Synthesis of a library of xylogluco-oligosaccharides for active-site mapping of xyloglucan endo-transglycosylase

Article abstract of DOI:10.1021/jo0525682

Complex oligosaccharides containing α-D-xylosyl-(1→6)-β-D- glucosyl residues and unsubstituted β-(1→4)-linked D-glucosyl units were readily synthesized using enzymatic coupling catalyzed by the Cel7B E197A glycosynthase from Humicola insolens. Constitutin

Full text of DOI:10.1021/jo0525682

Synthesis of a Library of Xylogluco-Oligosaccharides for Active-Site
Mapping of Xyloglucan endo-Transglycosylase
Re´gis Faure´,† Marc Saura-Valls,‡ Harry Brumer, III,§ Antoni Planas,‡ Sylvain Cottaz,† and
Hugues Driguez*,†
Centre de Recherche sur les Macromole´cules Ve´ge´tales (CERMAV-CNRS), BP53,
38041 Grenoble Cedex 9, France, Laboratory of Biochemistry, Institut Qu´ımic de Sarria`,
UniVersitat Ramon Llull, 08017 Barcelona, Spain, and School of Biotechnology,
Royal Institute of Technology, AlbaNoVa UniVersity Center, 106 91 Stockholm, Sweden
hugues.driguez@cermaV.cnrs.fr
ReceiVed December 13, 2005
Complex oligosaccharides containing R-D-xylosyl-(1f6)-â-D-glucosyl residues and unsubstituted â-(1f4)-
linked D-glucosyl units were readily synthesized using enzymatic coupling catalyzed by the Cel7B E197A
glycosynthase from Humicola insolens. Constituting this library required four key steps: (1) preparing
unprotected building blocks by chemical synthesis or enzymatic degradation of xyloglucan polymers;
(2) generating the donor synthon in the enzymatic coupling by temporarily introducing a lactosyl motif
on the 4-OH of the terminal glucosyl units of the xylogluco-oligosaccharides; (3) synthesizing the
corresponding R-fluorides, followed by their de-O-acetylation and the glycosynthase-catalyzed condensation
of these donors onto various acceptors; and (4) enzymatically releasing lactose or galactose from the
reaction product, affording the target molecules in good overall yields. These complex oligosaccharides
proved useful for mapping the active site of a key enzyme in plant cell wall biosynthesis and
modification: the xyloglucan endo-transglycosylase (XET). We also report some preliminary enzymatic
results regarding the efficiency of these compounds.
Introduction
The biosynthesis and the degradation of this major hemicellu-
losic polysaccharide are important in the plant life cycle and
for industrial applications.8
Xyloglucan is hydrolyzed in Vitro and in ViVo by microbial
and plant endo-â-1,4-glucanases belonging to glycoside hydro-
lase (GH) families9 5, 7, 12, 16, and 74. Hydrolysis yields
branched xylogluco-oligosaccharides (XGOs) in which the
reducing unit is usually a nonsubstituted glucosyl residue.10-13
Xyloglucan is a key structural polysaccharide in the cell walls
of many plant species1-4 and can also serve as a plant energy
reserve.5,6 It consists of a cellulose-like main chain of â-(1f4)-
linked D-glucosyl residues that is regularly substituted at C-6
with R-D-xylosyl and â-D-galactosyl-(1f2)-R-D-xylosyl resi-
dues. In the cell wall, some of the 2-hydroxyl groups of the
â-D-galactosyl units are substituted with R-L-fucosyl residues.7
† Centre de Recherche sur les Macromole´cules Ve´ge´tales (CERMAV-CNRS),
affiliated with the Joseph Fourier University, and member of the Institut de
Chimie Mole´culaire de Grenoble.
(7) Vincken, J. P.; York, W. S.; Beldman, G.; Voragen, A. G. J. Plant
Physiol. 1997, 114, 9-13.
(8) Rose, J. K.; Bennett, A. B. Trends Plant Sci. 1999, 4, 176-183.
(9) Couthino, P.; Henrissat, H. 1999, Carbohydrate-Active EnZYmes
server at URL http:/www.cazy.org.
‡ Universitat Ramon Llull.
§ Royal Institute of Technology.
(1) Fry, S. C. J. Exp. Bot. 1989, 40, 1-11.
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10.1021/jo0525682 CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/06/2006
J. Org. Chem. 2006, 71, 5151-5161
5151

Faure´ et al.
sequence variants of XET-like genes and found that each gene
is specifically regulated.23-26 Thus, corresponding enzymes may
have unique physiological roles based on substrate specificity
or catalytic mechanism. A better understanding of the funda-
mental mechanisms contributing to the assembly and growth
of the cell wall, in particular the cellulose-xyloglucan network,
may forward engineering of novel polysaccharide composites
with valuable physical properties by chemoenzymatic modifica-
tion of cellulose surface.13,27-29
The XET mechanism, which occurs in two steps, involves
attack on a glycosyl donor substrate to form a glycosyl-enzyme
intermediate that is degraded by glycosyl transfer to a suitable
acceptor substrate.30 In vitro studies have demonstrated that
XETs can use the xyloglucan polysaccharide as a donor in
combination with xyloglucan or short xyloglucan oligo-
saccharides, such as XXG 1 and XLLG 4 (Figure 1), as
acceptors.31-33 Although xyloglucan is used as the donor in
some XET assays,26 its polydispersive nature (in terms of
molecular mass and side-chain substitution) limits its use as a
substrate in detailed enzyme kinetic analysis. The tetradecasac-
charide XXXGXXXG 35 has been identified as a donor for the
mixed-function XET/xyloglucanase (EC 2.4.1.207/EC 3.2.1.151)
from nasturtium.34 Encouraged by this discovery, we developed
a new activity assay based on capillary electrophoresis to
evaluate low molecular mass xylogluco-oligosaccharides as XET
donors.35 In addition, we started synthesizing a library of well-
defined, low molecular mass donors for kinetic analysis and
subsite mapping studies of XETs.
FIGURE 1. Structure of some common xyloglucan derivatives (XGOs)
1-5.
Figure 1 gives the structures of some common XGOs. An
unambiguous letter code is used for the nomenclature of each
segment depending of the side chain: an unsubstituted D-Glcp
is assigned G, R-D-Xylp-(1f6)-â-D-Glcp segment is named X,
and â-D-Galp-(1f2)-R-D-Xylp-(1f6)-â-D-Glcp and R-L-Fucp-
(1f2)-â-D-Galp-(1f2)-R-D-Xylp-(1f6)-â-D-Glcp are respec-
tively called L and F.14
Results and Discussion
Glycosyl fluorides and nitrophenyl glycosides with the same
anomeric configuration as the natural substrate are potent
artificial donors in transglycosylation reactions catalyzed by
retaining glycoside hydrolases and transglycosylases.36,37 In a
preliminary study, we synthesized the XXXG â-2-chloro-4-
nitrophenyl and â-fluoride derivatives as potential donor
In 1984, XGO 5, but not 2 (Figure 1), was found to inhibit
auxin-stimulated growth of etiolated pea stem segments.15
A
few years later, the total chemical syntheses of 2 and 5 and its
precursors were described; however, poor overall yields pre-
cluded using chemical approaches to prepare larger oligosac-
charides for structure/activity relationship studies.16,17 Recent
discoveries of a group of enzymes involved in cell wall
modification have reopened the importance of accomplishing
these synthesis.
Plants have evolved a unique set of GH family 16 enzymes
to accomplish transient cell-wall modification in the absence
of xyloglucan hydrolysis. These enzymes, the xyloglucan endo-
transglycosylases (XETs, EC 2.4.1.207), can mediate molecular
grafting reactions between xyloglucan chains18-21 allowing
reorganization of the network as cell morphology changes.22
Plant genomic studies have identified numerous (30-40)
(23) Yokoyama, R.; Nishitani, K. Plant Cell Physiol. 2001, 42, 1025-
1033.
(24) Yokoyama, R.; Rose, J. K. C.; Nishitani, K. Plant Physiol. 2004,
134, 1088-1099.
(25) Aspeborg, H.; Schrader, J.; Coutinho, P. M.; Stam, M.; Kallas, A.;
Djerbi, S.; Nilsson, P.; Denman, S.; Amini, B.; Sterky, F.; Master, E.;
Sandberg, G.; Mellerowicz, E.; Sundberg, B.; Henrissat, B.; Teeri, T. T.
Plant Physiol. 2005, 137, 983-997.
(26) Rose, J. K. C.; Braam, J.; Fry, S. C.; Nishitani, K. Plant Cell Physiol.
2002, 43, 1421-1435.
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Plant Physiol. 1999, 121, 657-663.
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T. J. Am. Chem. Soc. 2004, 126, 5715-5721.
(14) Fry, S. C.; York, W. S.; Albersheim, P.; Darvill, A.; Hayashi, T.;
Joseleau, J. P.; Kato, Y.; Lorences, E. P.; Maclachlan, G. A.; McNeil, M.;
Mort, A. J.; Reid, J. S. G.; Seitz, H. U.; Selvendran, R. R.; Voragen, A. G.
J.; White, A. R. Physiol. Plantarum 1993, 89, 1-3.
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75, 295-297.
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235-245.
