentiation of
family 10 and 11 EXs. Both substrates were earlier
shown not to be hydrolyzed by the family 8 xylanase
from Pseudoalteromonas haloplanktis.21 The same
enzyme did not hydrolyze 6,8-difluoro-4-methylumbel-
liferyl xylobioside either.16 In this work we found out
that Umb-Xyl2 and Umb-Xyl3 are not hydrolyzed by
the appendage-dependent EX of family 5 from E.
chrysanthemi.
Both Umb-Xyl2 and Umb-Xyl3 are outstanding sub-
strates for rapid localization of EXs in electrophoretic
gels. An example of their use for this purpose was pre-
sented in our earlier article, describing the first synthesis
of the substrates.11 The fluorogenic substrates have a
great advantage in comparison with RBB-xylan22 or
the Congo Red stain.23 Indeed, whereas the detection
of EXs with RBB-xylan or Congo Red, including the
destaining and staining step, respectively, takes several
hours, the localization of EXs by Umb-Xyl2 and
Umb-Xyl3 is a matter of minutes. The disadvantage of
fluorogenic substrates is sometimes the rapid diffusion
of the released 4-methylumbelliferone in the gels, and
the need to use Umb-Xyl as the control substrate for
b-xylosidase, which may liberate the fluorescent aglycon
from the glycosides after a two or three step hydrolysis.
Umb-Cel may be recommended as the control substrate
for non-specific endo-b-(1!4)-glucanases hydrolyzing
xylans.24
tions of red (ripe) tomatoes, which indicates that EXs
observed on seeds of green unripe tomatoes have disap-
peared completely in the process of ripening. Examina-
tion of EXs on cuts of green tomato seeds showed
that the enzyme is confined to the seed coat and does
not occur in the seed interior, including the endosperm.
The identity of the enzyme detected with Umb-Xyl2 and
Umb-Xyl3 as an EX was confirmed in an experiment in
which isolated unripe tomato seed coats were incubated
with a 1.5% solution of 4-O-methylglucuronoxylan in
0.05 M sodium acetate buffer at pH 5.4. The EX bound
to the outer surface of the seed coat hydrolyzed the poly-
saccharide to Xyl, Xyl2, aldotetraouronic acid and
traces of Xyl3 (Fig. 2). TLC in an alkaline solvent sys-
tem25 confirmed the acidic nature of the third product.
Such product profile is characteristic for the action of
family 10 EXs.25 All so far identified plant EXs belong
to glycoside hydrolase family 10. When different parts
Localization of EXs in situ can be done easily using
agar gels replicas containing the fluorogenic substrates.
A successful detection of EXs on sections of fruiting
bodies of unripe tomatoes is shown in Figure 1. The sec-
tions were brought into contact with the gels containing
Umb-Xyl2 and Umb-Xyl3, Umb-Xyl and Umb-Cel.
Fluorescence was observed already after 5 min, but only
with the gels containing Umb-Xyl2 or Umb-Xyl3. No
evidence for the presence of EXs was obtained with sec-
Figure 2. Hydrolysis of beechwood 4-O-methylglucuronoxylan by the
EX(s) localized on unripe tomato seed coats (isolated from green
tomato seeds) followed by TLC on cellulose (E. Merck) in 3:2:2
EtOAc–AcOH–water. Reducing sugars were detected with the aniline–
hydrogen phthalate reagent. S1, standards Xyl–Xyl5; S2, glucose; C,
enzyme control; MeGlcAXyl3, aldotetraouronic acid.
Figure 1. Detection of endo-b-(1!4)-xylanase on a section of unripe (green) tomato (left panel) using Umb-Xyl2 (1) as a substrate after 10 and
20 min contact with the substrate-containing agar gel. The detection was followed under UV light. Umb-Xyl was used as a control for b-xylosidase
and Umb-Cel as a control for endo-b-(1!4)-glucanases.

544
M. Vrsˇanska´ et al. / Carbohydrate Research 343 (2008) 541–548
of other plants were tested for the presence of EX using
Umb-Xyl2 and Umb-Xyl3, the enzyme activity was re-
vealed only on seeds of the fruiting bodies of two other
plant species, potato and eggplant, which belong to-
gether with tomato to the Solanaceae family.
(500 mL) was added and the solution was washed in a
separatory funnel with a saturated soln of NaHCO3
(300 mL). The CHCl3 layer was washed once with water,
dried over Na2SO4, filtered, and concentrated under
diminished pressure. Purification by column chromato-
graphy on Silica gel 100 (120 · 2.5 cm; 3:4 n-hexane–
EtOAc or 3:2 toluene–EtOAc) yielded fractions containing
hexa-O-acetyl-b-xylobiose (3) and octa-O-acetyl-b-xylo-
triose (4) (Rf resp. 0.63 and 0.44 in 2:3 toluene–EtOAc),
which were separately pooled, evaporated and used as
starting compounds for further syntheses.
1. Experimental
1.1. General methods
1H NMR spectra were recorded in CDCl3 or D2O with a
Bruker WP360 or WP500, or a Bruker DPX Avance 300
spectrometer (Madison, WI); 13C NMR spectra were
recorded with the latter instrument. Chemical shifts are
expressed in parts per million (ppm) with reference to
Me4Si. IR spectra were recorded with a Beckman Accu-
lab 4 (Fullerton, CA) spectrometer. MS data were
recorded with a Hewlett–Packard 5988-A (EIMS,
ionization potential of 70 eV), or with an Agilent 1100
series MSD-VL as electrospray in positive mode from
1:1 0.5 mM aq ammonium acetate–CH3CN (ESIMS).
Melting points were determined with a Reichert Micro-
scope 269156 (Vienna, Austria) and are uncorrected.
Specific rotations were measured at 20 ꢁC after 20 h in
distilled water with a Perkin–Elmer 241 polarimeter.
TLC was performed with Silica gel 60F254 precoated
glass plates (E. Merck, Darmstadt, Germany) with
detection under UV light (254 nm) and subsequent dip-
ping of the plates in a 10% soln of H2SO4 in 2-propanol
and carbonization on a hot plate. Silica gel 100 (0.063–
0.200 mm, E. Merck), dried at 150–200 ꢁC under dimin-
ished pressure, was used for column chromatographic
separations. All reactions were carried out with mag-
netic stirring and at room temperature unless otherwise
indicated.
1.4. 2,3,20,30,40-Penta-O-acetyl-xylobiose (5)
To a soln of hexa-O-acetyl xylobiose (3, 1.64 g, 2 mmol)
in CH2Cl2 (5 mL), benzylamine (1 equiv, 218 lL) was
added. A second equiv of benzylamine was added after
2 h, and an extra 0.5 equiv after another 2 h (total
2.5 equiv). The reaction was completed after 12 h.
Washing with a 1 N HCl soln (2 · 10 mL) and with a
saturated NaHCO3 soln (10 mL), drying over anhyd
MgSO4 and purification by column chromatography
(eluent: 1:1 toluene–EtOAc) yielded 5 (863 mg, 1.75
mmol, 88%). It should be noted that substantial
amounts (up to 30%) of N-benzyl-penta-O-acetyl xylo-
biosylamine (11) may be formed. After separation by
column chromatography, this can be subsequently
hydrolyzed to 5 by stirring in the two-phase system
Et2O–1 N HCl, the reaction being complete after 6 h
and worked up as above. Penta-O-acetyl xylobiose (5):
white crystals; mp 171 ꢁC; Rf 0.40 (1:1 toluene–EtOAc);
IR (KBr): 1750, 1430, 1370, 1240, 1220, 1130,
1
1050 cmꢀ1; H NMR (500 MHz, CDCl3, a-anomer): d
5.44 (dd, 1H, J2,3 8.7, J3,4 9.6 Hz, H-3I), 5.33 (dd, 1H,
J1,2 2.8, J1,OH 3 Hz, H-1I), 5.08 (dd, 1H, J2,3 7.5, J3,4
7.5 Hz, H-3II), 4.87 (ddd, 1H, J4,5a 4.6, J4,5b 7.5 Hz,
H-4II), 4.79 (dd, 1H, H-2I), 4.79 (dd, 1H, J1,2 5.9 Hz,
H-2II), 4.57 (dd, 1H, H-1II), 4.10 (dd, 1H, J5a,5b
11.9 Hz, H-5aII), 3.85 (ddd, 1H, J4,5a 4.8, J4,5b 10 Hz,
H-4I), 3.80 (dd, 1H, J5a,5b 10.4 Hz, H-5bI), 3.70 (dd,
1H, H-5aI), 3.40 (dd, 1H, H-5bII), 2.79 (d, 1H, OH),
2.078 (s, 3H, CH3CO), 2.061 (s, 3H, CH3CO), 2.054
(s + s, 6H, CH3CO), 2.051 (s, 3H, CH3CO) ppm;
ESIMS: m/z 146 (50%), 188 (4%), 245 (4%), 313 (3%),
391 (8%), 510 [M+NH4]+ (100%), 515 [M+Na]+
(50%). N-Benzyl-penta-O-acetyl xylobiosylamine (11):
colourless oil; Rf 0.51 (1:1 toluene–EtOAc); IR (KBr):
1.2. Chemicals
The syrupy mixture of D-xylose and xylo-oligosaccha-
rides (22.2% D-xylose, 57.2% Xyl2 and 17.3% Xyl3)
was from Suntory Ltd. (Japan), obtained by courtesy
of Professor T. Yasui, (University of Tsukuba, Japan).