(17) Sakai, K.; Nakahara, Y.; Ogawa, T. Tetrahedron Lett. 1990, 31,
3035-3038.
(18) Fry, S. C.; Smith, R. C.; Renwick, K. F.; Martin, D. J.; Hodge, S.
K.; Matthews, K. J. Biochem. J. 1992, 282, 821-828.
(19) Nishitani, K.; Tominaga, R. J. Biol. Chem. 1992, 267, 21058-21064.
(20) Farkas, V.; Sulova, Z.; Stratilova, E.; Hanna, R.; Maclachlan, G.
Arch. Biochem. Biophys. 1992, 298, 365-370.
(21) Thompson, J. E.; Smith, R. C.; Fry, S. C. Biochem. J. 1997, 327,
699-708.
(22) Darley, C. P.; Forrester, A. M.; McQueen-Mason, S. J. Plant Mol.
Biol. 2001, 47, 179-195.
(29) Zhou, Q.; Greffe, L.; Baumann, M. J.; Malmstro¨m, E.; Teeri, T.
T.; Brumer, H., III. Macromolecules 2005, 38, 3547-3549.
(30) Sulova, Z.; Takacova, M.; Steele, N. M.; Fry, S. C.; Farkas, V.
Biochem. J. 1998, 330, 1475-1480.
(31) Lorences, E. P.; Fry, S. C. Physiol. Plantarum 1993, 88, 105-112.
(32) Tabuchi, A.; Kamisaka, S.; Hoson, T. Plant Cell Physiol. 1997,
38, 653-658.
(33) Johansson, P.; Brumer, H., III; Baumann, M. J.; Kallas, A. M.;
Henriksson, H.; Denman, S. E.; Teeri, T. T.; Jones, T. A. Plant Cell 2004,
16, 874-886.
(34) Fanutti, C.; Gidley, M. J.; Reid, J. S. G. Planta 1996, 200, 221-
228.
(35) Saura-Valls, M.; Faure´, R.; Raga`s, S.; Piens, K.; Brumer, H., III;
Teeri, T. T.; Cottaz, S.; Driguez, H.; Planas, A. Biochem. J. 2006, 395,
99-106.
(36) Okada, G.; Genghof, D. S.; Hehre, E. J. Carbohydr. Res. 1979, 71,
287-298.
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5152 J. Org. Chem., Vol. 71, No. 14, 2006

ActiVe-Site Mapping of Xyloglucan endo-Transglycosylase
SCHEME 1a
a Reagents and conditions: (i) (1) NIS, Et2O/CH2Cl2, (2) TMSOTf; (ii) (1) H2, Pd(OH)2-C, MeOH/THF; (2) Ac2O/pyridine, DMAP; (iii) (1) CAN,
toluene/CH3CN/H2O, (2) DAST, CH2Cl2, (3) hydrogen fluoride/pyridine.
substrates for recombinant Populus tremula x tremuloides
SCHEME 2a
(hybrid aspen) XET16A (PttXET16A). We observed no trans-
glycosylation with these derivatives as donors (unpublished data)
but found that the tetradecasaccharide XXXGXXXG is a donor
for PttXET16A.35 Assuming that this latter experimental result
indicates that substrate interactions with positive subsites of
PttXET16A are required, we prepared a range of xylogluco-
oligosaccharides that potentially spanned the catalytic residues
by binding both positive and negative subsites of PttXET16A.33
This library included combinations of the R-D-xylosyl-(1f6)-
â-D-glucosyl residues and unsubstituted â-(1f4)-linked D-
glucosyl units; these were coupled to either end of the XXXG
motif for enzyme subsite mapping studies using kinetic analysis.
The complexity and repetitive structure of XGOs prompted an
enzymatic approach using glycosynthases, glycosidase mutants
capable of performing oligosaccharide synthesis.38,39 Previously,
we demonstrated the efficiency of 6II-substituted R-cellobiosyl
fluorides as substrates for coupling reactions catalyzed by the
Cel7B E197A glycosynthase from Humicola insolens.40 Our
goal was to extend the utility of this enzyme in the synthesis of
XGO analogues. The required building blocks (R-fluoro-
glycoside donors and oligosaccharide acceptors) were prepared
either by chemical synthesis or by enzymatic hydrolysis of
xyloglucan.
We obtained glycoside acceptor 641 as described in the
literature in six steps from cellobiose and the glycoside donor
7 by benzylation of the known parent compound.42 Stereocon-
trolled and regioselective glycosylation of 6 with the phenyl
a Reagents and conditions: (i) MeONa/MeOH; (ii) (1) Cel7B E197A
glycosynthase, carbonate buffer, (2) Ac2O/pyridine, DMAP; (iii) (1) CAN,
thioxyloside 7 gave the expected trisaccharide 8 in moderate
yield (43%, Scheme 1). The structure of 8 was confirmed by
its quasi-quantitative conversion into the acetylated 9 and
complete assignment of its 1H NMR spectrum. Oxidative
deprotection of the anomeric position, followed by DAST
treatment, afforded an expected anomeric mixture of fluorides.43
Anomerization with HF/pyridine gave the pure R-fluoride 10
in 74% yield over the last three steps.
toluene/CH3CN/H2O, (2) DAST, CH2Cl2, (3) hydrogen fluoride/pyridine.
Because the multistep synthesis of XGOs achieved a poor
overall yield,17 we used specific enzymatic hydrolysis to
optimize the preparation of XXG 1 and XXXG 2 from tamarind
xyloglucan.35 According to the literature the corresponding
peracetylated R-fluoride 11 was prepared in good overall yield
from XXXG 2 (Scheme 1).35
An obvious approach to obtain well-defined XGOs was the
polycondensation of R-fluorides, obtained by de-O-acetylation
of 10 and 11, catalyzed by the Cel7B E197A glycosynthase
from H. insolens; however, as has been previously observed
for cellobiosyl fluoride,40 this reaction gave mostly insoluble
polymers with little formation of dimers and trimers (data not
shown). It also has been shown that 4-O-substituted glucosyl
or galactosyl units on the nonreducing end of the fluoride donors
prevent this uncontrolled polymerization.40,43,44 Based on this
information, we developed a new concept of enzymatic protec-
(38) Mackenzie, L. F.; Wang, K.; Warren, R. A. J.; Withers, S. G. J.
Am. Chem. Soc. 1998, 120, 5583-5584.
(39) Malet, C.; Planas, A. FEBS Lett. 1998, 440, 208-212.
(40) Fort, S.; Boyer, V.; Greffe, L.; Davies, G. J.; Moroz, O.; Chris-
tiansen, L.; Shu¨lein, M.; Cottaz, S.; Driguez, H. J. Am. Chem. Soc. 2000,
122, 5429-5437.
(41) Robina, I.; Gomez-Bujedo, S.; Fernandez-Bolanos, J. G.; Fuentes,
J. Synth. Commun. 1998, 28, 2379-2397.
(42) Purves, C. B. J. Am. Chem. Soc. 1929, 51, 3619-3627.
(43) Boyer, V.; Fort, S.; Frandsen, T. P.; Schu¨lein, M.; Cottaz, S.;
Driguez, H. Chem. Eur. J. 2002, 8, 1389-1394.
J. Org. Chem, Vol. 71, No. 14, 2006 5153

Faure´ et al.
SCHEME 3a
a Reagents and conditions: (i) Cel7B E197A glycosynthase, carbonate buffer; (ii) A. oryzae â-galactosidase, acetate buffer; (iii) (1) Ac2O/pyridine, DMAP,
(2) hydrazine acetate, DMF, (3) DAST, CH2Cl2, (4) hydrogen fluoride/pyridine.
tion/deprotection of the nonreducing glucosyl unit of the (xylo)-
gluco-oligosaccharide donors. This new approach involved
introducing a lactosyl unit (or alternatively a tetrahydropyranyl
(O-THP) group) on the 4ω-OH position of fluorides. After
coupling, the lactosyl unit can be wholly or partially removed
by â-galactosidase and â-glucosidase activities, while the 4ω-
O-THP can be removed by acid-catalyzed hydrolysis.
With this approach, the enzymatic condensation of R-lactosyl
fluoride 1245 in the presence of the compound arising from de-
O-acetylation of 9 gave the expected pentasaccharide isolated
in good yield after acetylation (89%). The pentasaccharide 13
was transformed into its corresponding R-fluoride 14 by
oxidative removal of the aglycon followed by DAST and HF/
pyridine treatments, as described above; the overall yield over
the three steps was 46% (Scheme 2).
galactosylation of 15 using the commercially available â-ga-
lactosidase from Aspergillus oryzae gave GXXXG 16 in 51%
yield from 15 (Scheme 3). XGOs obtained by enzymatic
condensation of 12 on acceptors 1 and 2 also gave corresponding
fluorides 17 and 18 in 46% and 56% overall yields using the
methodology already described for compound 11.35
Glycosynthase-catalyzed coupling of 4II-O-THP-cellobiosyl
fluoride 1944 and the XXXG acceptor 2 gave the nonasaccharide
GGXXXG 20 in 78% yield after acidic hydrolysis of the THP
group (Scheme 4). GGXXXG 20 can also act as a glycosynthase
substrate and be elongated by repeating the condensation with
4II-O-THP-cellobiosyl fluoride and acid hydrolysis of the THP
group; after these two steps, the undecasaccharide GGGGXXXG
21 was obtained in 86% yield. The coupling of 4II-O-THP-
cellobiosyl fluoride 19 with 16 likewise afforded GGGXXXG
22 in 76% yield after acidic deprotection.