Umb-Xyl and Umb-Cel were from Sigma (St. Louis,
MO, USA). Benzylamine, trichloroacetonitrile, 4-methyl-
umbelliferone, reagents and solvents were commercial;
CH2Cl2 was freshly distilled from P2O5 prior to use.
1750, 1500–1420, 1370, 1220, 1040, 940 cmꢀ1
;
1H
1.3. Preparation of fully acetylated xylo-oligosaccharides
NMR (500 MHz, CDCl3): d 7.33–7.25 (m, 5H, Ph),
5.12 (dd, 1H, J2,3 9.5, J3,4 9.5 Hz, H-3I), 5.07 (dd, 1H,
J2,3 7.5, J3,4 7.5 Hz, H-3II), 4.86 (ddd, 1H, J4,5a 4.3,
J4,5b 7.6 Hz, H-4II), 4.77 (dd, 1H, J1,2 9 Hz, H-2I),
4.75 (dd, 1H, J1,2 5.6 Hz, H-2II), 4.65 (d, 1H, H-1II),
4.09 (dd, 1H, J5a,5b 12 Hz, H-5aII), 4.01 (d, 1H, Jgem
13.8 Hz, CH2Ph), 3.97 (d, 1H, H-1I), 3.94 (dd, 1H,
J4,5a 5.5, J5a,5b 12 Hz, H-5aI), 3.84 (d, 1H, CH2Ph),
The syrupy mixture of D-xylose, Xyl2 and Xyl3 was
lyophilized and then dried to constant weight in a desic-
cator over P2O5. The dry mixture (10 g) was dissolved in
pyridine (200 mL) and Ac2O (200 mL) at 0 ꢁC. The acetyl-
ation was complete after 20 h. The mixture was poured
into ice-cold water and stirred for 2–3 h. CHCl3

M. Vrsˇanska´ et al. / Carbohydrate Research 343 (2008) 541–548
545
3.80 (ddd, 1H, J4,5b 10 Hz, H-4I), 3.39 (dd, 1H, H-5bII),
3.21 (dd, 1H, H-5bI), 2.056 (s + s + s, 9H, CH3CO),
2.033 (s, 3H, CH3CO), 2.030 (s, 3H, CH3CO) ppm.
1H, J5a,5b 12 Hz, H-5aII), 4.09 (dd, 1H, J4,5a 4.9, J5a,5b
12 Hz, H-5aI), 3.92 (ddd, 1H, J4,5b 8.5 Hz, H-4I), 3.51
(dd, 1H, H-5bI), 3.40 (dd, 1H, H-5bII), 2.40 (s, 3H,
CH3MUF), 2.091 (s, 3H, CH3CO), 2.081 (s, 3H,
CH3CO), 2.073 (s, 3H, CH3CO), 2.063 (s, 3H, CH3CO),
2.044 (s, 3H, CH3CO) ppm; EIMS: m/z 97 (100%), 139
(55%), 157 (55%), 199 (20%), 259 (14%) ½XylOAc3þꢁ, 273
(2%), 475 (2%) ½Xyl2OAc5þꢁ.