Enzymatic condensation of 12 on acceptor 2 gave Gal-
GXXXG 15 in 68% yield. Kinetically controlled enzymatic de-
De-O-acetylation of compound of 14 gave a pentasaccharide
donor that was coupled with the XXXG acceptor 2, removal of
the lactosyl moiety by treatment with the mixed-function
â-glucosidase/galactosidase from Agrobacterium sp.,46 afforded
(44) Fort, S.; Christiansen, L.; Schulein, M.; Cottaz, S.; Driguez, H. Isr.
J. Chem. 2000, 40, 217-221.
(45) Ju¨nneman, J.; Thiem, J.; Pedersen, C. Carbohydr. Res. 1993, 249,
91-94.
(46) Kempton, J. B.; Withers, S. G. Biochemistry 1992, 31, 9961-9969.
5154 J. Org. Chem., Vol. 71, No. 14, 2006

ActiVe-Site Mapping of Xyloglucan endo-Transglycosylase
SCHEME 4a
followed by de-lactosylation, as already described for 23, gave
the expected compound XGGXXXG 25 in 80% yield (Figure
2).
Under the same conditions, coupling of the fluoride resulting
from de-O-acetylation of 17 with XXXG 2 gave a tetradecasac-
charide which was either de-lactosylated to give XXGXXXG
26 in 79% yield or de-galactosylated as described for the
preparation of 16 to give GXXGXXXG 27 in 35% yield (Figure
3).
Using the same glycosynthase coupling strategy, de-O-
acetylation of the nonasaccharidyl fluoride 18 gave a new donor
which reacted with cellobiose GG 28, cellotetraose GGGG 29,
the trisaccharide XG 30,47 and the pentasaccharide XXG 135 to
give the corresponding 31-34 in excellent yields (Figure 4).
Structure and purity of the compounds were confirmed using
1
HR-ESMS, and H/13C NMR analysis or quantitative HPLC-
ESMS on 4-aminobenzoic acid ethyl ester derivatives.49 The
regio- and stereochemistry of the new glycosidic linkages
formed during the enzymatic condensations were confirmed by
enzymatic hydrolysis using wild-type Cel7B from H. insolens.
The ability of compounds 15 and 20-27 and 31-34 to act
as glycosyl donors in the transglycosylation reaction catalyzed
by PttXET16A48 was assessed by high-performance capillary
electrophoresis (HPCE)35 using the 8-aminonaphthalene-1,3,6-
trisulfonate conjugate of XXXG (XXXG-ANTS) as the acceptor
substrate.
Table 1 summarizes the preliminary results obtained under
standardized assay conditions, where are given the relative
reactivities of XGO donor substrates in PttXET16A-catalyzed
transglycosylation reactions using XXXG-ANTS as the acceptor
substrate. The new donors can be grouped into two classes
relative to the reference tetradecasaccharide XXXGXXXG 35,
in which the glycosidic bond of the internal unsubstituted
a Reagents and conditions: (i) (1) Cel7B E197A glycosynthase, carbonate
buffer; (2) 1 M HCl; (ii) (1) Cel7B E197A glycosynthase, carbonate buffer
+ 19; (2) 1 M HCl.
the decasaccharide XGXXXG 23 in 66% yield over the two
steps. When this sequence of reactions was repeated with
decasaccharide 23 as an acceptor, the tridecasaccharide
XGXGXXXG 24 was isolated in 87% overall yield. Condensa-
tion of the same pentasaccharide donor and GXXXG 16
FIGURE 2. Structure of XGOs 23-25.
FIGURE 3. Structure of XGOs 26 and 27.
J. Org. Chem, Vol. 71, No. 14, 2006 5155

Faure´ et al.
FIGURE 4. Structure of cello-oligomers 28 and 29 and XGOs 30-35.
TABLE 1. Relative Reactivities of XGO Donor Substrates in
PttXET16A-Catalyzed Transglycosylation Reactions Using
XXXG-ANTS as the Acceptor Substrate
the H. insolens Cel7B E197A glycosynthase-assisted synthesis
has proven to be a powerful approach for the preparation of a
library of xyloglucan oligosaccharides from tamarin seed
xyloglucan. A block synthesis strategy has been achieved by
employing lactosyl or 4II-O-THP-cellobiosyl building blocks as
temporary protecting group, allowing the oligomerization of
xyloglucan substructures catalyzed by the Cel7B E197A gly-
cosynthase. The variety and the complexity of these well-defined
xyloglucan oligomers constitute and unprecedented library of
oligosaccharides useful for the biochemical characterization of
xyloglucan endo-transglycosylases (XET), an important class
of enzymes involved in the cell wall polysaccharide biosynthesis
and modification in plants, and of practical importance in the
chemoenzymatic surface modification of cellulose for the
production of new composite biomaterials.
initial ratesa
reactivity relative to
donor
V0/[E] (103 s-1
)
XXXGXXXG 35b (%)
XXXGGG 31
XXXGXG 33
n.r.c
n.r.
XXXGXXG 34
XXXGGGGG 32
XXXGXXXG 35
GXXGXXXG 27
XGXGXXXG 24
GGGGXXXG 21
GGGXXXG 22
XGGXXXG 25
XXGXXXG 26
XGXXXG 23
28.8
n.r.
37
78.4
95.0
40.0
40.8
20.4
31.5
20.9
1.0
100
121
51
52
26
40
27
1.3
0.6
0.5
GGXXXG 20
GalGXXXG 15
0.5
0.4
Experimental Section
a Initial rates V0/[E] were calculated from the peak areas of products in
the electropherograms when monitoring the reaction for 15-30 min, using
Man-ANTS (ANTS-derivatized mannose) as an internal standard.35 b Rela-
tive reactivity: initial rate of transglycosylation of various donors relative
to the rate of donor XXXGXXXG (35). c n.r., no reaction detected.
Phenyl 2,3,4-Tri-O-benzyl-1-thio-â-D-xylopyranoside (7). To
a solution of phenyl 1-thio-â-D-xylopyranoside42 (0.97 g, 4.0 mmol)
in DMF (30 mL) were added NaH (0.55 g, 24 mmol, 6 equiv) and
a catalytic amount of Bu4NI (10 mg). The mixture was vigorously
stirred for 15 min at 0 °C, and then BnBr (4.3 mL, 36 mmol, 9
equiv) was added dropwise. After the solution was stirred for 30
min at 0 °C and 12 h at rt, MeOH and then Et3N (2 mL) were
added, and the resulting mixture was evaporated. The residue was
dissolved in CH2Cl2, washed with water, dried, and concentrated
under reduce pressure. Flash chromatography (cyclohexane/Et2O,
9:1 v/v) of the residue gave the expected phenyl thioxyloside 7
glucosyl (G) residue is cleaved to yield the transfer product
XXXGXXXG-ANTS.35 Those designed to probe the contribu-
tion of the positive enzyme subsites to XET catalysis have the
general structure XXXG-nnnn; those designed to target negative
subsites have the general structure nnnn-XXXG. We identified
compounds GalGXXXG 15 and GGXXXG 20 as the smallest
XET donors described so far, providing new insight into the
substrate specificity of PttXET16A. More detailed comparative
kinetic analysis for this new series of compounds will allow
mapping of both donor and acceptor subsites of the binding
cleft of the enzyme.
(1.71 g, 3.3 mmol) in 83% yield, which crystallized in EtOH: mp
1
51-53 °C; [R]25 ) +7 (c ) 1.0 in CHCl3); H NMR (CDCl3,
D
300 MHz) δ 7.57-7.28 (m, 20H, SPh and CH2Ph), 4.94, 4.90,
4.87, 4.79, 4.74 (5d, 2JH,H ) 11.0, 10.5, 11.0, 10.5, 11.7 Hz, 5H, 5
2
CH2Ph), 4.72 (d, J1,2 ) 9.4 Hz, 1H, H-1), 4.65 (d, JH,H ) 11.7
Hz, 1H, CH2Ph), 4.10 (dd, J4,5a ) 4.6 and J5a,5b ) 11.5 Hz, 1H,
H-5a), 3.72-3.63 (m, 2H, H-3 and H-4), 3.49 (t, J2,3 ) 8.6 Hz,
1H, H-2), 3.29 (dd, J4,5b ) 9.7 Hz, 1H, H-5b); 13C NMR (CDCl3,
75 MHz) δ 131.8-127.5 (SPh and CH2Ph), 88.3 (C-1), 85.2 (C-3
or C-4), 80.4 (C-2), 77.7 (C-3 or C-4), 75.5, 75.3, 73.1 (CH2Ph),
67.4 (C-5); DCI-MS m/z 530 [M + NH4]+. Anal. Calcd for
C32H32O4S: C, 74.97; H, 6.29; S, 6.25. Found: C, 75.00; H, 6.38;
S, 6.55.