1.5. 2,3,20,30,40-Penta-O-acetyl-a-D-xylobiosyl
trichloroacetimidate (7)
To a soln of 5 (685 mg, 1.39 mmol) and trichloroaceto-
nitrile (5 equiv, 700 lL) in CH2Cl2 (20 mL), 1,8-diazabi-
cyclo[5,4,0]undec-7-ene (0.2 equiv, 41 lL) was added
and stirring was continued for 30 min. Concentration
under diminished pressure and purification by column
chromatography (dried Silica gel 100; eluent: 7:3 tolu-
ene–EtOAc) yielded 7 (701 mg, 1.101 mmol, 79%) as a
white powder; mp 159 ꢁC; Rf 0.56 (1:1 toluene–EtOAc);
IR (KBr): 1760, 1680, 1440, 1370, 1340, 1230,
1.7. Preparation of 4-methylumbelliferyl b-xylobioside (1)
To a mixture of 9 (3.5 g, 5.385 mmol) in MeOH
(20 mL), a chip of sodium (0.1 equiv) was added. The
reaction was completed after 1 h. Silica gel 100
(200 mg) was added and stirring was continued for
5 min. Filtration and concentration under diminished
pressure yielded 1 (2.063 g, 87%) as a slightly yellowish
1040 cmꢀ1 1H NMR (500 MHz, CDCl3): d 8.60 (s,
;
1H, CNHCCl3), 6.40 (d, 1H, J1,2 3.7 Hz, H-1I), 5.51
(dd, 1H, J2,3 9.6, J3,4 9.6 Hz, H-3I), 5.07 (dd, 1H, J2,3
8.0, J3,4 7.8 Hz, H-3II), 4.98 (dd, 1H, H-2I), 4.80 (ddd,
1H, J4,5a 4.8, J4,5b 7.8 Hz, H-4II), 4.77 (dd, 1H, J1,2
6.5 Hz, H-2II), 4.53 (d, 1H, H-1II), 3.96 (dd, 1H, J4,5a
5.0, J5a,5b 12 Hz, H-5aI), 3.88 (ddd, 1H, J4,5b 10 Hz,
H-4I), 4.05 (dd, 1H, J5a,5b 12 Hz, H-5aII), 3.77 (dd,
1H, H-5bI), 3.38 (dd, 1H, H-5bII), 2.060 (s, 3H,
CH3CO), 2.053 (s, 3H, CH3CO), 2.032 (s, 3H, CH3CO),
2.030 (s, 3H, CH3CO), 2.005 (s, 3H, CH3CO) ppm. This
compound is moderately stable and should be used
within days.
powder; mp >200 (dec); ½aꢁD ꢀ82.5 (c 1.0, water),
20
lit.12 [a]D ꢀ81.6 (c 1.0, water); Rf 0.53 (1:1 toluene–
1
MeOH); H NMR (500 MHz, D2O): d 7.47 (dd, 1H,
J5,6 3.8, J6,8 8.8 Hz, H-6MUF), 6.93 (d, 1H, H-5MUF),
6.83 (d, 1H, H-8MUF), 6.04 (s, 1H, H-3MUF), 5.05 (d,
1H, J1,2 7.5 Hz, H-1I), 4.45 (d, 1H, J1,2 7.8 Hz, H-1II),
4.14 (dd, 1H, J4,5a 4.9, J5a,5b 11.2 Hz, H-5aI), 3.96 (dd,
1H, J4,5a 5.4, J5a,5b 11.6 Hz, H-5aII), 3.83 (ddd, 1H,
J3,4 9, J4,5b 9 Hz, H-4I), 3.68 (dd, 1H, J2,3 8 Hz, H-3I),
3.60 (ddd, 1H, J3,4 8, J4,5b 9 Hz, H-4II), 3.59 (dd, 1H,
H-5bI), 3.57 (dd, 1H, H-5bII), 3.42 (dd, 1H, J2,3 8 Hz,
H-3II), 3.29 (dd, 1H, H-2I), 3.26 (dd, 1H, H-2II), 2.27
1
(s, 3H, CH3MUF) ppm. The H resonances at the carbo-
1.6. Preparation of 4-methylumbelliferyl 2,3,20,30,40-
penta-O-acetyl-b-xylobioside (9)
hydrate regions matched previously reported data, and
the 13C NMR spectrum was identical.12,17 This com-
pound is hygroscopic and should be kept in a desiccator.