4-Methoxyphenyl [(2,3,4-Tri-O-benzyl-R-D-xylopyranosyl)-
(1f6)]-(2,3-di-O-benzyl-â-D-glucopyranosyl)-(1f4)-2,3,6-tri-O-
benzyl-â-D-glucopyranoside (8). A solution of phenyl thioxyloside
7 (616 mg, 1.20 mmol, 1.2 equiv) and diol acceptor 6 (899 mg,
1.00 mmol) in Et2O/CH2Cl2 (7:3 v/v, 65 mL) was stirred 15 min at
Conclusion
A combination of enzymatic and chemical steps, exploiting
the versatility of wild-type glycoside hydrolases together with
(47) Mori, M.; Eda, S.; Katoˆ, K. Agric. Biol. Chem 1979, 43, 145-149.
(48) Kallas, A. M.; Piens, K.; Denman, S. E.; Henriksson, H.; Faldt, J.;
Johansson, P.; Brumer, H., III; Teeri, T. T. Biochem. J. 2005, 390, 105-
113.
(49) Dumon, C.; Samain, E.; Priem, B. Biotechnol. Prog. 2004, 20, 412-
419.
5156 J. Org. Chem., Vol. 71, No. 14, 2006

ActiVe-Site Mapping of Xyloglucan endo-Transglycosylase
0 °C under argon, sheltered from light, in the presence of
N-iodosuccinimide (345 mg, 1.50 mmol, 1.5 equiv). Then a solution
of TMSOTf (93 µL, 0.50 mmol, 0.5 equiv) in CH2Cl2 was added.
After being stirred for 3 h at 0 °C, under the same conditions, the
mixture was diluted with CH2Cl2, filtered through diatomaceous
earth, washed with saturated aqueous sodium hydrogen carbonate
then 10% (m/v) aqueous sodium thiosulfate, dried, and evaporated.
Flash chromatography (cyclohexane/EtOAc, 3:1 v/v), followed by
column chromatography using silica gel Si 60 (63-200 µm; toluene/
(152 mg, 176 µmol) in 95% yield, characterized only by CI-MS
(m/z ) 872 [M + NH4]+).
In a plastic vessel, a solution of the R/â-fluorides (101 mg, 120
µmol) in hydrogen fluoride/pyridine (7:3 v/v, 1 mL) was stirred at
-50 °C for 15 min, and then the temperature of the cooling bath
was raised to -10 °C for 2.5 h. The solution was diluted with
dichloromethane, and then poured into a plastic beaker containing
an ice-cooled solution of ammonia in water (3 M). The organic
layer was washed with saturated aqueous sodium hydrogen carbon-
ate (three times), dried, and concentrated under reduced pressure.
Flash chromatography (cyclohexane/EtOAc, 2:1, 3:2 then 1:1 v/v)
using silica gel neutralized with triethylamine of the residue gave
EtOAc, 9:1 v/v), gave the trisaccharide 8 (555 mg, 0.43 mmol) in
1
43% yield: [R]25 ) +52 (c ) 1.2 in CHCl3); H NMR (CDCl3,
D
400 MHz) δ 7.36-7.20 (m, 40H, CH2Ph), 7.02-6.77 (m, 4H, C6H4-
OCH3), 4.97-4.37 (m, 16H, CH2Ph), 4.83 (d, J1,2 ) 7.7 Hz, 1H,
H-1 of GlcI), 4.52 (d, J1,2 ) 3.8 Hz, 1H, H-1 of XylII), 4.49 (d, J1,2
) 7.8 Hz, 1H, H-1 of GlcII), 4.00-3.09 (m, 17H, H-2 to H-6 of
Glc and H-2 to H-5 of Xyl), 3.76 (s, 3H, C6H4OCH3); 13C NMR
(CDCl3, 100 MHz) δ 155.3, 151.6 (C-1 and C-4 of C6H4OCH3),
139.1-138.5 (C-1 of CH2Ph), 128.5-127.3 (others C aromatic of
CH2Ph), 118.5, 114.5 (others C aromatic of C6H4OCH3), 102.8 (C-1
of GlcI), 102.4 (C-1 of GlcII), 98.3 (C-1 of XylII), 84.4, 82.7, 81.8,
81.5, 81.4, 79.4, 77.8, 76.8, 75.8, 75.1, 72.0 (C-2 to C-5 of Glc
and C-2 to C-4 of Xyl), 75.7, 75.4, 75.2, 75.0 (broad), 73.8, 73.3,
73.2 (CH2Ph), 70.9, 68.1, 60.2 (C-5 of XylII and C-6 of GlcI,II),
55.6 (C6H4OCH3); DCI-MS m/z 1318 [M + NH4]+; ES-HRMS m/z
calcd for C80H84O16Na [M + Na]+ 1323.5657, found 1323.5647.
4-Methoxyphenyl (2,3,4-Tri-O-acetyl-R-D-xylopyranosyl)-
(1f6)-(2,3,4-tri-O-acetyl-â-D-glucopyranosyl)-(1f4)-2,3,6-tri-O-
acetyl-â-D-glucopyranoside (9). To a solution of trisaccharide 8
(699 mg, 0.54 mmol) in MeOH/THF (1:1 v/v, 25 mL) was added
a catalytic amount of Pd(OH)2-C. After vigorous stirring for 12 h
under H2 (1 atm) at rt, the reaction mixture was filtered through
diatomaceous earth and the solution concentrated under reduced
pressure and acetylated (acetic anhydride/pyridine, 1:2 v/v, 15 mL)
in the presence of a catalytic amount of (dimethylamino)pyridine
(DMAP). After 12 h of stirring at rt, the reaction was quenched by
addition of MeOH at 0 °C and concentrated under reduced pressure.
Flash chromatography (EtOAc/petroleum ether, 2:3 v/v) of the
the expected fluoride 10 (99 mg, 116 µmol) in 98% yield: [R]25
D
) +67 (c ) 1.8 in CHCl3); 1H NMR (CDCl3, 400 MHz) see Table
B in the Supporting Information; 13C NMR (CDCl3, 100 MHz) δ
170.2-169.0 (COCH3), 103.6 (d, JC,F ) 229.7 Hz, C-1 of GlcI),
100.1 (C-1 of GlcII), 96.1 (C-1 of XylII), 74.6 (C-4 of GlcI), 72.9,
72.8, 71.7, 70.8, 70.7 (d, JC,F ) 4.1 Hz), 70.3 (d, JC,F ) 24.6 Hz),
69.2 (broad), 69.1, 69.0 (C-2 to C-5 of Glc and C-2 to C-4 of Xyl),
67.6 (C-6 of GlcII), 61.1 (C-6 of GlcI), 58.9 (C-5 of XylII), 20.9-
20.5 (COCH3); FAB-MS m/z ) 893 [M + K]+; ES-HRMS m/z
calcd for C35H47O23FNa [M + Na]+ 877.2390, found 877.2383.
4-Methoxyphenyl (2,3,4,6-Tetra-O-acetyl-â-D-galactopyrano-
syl)-(1f4)-(2,3,6-tri-O-acetyl-â-D-glucopyranosyl)-(1f4)-[(2,3,4-
tri-O-acetyl-R-D-xylopyranosyl)-(1f6)]-(2,3-di-O-acetyl-â-D-
glucopyranosyl)-(1f4)-2,3,6-tri-O-acetyl-â-D-glucopyranoside (13).
Compound 9 (192 mg, 0.2 mmol) was treated in methanol (30 mL)
with sodium methoxide (1 M in methanol, 100 µL) for 5 h at 0 °C.
The solution was neutralized with Amberlite IRN 120 (H+), filtered,
and then concentrated under reduced pressure. The residue dissolved
in deionized water was freeze-dried to give the expected compound
in quantitative yield (116 mg). This compound was only character-
ized by mass spectroscopy: FAB-MS m/z ) 581 [M + H]+; ES-
HRMS m/z calcd for C24H36O16Na [M + Na]+ 603.1901, found
603.1914.
To this trisaccharide (103 mg, 0.18 mmol) in carbonate/
bicarbonate buffer (0.1 m, pH 10, 5 mL) were added H. insolens
Cel7B E197A glycosynthase (2 mg) and lactosyl fluoride 12 (80
mg, 0.23 mmol, 1.3 equiv). The solution was placed in a rotative
shaker for 20 h at 37 °C and then freeze-dried. The residue was
acetylated (acetic anhydride/pyridine, 1:2 v/v, 24 mL) in the
presence of a catalytic amount of DMAP. After 12 h of stirring at
rt, the reaction was quenched by addition of MeOH at 0 °C,
concentrated under reduced pressure, and coevaporated with toluene.
Flash chromatography (toluene/acetone, 4:1 v/v) of the residue gave
residue gave the acetylated 9 (497 mg, 0.52 mmol) in 97% yield:
1
[R]25 ) +32 (c ) 1.0 in CHCl3); H NMR (CDCl3, 400 MHz)
D
see Table A in the Supporting Information; 13C NMR (CDCl3, 100
MHz) δ 170.3-169.1 (COCH3), 155.8, 150.9 (C-1 and C-4 of C6H4-
OCH3), 118.7, 114.5 (others C aromatic of C6H4OCH3), 100.0,
100.0 (C-1 of GlcI,II), 96.1 (C-1 of XylII), 75.4 (C-4 of GlcI), 73.0,
72.8, 72.7, 72.7, 71.7, 71.4, 70.9, 69.4, 69.0, 68.9 (C-2 to C-5 of
Glc and C-2 to C-4 of Xyl), 67.2 (C-6 of GlcII), 61.8 (C-6 of GlcI),
59.0 (C-5 of XylII), 55.7 (C6H4OCH3), 20.8-20.5 (COCH3); DCI-
MS m/z ) 976 [M + NH4]+; ES-HRMS m/z calcd for C42H54O25-
Na [M + Na]+ 981.2852, found 981.2860.
the pentasaccharide 13 (237 mg, 0.15 mmol) in 87% yield: [R]25
D
) +5 (c ) 1.0 in CHCl3); 1H NMR (CDCl3, 400 MHz) see Table
C in the Supporting Information; 13C NMR (CDCl3, 100 MHz) δ
170.4-168.8 (COCH3), 155.8, 150.8 (C-1 and C-4 of C6H4OCH3),
118.7, 114.5 (others C aromatic of C6H4OCH3), 101.3 (C-1 of Gal),
100.5 (C-1 of GlcII), 100.3 (C-1 of GlcIII), 99.7 (C-1 of GlcI), 97.1
(C-1 of XylII), 76.8, 75.8, 74.7, 74.3, 73.4, 73.1, 72.5, 72.5, 72.1,
72.0, 71.9, 71.6, 71.1, 70.6, 70.5, 69.0, 68.9, 68.6, 66.6 (C-2 to
C-5 of Glc and Gal, and C-2 to C-4 of Xyl), 64.8 (C-6 of GlcII),
62.1, 62.0 (C-6 of GlcI,III), 60.8 (C-6 of Gal), 59.0 (C-5 of XylII),
55.6 (C6H4OCH3), 21.0-20.4 (CO CH3); MALDI-TOF-MS m/z )
1557 [M + Na]+; ES-HRMS m/z calcd for C66H86O41Na [M +
Na]+ 1557.4542, found 1557.4541.
(2,3,4-Tri-O-acetyl-R-D-xylopyranosyl)-(1f6)-(2,3,4-tri-O-
acetyl-â-D-glucopyranosyl)-(1f4)-2,3,6-tri-O-acetyl-R-D-glu-
copyranosyl Fluoride (10). A mixture of acetylated glycoside 9
(410 mg, 430 µmol) and ammonium cerium nitrate (CAN, 2.34 g,
4.3 mmol, 10 equiv) in toluene/acetonitrile/water (1:1.4:1 v/v/v,
34 mL) was stirred at rt for 1.5 h. The reaction mixture was diluted
with CH2Cl2 and then successively washed with brine, saturated
aqueous sodium hydrogen carbonate, and water, dried, concentrated,
and purified by flash chromatography (EtOAc/petroleum ether, 3:2
v/v) to give the expected hydroxy compound (290 mg, 340 µmol)
in 79% yield characterized only by mass spectroscopy: CI-MS m/z
) 870 [M + NH4]+; ES-HRMS m/z calcd for C35H48O24Na [M +
Na]+ 875.2433, found 875.2429.
Diethylaminosulfur trifluoride (DAST, 124 µL, 940 µmol, 5
equiv) was added dropwise to a stirred solution of the hydroxy
compound (160 mg, 188 µmol) in CH2Cl2 (8 mL) at -30 °C. The
solution was stirred for 2 h, and then the temperature of the cooling
bath was raised to rt and the solution was concentrated under
reduced pressure. Flash chromatography (EtOAc/petroleum ether,
1:2 then 1:1 v/v) using silica gel neutralized with triethylamine of
the residue gave corresponding fluoride as a mixture of R/â anomers
(2,3,4,6-Tetra-O-acetyl-â-D-galactopyranosyl)-(1f4)-(2,3,6-
tri-O-acetyl-â-D-glucopyranosyl)-(1f4)-[(2,3,4-tri-O-acetyl-R-D-
xylopyranosyl)-(1f6)]-(2,3-di-O-acetyl-â-D-glucopyranosyl)-
(1f4)-2,3,6-tri-O-acetyl-R-D-glucopyranosyl Fluoride (14). A
mixture of acetylated glycoside 13 (223 mg, 145 µmol) and CAN
(797 mg, 1.450 mmol, 10 equiv) in toluene/acetonitrile/water (1:
1.4:1 v/v/v, 15 mL) was stirred at rt for 2 h. The reaction mixture
was diluted with CH2Cl2 and then successively washed with brine,
saturated aqueous sodium hydrogen carbonate, and water, dried,
concentrated, and purified by flash chromatography (toluene/
acetone, 3:1 v/v) to give the expected C1-hydroxy compound (161
J. Org. Chem, Vol. 71, No. 14, 2006 5157

Faure´ et al.
mg, 113 µmol; MALDI-TOF-MS m/z ) 1451 [M + Na]+) in 78%
yield. To a solution of this C-1 hydroxy compound (160 mg, 112
µmol) in CH2Cl2 (5 mL) at -30 °C was added DAST (75 µL, 550
µmol, 5 equiv) dropwise. After the mixture was stirred for 2 h, the
temperature of the cooling bath was raised to rt, and then the
reaction was quenched by addition of MeOH and the solution
concentrated under reduced pressure. Flash chromatography (toluene/
acetone, 4:1 v/v) using silica gel neutralized with triethylamine of
the residue gave the expected mixture of R/â fluorides (103 mg,
72 µmol; MALDI-TOF-MS m/z ) 1453 [M + Na]+) in 64% yield.
In a plastic vessel, a solution of these R/â-fluorides (60 mg, 42
µmol) in hydrogen fluoride/pyridine (7:3 v/v, 0.75 mL) was stirred
at -50 °C for 15 min, and then the temperature of the cooling
bath was raised to -10 °C for 2.5 h. The solution was diluted with
dichloromethane and then poured into a plastic beaker containing
an ice-cooled solution of ammoniac (3 M). The organic layer was
washed with saturated aqueous sodium hydrogencarbonate (three
times), dried, and concentrated under reduced pressure. Flash
chromatography (toluene/acetone, 3.5:1 v/v) using silica gel neutral-
ized with triethylamine of the residue gave the expected fluoride
(1f4)-[(2,3,4-tri-O-acetyl-R-D-xylopyranosyl)-(1f6)]-(2,3-di-O-
acetyl-â-D-glucopyranosyl)-(1f4)-2,3,6-tri-O-acetyl-R-D-
glucopyranosyl Fluoride (17). H. insolens Cel7B E197A glycosyn-
thase (3 mg) was added to a solution of lactosyl fluoride 12 (151
mg, 0.44 mmol, 1.3 equiv) and pentasaccharide 1 (259 mg, 0.34
mmol) in carbonate/bicarbonate buffer (0.1 m, pH 10, 6 mL). The
solution was placed in a rotative shaker for 20 h at 37 °C and then
freeze-dried. The residue was acetylated (acetic anhydride/pyridine,
1:2 v/v, 24 mL) in the presence of a catalytic amount of DMAP.
After 12 h of stirring at rt, the reaction was quenched by addition
of MeOH at 0 °C, and the resulting solution was concentrated under
reduced pressure and coevaporated with toluene. Flash chroma-
tography (toluene/acetone, 3.5:1 v/v) of the residue gave the
expected acetylated heptasaccharide (500 mg, 0.26 mmol) in 75%
yield characterized by MALDI-TOF-MS: m/z ) 1997 [M + Na]+.
A mixture of this compound (479 mg, 243 µmol) and hydrazine
acetate (34 mg, 369 µmol, 1.5 equiv) in DMF (5 mL) was stirred
at 50 °C for 25 min. The solution was diluted with ethyl acetate,
washed with brine (two times), dried, concentrated, and coevapo-
rated with toluene. Flash chromatography (toluene/acetone, 3:1 v/v)
of the residue gave the expected C-1 hydroxy compound (345 mg,
179 µmol; MALDI-TOF-MS m/z ) 1955 [M + Na]+) in 74% yield.
To a solution of this hydroxy compound (340 mg, 176 µmol) in
CH2Cl2 (10 mL) at -30 °C was added DAST (120 µL, 880 µmol,
5 equiv) dropwise. After the solution was stirred for 2 h, the
temperature of the cooling bath was raised to rt, and then the
reaction was quenched by addition of MeOH and the solution was
concentrated under reduced pressure. Flash chromatography (toluene/
acetone, 3:1 v/v) using silica gel neutralized with triethylamine of
the residue gave the expected R/â-fluorides (328 mg, 169 µmol;
MALDI-TOF-MS m/z ) 1957 [M + Na]+) in 96% yield. In a
plastic vessel, a solution of the R/â-fluorides (310 mg, 160 µmol)
in hydrogen fluoride/pyridine (7:3 v/v, 3 mL) was stirred at -50
°C for 15 min, and then the temperature of the cooling bath was
raised to -10 °C for 2.5 h. The solution was diluted with
dichloromethane and then poured into a plastic beaker containing
an ice-cooled solution of ammonia in water (3 M). The organic
layer was washed with saturated aqueous sodium hydrogen carbon-
ate (three times), dried, and concentrated under reduced pressure.