Anal. Calcd for C20H24O11ÆH2O: C, 52.40; H, 5.72.
Found: C, 52.60; H, 5.65.
A mixture of 7 (780 mg, 1.23 mmol) and 4-methylumbel-
liferone (1.5 equiv, 338 mg, 1.84 mmol) in CH2Cl2
˚
(40 mL) in the presence of 4 A molecular sieves (0.5 g)
was stirred for 2 h and then brought to ꢀ15 ꢁC (cooling
bath benzylalcohol–solid CO2). A soln of BF3ÆOEt2
(15 lL) in CH2Cl2 (1 mL) was added dropwise during
5 min. The reaction was completed after 1 h at
ꢀ15 ꢁC. Solid NaHCO3 (100 mg) was added and the
mixture was brought to room temperature. Washing
with water (20 mL), with a saturated NaHCO3 soln
(5 · 20 mL), drying over anhyd MgSO4 and purification
by column chromatography (eluent: 7:3 toluene–EtOAc)
yielded 9 (456 mg, 701 lmol, 57%) as white crystals; mp
99 ꢁC, lit.12 mp 108.5–110 ꢁC (from MeOH); Rf 0.38 (1:1
toluene–EtOAc); IR (KBr): 1760–1750, 1620, 1430,
1.8. 2,3,20,30,200,300,400-Hepta-O-acetyl-xylotriose (6)
To a soln of 4 (2.05 g, 2.73 mmol) in CH2Cl2 (10 mL),
benzylamine (1 equiv, 313 lL) was added. A second
equiv of benzylamine was added after 2 h, and an extra
0.5 equiv after another 2 h (total 2.5 equiv). The reaction
was completed after 22 h. Washing with 1 N HCl soln
(2 · 30 mL) and with
a saturated NaHCO3 soln
(30 mL), drying over anhyd MgSO4 and purification
by column chromatography (2:3 toluene–EtOAc)
yielded amorphous 6 (1.685 g, 2.38 mmol, 87%); Rf
0.36 (3:7 toluene–EtOAc); IR (KBr): 1760, 1440, 1380,
1380, 1230, 1140, 1060 cmꢀ1
;
1H NMR (360 MHz,
1260, 1220, 1050 cmꢀ1 1H NMR (500 MHz, CDCl3,
;
CDCl3): d 7.53 (d, 1H, J5,6 8.7 Hz, H-5MUF), 6.96 (d,
1H, J6,8 2.5 Hz, H-8MUF), 6.92 (dd, 1H, H-6MUF), 6.20
(s, 1H, H-3MUF), 5.22 (dd, 1H, J2,3 8.0, J3,4 8.0 Hz,
H-3II), 5.16 (s, 1H, J1,2 6.3 Hz, H-1I), 5.16 (dd, 1H,
J2,3 10.0 Hz, H-2I), 5.12 (dd, 1H, J3,4 10.5 Hz, H-3I),
4.91 (ddd, 1H, J4,5a 4.8, J4,5b 8.0 Hz, H-4II), 4.84 (dd,
1H, J1,2 6.2 Hz, H-2II), 4.59 (d, 1H, H-1II), 4.12 (dd,
major a-anomer): d 5.41 (dd, 1H, J2,3 9, J3,4 9 Hz,
H-3I), 5.31 (dd, 1H, J1,2 2.3, J1,OH 4 Hz, H-1I), 5.08
(dd, 1H, J2,3 7.7, J3,4 7.6 Hz, H-3III), 5.05 (dd, 1H, J2,3
8.0, J3,4 8.0 Hz, H-3II), 4.87 (ddd, 1H, J4,5a 4.7, J4,5b
7.6 Hz, H-4III), 4.79 (dd, 1H, J1,2 5.9 Hz, H-2III), 4.75
(dd, 1H, J1,2 6.7 Hz, H-2II), 4.74 (dd, 1H, H-2I), 4.55
(d, 1H, H-1III), 4.58 (d, 1H, H-1II), 4.08 (dd, 1H, J5a,5b

546
M. Vrsˇanska´ et al. / Carbohydrate Research 343 (2008) 541–548
12 Hz, H-5aIII), 3.94 (ddd, 1H, J4,5a 5, J4,5b 10 Hz, H-4I),
3.80 (dd, 1H, J5a,5b 11 Hz, H-5aI), 3.79 (ddd, 1H, J4,5a
5.1, J4,5b 9 Hz, H-4II), 3.67 (dd, 1H, J5a,5b 11 Hz,
H-5aII), 3.39 (dd, 1H, H-5bIII), 3.32 (dd, 1H, H-5bI),
3.32 (dd, 1H, H-5bII), 3.10 (d, 1H, OH), 2.071 (s, 3H,
CH3CO), 2.053 (s, 3H, CH3CO), 2.046 (s, 3H, CH3CO),
2.038 (s, 3H, CH3CO), 2.028 (s, 3H, CH3CO), 2.026 (s,
3H, CH3CO), 2.023 (s, 3H, CH3CO) ppm; separately
resolved resonances of minor b-anomer: 5.14 (dd, 1H,
J2,3 8, J3,4 9 Hz, H-3I), 4.63 (dd, 1H, J1,2 8 Hz, H-2I),
4.57 (d, 1H, J1,2 6.7 Hz, H-1II), 3.60 (d, 1H, J1,OH
8.5 Hz, H-OH) ppm; ratio of a/b: 1.8/1.0; ESIMS: m/z
315 (10%), 726 ([M+NH4]+, 100%); 731 ([M+Na]+,
36%). It should be noted that substantial amounts (up
to 30%) of N-benzyl-hepta-O-acetyl xylotriosylamine
(12), colourless oil, Rf 0.84 (3:7 toluene–EtOAc), may
be formed. After separation by column chromato-
graphy, this can be subsequently hydrolyzed to 6 by stir-
ring in the two-phase system Et2O–1 N HCl, the reaction
being complete after 6 h and worked up as above.
for 2 h and then brought to ꢀ15 ꢁC (cooling bath benzyl-
alcohol–solid CO2). A soln of BF3ÆOEt2 (0.1 equiv,
10 lL) in CH2Cl2 (1 mL) was added dropwise during
5 min. The reaction was completed after 1 h at
ꢀ15 ꢁC. Solid NaHCO3 (200 mg) was added and the
mixture was brought to room temperature. Washing
with water (20 mL), with a saturated NaHCO3 soln
(5 · 20 mL), drying over anhyd MgSO4 and purification
by column chromatography (55:45 toluene–EtOAc)
yielded 10 (415 mg, 0.479 mmol, 56%) as white crystals;
mp 121 ꢁC; Rf 0.39 (2:3 toluene–EtOAc); IR (KBr):
1760–1740, 1620, 1510–1500, 1430, 1370, 1250–1240,
1
1150, 1120, 1060–1030, 870 cmꢀ1; H NMR (500 MHz,
CDCl3): d 7.51 (d, 1H, J5,6 8.7 Hz, H-5MUF), 6.94 (d,
1H, J6,8 2.4 Hz, H-8MUF), 6.91 (dd, 1H, H-6MUF), 6.18
(q, 1H, J3,CH3 1 Hz, H-3MUF), 5.20 (dd, 1H, J2,3 8.1,
J3,4 8.1 Hz, H-3I), 5.17 (d, 1H, J1,2 6.5 Hz, H-1I), 5.11
(dd, 1H, H-2I), 5.09 (dd, 1H, J2,3 7.7, J3,4 7.6 Hz,
H-3III), 5.08 (dd, 1H, J2,3 8.4, J3,4 8.7 Hz, H-3II), 4.87
(ddd, 1H, J4,5a 4.7, J4,5b 7.6 Hz, H-4III), 4.79 (dd, 1H,
J1,2 5.8 Hz, H-2III), 4.78 (dd, 1H, J1,2 6.5 Hz, H-2II),
4.56 (d, 1H, H-1III), 4.51 (d, 1H, H-1II), 4.09 (dd, 1H,
J4,5a 4.8, J5a,5b 12.1 Hz, H-5aI), 4.06 (dd, 1H, J5a,5b
12.1 Hz, H-5aIII), 3.96 (dd, 1H, J4,5a 5.1, J5a,5b
11.9 Hz, H-5aII), 3.88 (ddd, 1H, J4,5b 8.4 Hz, H-4I),
3.82 (ddd, 1H, J4,5b 9.0 Hz, H-4II), 3.50 (dd, 1H,
H-5bI), 3.39 (dd, 1H, H-5bIII), 3.33 (dd, 1H, H-5bII),
2.40 (d, 3H, CH3MUF), 2.081 (s, 3H, CH3CO), 2.066 (s,
3H, CH3CO), 2.058 (s + s, 6H, CH3CO), 2.052 (s, 3H,
CH3CO), 2.036 (s, 3H, CH3CO), 2.027 (s, 3H, CH3CO)
ppm. ESIMS: m/z 392 (22%), 883 ([M+O+H]+ or
[M+16+1]+, 100%), 889 ([M+Na]+, 82%); MW 866.