Flash chromatography (cyclohexane/acetone, 3:2 v/v) using silica
gel neutralized with triethylamine of the residue gave the expected
fluoride 17 (203 mg, 105 µmol) in 65% yield: [R]25D ) +34 (c )
1.0 in CHCl3); 1H NMR (CDCl3, 400 MHz) see Table E in
Supporting Information; 13C NMR (CDCl3, 100 MHz) δ 170.5-
168.8 (COCH3), 103.6 (d, JC,F ) 229.5 Hz, C-1 of GlcI), 101.4
(C-1 of Gal), 100.5, 100.3, 100.0 (C-1 of GlcII,III,IV), 97.1 (C-1 of
XylIII), 97.0 (C-1 of XylII), 76.0, 75.6, 75.0, 74.7, 74.6, 74.5, 73.1,
72.7, 72.5, 72.4, 72.0, 71.9, 71.8, 71.1, 70.6, 70.6, 70.5, 70.4, 70.3,
69.5, 69.1, 69.0 (broad), 68.9, 68.6, 66.5 (C-2 to C-5 of Glc and
Gal, and C-2 to C-4 of Xyl), 65.5 (C-6 of GlcIII), 65.1 (C-6 of
GlcII), 62.2 (C-6 of GlcIV), 61.3 (C-6 of GlcI), 60.7 (C-6 of Gal),
59.1, 58.9 (C-5 of Xyl), 20.6-20.5 (COCH3); MALDI-TOF-MS
m/z ) 1957 [M + Na]+; ES-HRMS m/z calcd for C80H107O53FNa
[M + Na]+ 1957.5559, found 1957.5557.
14 (56 mg, 39 µmol) in 93% yield: [R]25 ) +30 (c ) 1.0 in
D
CHCl3); 1H NMR (CDCl3, 400 MHz) see Table D in the Supporting
Information; 13C NMR (CDCl3, 100 MHz) δ 170.4-168.8 (COCH3),
103.6 (d, JC,F ) 229.8 Hz, C-1 of GlcI), 101.3 (C-1 of Gal), 100.4
(C-1 of GlcII), 100.2 (C-1 of GlcIII), 97.1 (C-1 of Xyl), 75.9, 75.7,
74.7, 74.3, 73.1, 72.5, 72.2, 72.0, 71.6, 71.1, 70.7, 70.5, 70.4, 70.4,
69.4, 69.0, 68.9, 68.6, 66.6 (C-2 to C-5 of Glc and Gal, and C-2 to
C-4 of Xyl), 65.1 (C-6 of GlcII), 62.1 (C-6 of GlcIII), 61.2 (C-6 of
GlcI), 60.9 (C-6 of Gal), 59.1 (C-5 of XylII), 20.9-20.4 (COCH3);
MALDI-TOF-MS m/z ) 1453 [M + Na]+; ES-HRMS m/z calcd
for C59H79O39FNa [M + Na]+ 1453.4080, found 1453.4081.
â-D-Galactopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-[R-D-
xylopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-xylopyra-
nosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-xylopyranosyl-
(1f6)]-â-D-glucopyranosyl-(1f4)-D-glucopyranose (GalGXXXG
15). H. insolens Cel7B E197A glycosynthase (5 mg) was added to
a solution of lactosyl fluoride 12 (0.31 g, 0.89 mmol, 1.2 equiv)
and heptasaccharide 2 (0.79 g, 0.74 mmol) in carbonate/bicarbonate
buffer (0.1 m, pH 10, 15 mL). The solution was placed in a rotative
shaker for 20 h at 37 °C and then freeze-dried. The reaction mixture
was diluted in acetonitrile/water (1:1 v/v, 10 mL), silica gel was
added, and the suspension was evaporated. The powder was
deposited at the top of a silica gel column and a gradient (acetonitrile
then acetonitrile/water, 95:5, 9:1, 85:15, 8:2 and 75:25 v/v) was
used to elute 15 (0.70 g) in 68% yield: MALDI-TOF-MS m/z )
1409 [M + Na]+; ES-HRMS m/z calcd for C51H86O43Na [M +
Na]+ 1409.4441, found 1409.4439; LC/ES-MS analysis gave m/z
1558 [M(ABEE) + Na]+; tR ) 16.36 min.
â-D-Glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-
glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-glucopy-
ranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-glucopyranosyl-
(1f4)-D-glucopyranose (GXXXG 16). A solution of nonasaccharide
15 (56.0 mg, 40.4 µmol) in acetate buffer (0.1 m, pH 4.6, 2 mL)
was treated with commercially available â-galactosidase (A. oryzae,
5 mg, 40 units) for 2.5 h at 30 °C. The reaction mixture was diluted
in acetonitrile/water (1:1 v/v, 10 mL), silica gel was added, and
the suspension was evaporated. The powder was deposited at the
top of a silica gel column, and a gradient (acetonitrile then
acetonitrile/water, 95:5, 9:1, 85:15, 8:2 and 75:25 v/v) was used to
elute the octasaccharide 16 (25.0 mg, 20.4 µmol) in 51% yield:
1H NMR (D2O, 400 MHz) δ ) 5.22 (d, J1,2 ) 3.7 Hz, 0.4H, H-1
(2,3,4,6-Tetra-O-acetyl-â-D-galactopyranosyl)-(1f4)-(2,3,6-
tri-O-acetyl-â-D-glucopyranosyl)-(1f4)-[(2,3,4-tri-O-acetyl-R-D-
xylopyranosyl)-(1f6)]-(2,3-di-O-acetyl-â-D-glucopyranosyl)-
(1f4)-[(2,3,4-tri-O-acetyl-R-D-xylopyranosyl)-(1f6)]-(2,3-di-O-
acetyl-â-D-glucopyranosyl)-(1f4)-[(2,3,4-tri-O-acetyl-R-D-
xylopyranosyl)-(1f6)]-(2,3-di-O-acetyl-â-D-glucopyranosyl)-
(1f4)-2,3,6-tri-O-acetyl-R-D-glucopyranosyl Fluoride (18). H.
insolens Cel7B E197A glycosynthase (5 mg) was added to a
solution of lactosyl fluoride 12 (0.31 g, 0.89 mmol, 1.2 equiv) and
heptasaccharide 2 (0.79 g, 0.74 mmol) in carbonate/bicarbonate
buffer (0.1 m, pH 10, 15 mL). The solution was placed in a rotative
shaker for 20 h at 37 °C and then freeze-dried. The residue was
acetylated (acetic anhydride/pyridine, 1:2 v/v, 75 mL) in the
presence of a catalytic amount of DMAP. After 12 h of stirring at
rt, the reaction was quenched by addition of MeOH at 0 °C,
of GlcIR), 4.95 (d, J1,2 ) 3.7 Hz, 3H, H-1 of Xyl), 4.66 (d, J1,2
)
7.9 Hz, 0.6H, H-1 of GlcIâ), 4.58-4.54 (m, 3H, H-1 of GlcII,III,IV),
4.51 (d, J1,2 ) 7.8 Hz, 1H, H-1 of GlcV); MALDI-TOF-MS m/z )
1247 [M + Na]+; ES-HRMS m/z calcd for C45H76O38Na [M +
Na]+ 1247.3912, found 1247.3910.
(2,3,4,6-Tetra-O-acetyl-â-D-galactopyranosyl)-(1f4)-(2,3,6-
tri-O-acetyl-â-D-glucopyranosyl)-(1f4)-[(2,3,4-tri-O-acetyl-R-D-
xylopyranosyl)-(1f6)]-(2,3-di-O-acetyl-â-D-glucopyranosyl)-
5158 J. Org. Chem., Vol. 71, No. 14, 2006

ActiVe-Site Mapping of Xyloglucan endo-Transglycosylase
concentrated under reduced pressure, and coevaporated with toluene.
Flash chromatography (toluene/acetone, 4:1 v/v) of the residue gave
the expected acetylated nonasaccharide (1.25 g, 0.50 mmol) in 68%
yield, characterized by MALDI-TOF-MS: m/z ) 2501 [M + Na]+.
The acetylated nonasaccharide was processed to give fluoride
18, as already described for 17. C-1 hydroxy compound was
obtained from acetylated nonasaccharide (820 mg, 335 µmol) and
purified by flash chromatography (toluene/acetone, 3:1 v/v) to give
the expected mixture of free anomers (597 mg, 245 µmol; MALDI-
TOF-MS m/z ) 2459 [M + Na]+) in 73% yield. These hydroxy
compounds (464 mg, 190 µmol) were fluorinated, and the expected
R/â-fluorides (443 mg, 182 µmol; MALDI-TOF-MS m/z ) 2461
[M + Na]+) were obtained in 95% yield, after flash chromatography
(cyclohexane/acetone, 3:2 v/v). Anomerization of fluorides (477
mg, 195 µmol) gave, after flash chromatography (cyclohexane/
acetone, 4:1, 3:1, 2:1, 3:2 then 55-45 v/v), the fluoride 18 (381
C63H106O53Na [M + Na]+ 1733.5497, found 1733.5498; LC/ES-
MS analysis gave m/z 1882 [M(ABEE) + Na]+; tR ) 16.09 min.
â-D-Glucopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-â-D-
glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-glucopy-
ranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-glucopyranosyl-
(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-D-
glucopyranose (GGGXXXG 22). O-THP-cellobiosyl fluoride 19
(2.7 mg, 6.4 µmol, 1.3 equiv) and octasaccharide 16 (6.0 mg, 4.9
µmol) in carbonate/bicarbonate buffer (0.1 m, pH 10, 1 mL) were
incubated with H. insolens Cel7B E197A glycosynthase (1 mg)
for 24 h at 37 °C. The solution was diluted with water (4 mL), and
HCl (1 M) was added to lower the pH to 1-2. After 5 min, the
solution was neutralized with Et3N. The process was repeated twice
until complete hydrolysis of the tetrahydropyranyl group. Silica gel
was added, and the suspension was evaporated. The powder was
deposited at the top of a silica gel column and a gradient (acetonitrile
then acetonitrile/water, 95:5, 9:1, 85:15, 8:2 and 75:25 v/v) was
used to elute the decasaccharide 22 (5.7 mg, 3.7 µmol) in 76%
yield: MALDI-TOF-MS m/z ) 1571 [M + Na]+; ES-HRMS m/z
calcd for C57H96O48Na [M + Na]+ 1571.4969, found 1571.4978.
R-D-Xylopyranosyl-(1f6)-â-D-glucopyranosyl-(1f4)-â-D-glu-
copyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-glucopyra-
nosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-glucopyranosyl-
(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-D-
glucopyranose (XGXXXG 23). The peracetylated fluoride 14 (105
mg, 73 µmol) was treated in methanol (10 mL) with sodium
methoxide (1 M in methanol, 37 µL) for 5 h at 0 °C. Sodium
methoxide (37 µL) was added again, and the reaction mixture was
stirred for another 12 h at 0 °C. The solution was neutralized with
Amberlite IRN 120 (H+), filtered, and concentrated under reduced
pressure. The residue dissolved in deionized water was freeze-dried
to give the expected pentasaccharide (56 mg, 69 µmol) in 95%
yield, characterized by FAB-MS m/z ) 823 [M + Na]+.
To this free fluoride (13.2 mg, 16.5 µmol, 1.1 equiv) were added
heptasaccharide 2 (15.9 mg, 15.0 µmol) in phosphate buffer (0.1
m, pH 7, 1 mL) and then H. insolens Cel7B E197A glycosynthase
(2 mg). The solution was placed in a rotative shaker for 20 h at 37
°C, and then H. insolens Cel7B E197A glycosynthase (0.5 mg)
was added again. After 12 h at 37 °C, recombinant â-glucosidase/
galactosidase (Agrobacterium sp., 0.2 mg) was added. The reaction
mixture was kept at 37 °C for 24 h, and another quantity of
â-glucosidase/galactosidase (0.1 mg) was added. After 20 h at 37
°C, the solution was diluted with acetonitrile/water (1:1 v/v, 10
mL), silica gel was added, and the suspension was evaporated. The
powder was deposited at the top of a silica gel column, and a
gradient (acetonitrile then acetonitrile/water, 95:5, 9:1, 85:15, 8:2
and 75:25 v/v) was used to elute the decasaccharide 23 (15.0 mg,
9.9 µmol) in 66% yield: MALDI-TOF-MS m/z ) 1541 [M + Na]+;
ES-HRMS m/z calcd for C56H94O47Na [M + Na]+ 1541.4863, found
1541.4857; LC/ES-MS analysis gave m/z 1690 [M(ABEE) + Na]+;
tR ) 16.47 min.
mg, 156 µmol) in 80% yield: [R]25 ) +30 (c ) 1.0 in CHCl3);
D
1H NMR (CDCl3, 400 MHz) δ 5.59 (dd, J1,2 ) 2.6 and JH,F ) 53.1
Hz, 1H, H-1 of GlcI), 5.01, 4.97 (m, H-1 of Xyl), 4.82, 4.76, 4.69
(m, H-1 of GlcIII,IV,V), 4.50 (d, J1,2 ) 8.0 Hz, 1H, H-1 of GlcII),
4.36 (d, J1,2 ) 7.9 Hz, 1H, H-1 of Gal), 2.09-1.91 (m, 75H,
COCH3); 13C NMR (CDCl3, 100 MHz) δ 170.6-168.8 (COCH3),
103.6 (d, JC,F ) 229.5 Hz, C-1 of GlcI), 101.4 (C-1 of Gal), 100.6,
100.4, 100.2, 100.0 (C-1 of GlcII,III,IV,V), 97.1, 97.1, 96.8 (C-1 of
Xyl), 75.9, 75.6, 75.3, 75.2, 74.6, 74.5 (broad), 73.2, 73.0, 72.7,
72.5, 72.4, 72.0, 72.0, 71.9, 71.5, 71.1, 70.6, 70.5, 70.5, 70.4, 70.4,
70.3, 70.3, 70.2, 69.4, 69.1, 69.1, 69.0, 68.9, 68.8, 68.5, 66.5 (C-2
to C-5 of Glc and Gal, and C-2 to C-4 of Xyl), 65.6, 65.4, 65.2
(C-6 of GlcII,III,IV), 62.1, 61.2, 60.7 (C-6 of GlcI,V and Gal), 59.0,
58.9, 58.9 (C-5 of Xyl), 20.9-20.2 (COCH3); MALDI-TOF-MS
m/z ) 2461 [M + Na]+; ES-HRMS m/z calcd for C101H135O67FNa
[M + Na]+ 2461.7038, found 2461.7006.
â-D-Glucopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-[R-D-
xylopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-xylopyra-
nosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-xylopyranosyl-
(1f6)]-â-D-glucopyranosyl-(1f4)-D-glucopyranose (GGXXXG
20). O-THP-cellobiosyl fluoride 19 (26.2 mg, 61.2 µmol, 1.3 equiv)
and heptasaccharide 2 (50.0 mg, 47.0 µmol) in carbonate/bicarbon-
ate buffer (0.1 m, pH 10, 1.5 mL) were incubated with H. insolens
Cel7B E197A glycosynthase (2 mg) for 20 h at 37 °C. The solution
was diluted with water (3.5 mL), and HCl (1M) was added to lower
the pH to 1-2. After 5 mn, the solution was neutralized with Et3N.
The process was repeated twice until complete hydrolysis of the
tetrahydropyranyl group. Silica gel was added and the suspension
was evaporated. The powder was deposited at the top of a silica
gel column and a gradient (acetonitrile then acetonitrile/water, 95:
5, 9:1, 85:15, 8:2 and 75:25 v/v) was used to elute the nonasac-
charide 20 (50.6 mg, 36.5 µmol) in 78% yield: MALDI-TOF-MS
m/z ) 1409 [M + Na]+; ES-HRMS m/z calcd for C51H86O43Na
[M + Na]+ 1409.4441, found 1409.4482; LC/ES-MS analysis gave
m/z 1558 [M(ABEE) + Na]+; tR ) 16.47 min.
â-D-Glucopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-â-D-
glucopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-[R-D-xylopy-
ranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-xylopyranosyl-
(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-
â-D-glucopyranosyl-(1f4)-D-glucopyranose (GGGGXXXG 21).
O-THP-cellobiosyl fluoride 19 (5.8 mg, 13.6 µmol, 1.3 equiv) and
nonasaccharide 20 (14.5 mg, 10.5 µmol) in carbonate/bicarbonate
buffer (0.1 m, pH 10, 1.5 mL) were incubated with H. insolens
Cel7B E197A glycosynthase (1 mg) for 20 h at 37 °C. The solution
was diluted with water (3.5 mL), and HCl (1 M) was added to
lower the pH to 1-2. After 5 min, the solution was neutralized
with Et3N. The process was repeated twice until complete hydrolysis
of the tetrahydropyranyl group. Silica gel was added, and the
suspension was evaporated. The powder was deposited at the top
of a silica gel column, and a gradient (acetonitrile then acetonitrile/
water, 95:5, 9:1, 85:15, 8:2 and 75:25 v/v) was used to elute the
undecasaccharide 21 (15.3 mg, 8.9 µmol) in 86% yield: MALDI-
TOF-MS m/z ) 1733 [M + Na]+; ES-HRMS m/z calcd for
R-D-Xylopyranosyl-(1f6)-â-D-glucopyranosyl-(1f4)-â-D-glu-
copyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-glucopyra-
nosyl-(1f4)-â-D-glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-
â-D-glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-
glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-
glucopyranosyl-(1f4)-D-glucopyranose (XGXGXXXG 24). A
mixture of deacetylated fluoride (5.2 mg, 6.4 µmol, 1.3 equiv)
obtained from 14 as described for the preparation of 23, and
decasaccharide 23 (7.5 mg, 4.9 µmol) in phosphate buffer (0.1 m,
pH 7, 1 mL) was incubated with H. insolens Cel7B E197A
glycosynthase (1.5 mg). The solution was placed in a rotative shaker
for 20 h at 37 °C, and recombinant â-glucosidase/galactosidase
(Agrobacterium sp., 0.2 mg) was added. After 24 h at 37 °C, another
quantity of â-glucosidase/galactosidase (0.1 mg) was added. After
20 h at 37 °C, the solution was diluted with acetonitrile/water (1:1
v/v, 10 mL), silica gel was added, and the suspension was
evaporated. The powder was deposited at the top of a silica gel
column, and a gradient (acetonitrile then acetonitrile/water, 95:5,
J. Org. Chem, Vol. 71, No. 14, 2006 5159

Faure´ et al.