1.9. 2,3,20,30,200,300,400-Hepta-O-acetyl-a-D-xylotriosyl
trichloroacetimidate (8)
To a soln of 6 (576 mg, 0.813 mmol) and trichloroaceto-
nitrile (5 equiv, 410 lL) in CH2Cl2 (15 mL), 1,8-diaza-
bicyclo[5,4,0]undec-7-ene (0.2 equiv, 25 lL) was added
and stirring was continued for 30 min. Concentration
under diminished pressure and purification by column
chromatography (dried Silica gel 100; eluent: 1:1 tolu-
ene–EtOAc) yielded 8 (551 mg, 0.646 mmol, 80%) as a
white powder, mp 92 ꢁC; Rf 0.66 (3:7 toluene–EtOAc);
IR (KBr): 1760, 1690, 1450, 1430, 1380, 1250, 1220,
1050 cmꢀ1
;
1H NMR (500 MHz, CDCl3): d 8.63 (s,
1.11. Preparation of 4-methylumbelliferyl b-xylotrioside
(2)
1H, CNHCCl3), 6.42 (d, 1H, J1,2 3.7 Hz, H-1I), 5.47
(dd, 1H, J2,3 9.6, J3,4 9.6 Hz, H-3I), 5.08 (dd, 1H, J2,3
7.6, J3,4 7.6 Hz, H-3III), 5.06 (dd, 1H, J2,3 8.5, J3,4
8.3 Hz, H-3II), 4.99 (dd, 1H, H-2I), 4.87 (ddd, 1H,
J4,5a 4.7, J4,5b 7.6 Hz, H-4III), 4.79 (dd, 1H, J1,2
5.8 Hz, H-2III), 4.74 (dd, 1H, J1,2 6.7 Hz, H-2II), 4.56
(d, 1H, H-1III), 4.50 (d, 1H, H-1II), 4.09 (dd, 1H, J5a,5b
12 Hz, H-5aIII), 3.95 (dd, 1H, J4,5a 5.0, J5a,5b 12 Hz,
H-5aI), 3.87 (ddd, 1H, J4,5b 10 Hz, H-4I), 3.85 (dd, 1H,
J4,5a 5.0, J5a,5b 12 Hz, H-5aII), 3.82 (ddd, 1H, J4,5b
8.9 Hz, H-4II), 3.75 (dd, 1H, H-5bI), 3.39 (ddd, 1H,
H-5bIII), 3.34 (dd, 1H, H-5bII), 2.059 (s, 3H, CH3CO),
2.053 (s + s, 6H, CH3CO), 2.031 (s, 3H, CH3CO),
2.029 (s, 3H, CH3CO), 2.012 (s, 3H, CH3CO), 2.004
(s, 3H, CH3CO) ppm.
Compound 10 (415 mg, 479 lmol) was taken in a soln of
sodium methanolate in MeOH (0.1 equiv, 11 mL of a
4.35 mM soln). The reaction was completed after 2 h.
Silica gel 100 (100 mg) was added and stirring was con-
tinued for 5 min. Filtration and concentration under
diminished pressure yielded 2 (267 mg, 97%) as a slightly
yellowish powder; mp >200 ꢁC (dec); Rf 0.40 (1:1 tolu-
20
ene–MeOH); ½aꢁD ꢀ97.5 (c 0.60, water); 1H NMR
(300 MHz, D2O): d 7.62 (dd, 1H, J5,6 3.8, J6,8 8.8 Hz,
H-6MUF), 7.02 (d, 1H, H-5MUF), 6.97 (d, 1H,
H-8MUF), 6.16 (s, 1H, H-3MUF), 5.18 (d, 1H, J1,2
7.6 Hz, H-1I), 4.58 (d, 1H, J1,2 7.8 Hz, H-1II), 4.53 (d,
1H, J1,2 7.8 Hz, H-1III), 4.24 (dd, 1H, J4,5a 5.0, J5a,5b
11.5 Hz, H-5aI), 4.19 (dd, 1H, J4,5a 5.4, J5a,5b 11.7 Hz,
H-5aII), 4.04 (dd, 1H, J4,5a 5.4, J5a,5b 11.5 Hz, H-5aIII),
3.94 (ddd, 1H, J3,4 9.4, J4,5b 9.4 Hz, H-4I), 3.88 (ddd,
1H, J3,4 9.4, J4,5b 9.4 Hz, H-4II), 3.78 (dd, 1H, J2,3
10.2 Hz, H-3I), 3.69 (ddd, 1H, J3,4 9.3, J4,5b 9.4 Hz,
H-4III), 3.69 (dd, 1H, H-2I), 3.65 (dd, 1H, H-5aI), 3.63
(dd, 1H, J2,3 9.0 Hz, H-3II), 3.49 (dd, 1H, J2,3 9.3 Hz,
1.10. 4-Methylumbelliferyl 2,3,20,30,200,300,400-hepta-O-
acetyl-b-xylotrioside (10)
A mixture of 8 (730 mg, 0.865 mmol) and 4-methyl-
umbelliferone (1.5 equiv, 226 mg) in CH2Cl2 (20 mL) in
˚
the presence of 4 A molecular sieves (0.5 g) was stirred

M. Vrsˇanska´ et al. / Carbohydrate Research 343 (2008) 541–548
547
H-3III), 3.46 (dd, 1H, H-5bII), 3.38 (dd, 1H, H-5bIII),
3.35 (dd, 1H, H-2II), 3.35 (dd, 1H, H-2III), 2.35 (s, 3H,
tute, Norwich, UK) and Professor J. F. Preston (Univer-
sity of Gainesville, FL, USA) for pure xylanases gifts,
Dr. J. Alfo¨ldi (Institute of Chemistry, Slovak Academy
of Sciences, Bratislava, Slovakia) and Professor Dr. J.
Van der Eycken (Laboratory for Organic Synthesis,
Ghent University, Belgium) for the NMR measure-
ments. This work was supported by grant VEGA No.
2/6130/26 from the Slovak Grant Agency for Science,
grant GLYCOBIOS from the Slovak Academy of
Sciences and by the F.W.O., Belgium.
1
CH3MUF) ppm. H and 13C NMR chemical shifts are
1
compiled in Table 1. The H resonances of the carbo-
hydrate regions matched previously reported data, and
the 13C NMR spectrum was identical.17 This compound
is hygroscopic and should be kept in a desiccator. Anal.
Calcd for C25H32O15ÆH2O: C, 50.84; H, 5.80. Found: C,
51.04; H, 5.64.
1.12. Enzymes
The following EXs were used in the present work: family
10 EXs were from Cryptococcus albidus26 and from
Streptomyces lividans (XlnA),27 family 11 EXs were
from Schizophyllum commune,28 Thermomyces lanugino-
sus29 and from S. lividans (Xln C).30 Family 5 EX from
Erwinia chrysanthemi31 was provided by J.F. Preston
(University of Florida, Gainesville, FL, USA). The
activity of endo-b-(1!4)-xylanase was determined on
4-O-methylglucuronoxylan.26 One unit of activity is de-
fined as the amount of enzyme, which liberates 1 lmol
of D-xylose equiv in 1 min.
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