9:1, 85:15, 8:2 and 75:25 v/v) was used to elute the expected
compound 24 (8.5 mg, 4.3 µmol) in 87% yield: MALDI-TOF-
MS m/z ) 1997 [M + Na]+; ES-HRMS m/z calcd for C73H122O61-
Na [M + Na]+ 1997.6342, found 1997.6124; LC/ES-MS analysis
gave m/z 2146 [M(ABEE) + Na]+; tR ) 15.82 min.
5, 9:1, 85:15, 8:2, 75:25 and 7:3 v/v) was used to elute the lactosyl
protected compound, which was freeze-dried. A solution of the
residue in acetate buffer (0.1 m, pH 4.6, 2 mL) was treated by
commercially available â-galactosidase (A. oryzae, 3 mg, 24 units)
for 2.5 h at 30 °C. The reaction mixture was diluted in acetonitrile/
water (1:1 v/v, 10 mL), silica gel was added, and the suspension
was evaporated. The powder was deposited at the top of a silica
gel column and a gradient (acetonitrile then acetonitrile/water, 95:
5, 9:1, 85:15, 8:2, and 75:25 v/v) was used to elute the expected
compound 27 (9.4 mg, 4.8 µmol) in 35% yield: MALDI-TOF-
MS m/z ) 1997 [M + Na]+; ES-HRMS m/z calcd for C73H122O61-
Na [M + Na]+ 1997.6342, found 1997.6304; LC/ES-MS analysis
gave m/z 2146 [M(ABEE) + Na]+; tR ) 15.83 min.
R-D-Xylopyranosyl-(1f6)-â-D-glucopyranosyl-(1f4)-[R-D-xy-
lopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-xylopyrano-
syl-(1f6)]-â-D-glucopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-
â-D-glucopyranosyl-(1f4)-D-glucopyranose (XXXGGG 31).
Compound 18 (200 mg, 82 µmol) was de-O-acetylated as already
described for 14 to give the corresponding free fluoride (114 mg)
in quantitative yield, characterized by MALDI-TOF-MS m/z ) 1411
[M + Na]+.
R-D-Xylopyranosyl-(1f6)-â-D-glucopyranosyl-(1f4)-â-D-glu-
copyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-[R-D-xylopyrano-
syl-(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-
â-D-glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-
glucopyranosyl-(1f4)-D-glucopyranose (XGGXXXG 25). A
mixture of deacetylated fluoride (10.8 mg, 13.5 µmol, 1.1 equiv)
obtained from 14 as described for the preparation of 23 and
octasaccharide 16 (15.0 mg, 12.2 µmol) in phosphate buffer (0.1
m, pH 7, 1 mL) was incubated with H. insolens Cel7B E197A
glycosynthase (2 mg). The solution was placed in a rotative shaker
for 14 h at 37 °C, and then recombinant â-glucosidase/galactosidase
(Agrobacterium sp., 0.2 mg) was added. The reaction mixture was
kept at 37 °C for 24 h, and another quantity of â-glucosidase/
galactosidase (0.1 mg) was added. After 24 h at 37 °C, the solution
was diluted with acetonitrile/water (1:1 v/v, 10 mL), silica gel was
added, and the suspension was evaporated. The powder was
deposited at the top of a silica gel column, and a gradient
(acetonitrile then acetonitrile/water, 95:5, 9:1, 85:15, 8:2, 75:25,
and 7:3 v/v) was used to elute the undecasaccharide 25 (15.9 mg,
9.8 µmol) in 80% yield: MALDI-TOF-MS m/z ) 1703 [M + Na]+;
ES-HRMS m/z calcd for C62H104O52Na [M + Na]+ 1703.5391,
found 1703.5394; LC/ES-MS analysis gave m/z 1852 [M(ABEE)
+ Na]+; tR ) 16.08 min.
R-D-Xylopyranosyl-(1f6)-â-D-glucopyranosyl-(1f4)-[R-D-xy-
lopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-â-D-glucopyrano-
syl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-
[R-D-xylopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-
xylopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-D-
glucopyranose (XXGXXXG 26). Compound 17 (117 mg, 60
µmol) was de-O-acetylated as already described for 14 to give the
corresponding free fluoride (61 mg, 56 µmol) in 92% yield,
characterized by MALDI-TOF-MS m/z ) 1117 [M + Na]+.
To this deprotected fluoride (15.0 mg, 13.7 µmol) were added
heptasaccharide 2 (16.0 mg, 15.1 µmol, 1.1 equiv) in phosphate
buffer (0.1 m, pH 7, 1.5 mL) and then H. insolens Cel7B E197A
glycosynthase (3 mg). The solution was placed in a rotative shaker
for 12 h at 37 °C, and then recombinant â-glucosidase/galactosidase
(Agrobacterium sp., 0.2 mg) was added. The reaction mixture was
kept at 37 °C for 24 h, and another quantity of â-glucosidase/
galactosidase (0.1 mg) was added. After 24 h at 37 °C, the solution
was diluted with acetonitrile/water (1:1 v/v, 10 mL), silica gel was
added, and the suspension was evaporated. The powder was
deposited at the top of a silica gel column and a gradient (acetonitrile
then acetonitrile/water, 95:5, 9:1, 85:15, 8:2 and 75:25 v/v) was
used to elute the undecasaccharide 26 (19.7 mg, 10.9 µmol) in 79%
yield: MALDI-TOF-MS m/z ) 1835 [M + Na]+; ES-HRMS m/z
calcd for C67H112O56Na [M + Na]+ 1835.5814, found 1835.5812;
LC/ES-MS analysis gave m/z 1984 [M(ABEE) + Na]+; tR ) 16.07
min.
To this deprotected fluoride (20.8 mg, 15.0 µmol) were added
cellobiose 28 (6.7 mg, 19.5 µmol, 1.3 equiv) in carbonate/
bicarbonate buffer (0.1 m, pH 10, 1 mL) and then H. insolens Cel7B
E197A glycosynthase (2 mg). The solution was placed in a rotative
shaker for 12 h at 37 °C, and then the solution was diluted with
acetonitrile/water (1:1 v/v, 10 mL), silica gel was added, and the
suspension was evaporated. The powder was deposited at the top
of a silica gel column, and a gradient (acetonitrile then acetonitrile/
water, 95:5, 9:1, 85:15, 8:2, 75:25, and 7:3 v/v) was used to eluted
the expected lactosyl protected nonasaccharide (19.1 mg, 11.1 µmol;
MALDI-TOF-MS m/z ) 1733 [M + Na]+; ES-HRMS m/z calcd
for C63H106O53Na [M + Na]+ 1733.5497, found 1733.5525; LC/
ES-MS analysis gave m/z 1882 [M(ABEE)+ + Na]; tR ) 16.29
min) in 74% yield. A solution of this undecasaccharide (17.4 mg,
10.2 µmol) in phosphate buffer (0.1 m, pH 6.8, 2 mL) was treated
at 37 °C by successive additions of recombinant â-glucosidase/
galactosidase (Agrobacterium sp., 1 mg) over 1 week. The solution
was diluted with acetonitrile/water (1:1 v/v, 10 mL), silica gel was
added, and the suspension was evaporated. The powder was
deposited at the top of a silica gel column and a gradient (acetonitrile
then acetonitrile/water, 95:5, 9:1, 85:15, 8:2 and 75:25 v/v) was
used to elute the nonasaccharide 31 (10.5 mg, 7.6 µmol) in 75%
yield: MALDI-TOF-MS m/z ) 1409 [M + Na]+; ES-HRMS m/z
calcd for C51H86O43Na [M + Na]+ 1409.4441, found 1409.4439;
LC/ES-MS analysis gave m/z 1558 [M(ABEE) + 2Na]2+; tR
16.81 min.
)
R-D-Xylopyranosyl-(1f6)-â-D-glucopyranosyl-(1f4)-[R-D-xy-
lopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-xylopyrano-
syl-(1f6)]-â-D-glucopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-
â-D-glucopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-â-D-
glucopyranosyl-(1f4)-D-glucopyranose (XXXGGGGG 32). A
mixture of deacetylated fluoride (20.8 mg, 15.0 µmol) obtained
from 18 as described for the preparation of 31, and cellotetraose
29 (13.0 mg, 19.5 µmol, 1.3 equiv) in phosphate buffer (0.1 m, pH
7, 1.5 mL) were incubated with H. insolens Cel7B E197A
glycosynthase (3 mg). The solution was placed in a rotative shaker
for 12 h at 37 °C, and then recombinant â-glucosidase/galactosidase
(Agrobacterium sp., 0.2 mg) was added. The reaction mixture was
kept at 37 °C for 24 h, and another quantity of â-glucosidase/
galactosidase (0.1 mg) was added. After 20 h at 37 °C, the solution
was diluted with acetonitrile/water (1:1 v/v, 10 mL), silica gel was
added, and the suspension was evaporated. The powder was
deposited at the top of a silica gel column, and a gradient
(acetonitrile then acetonitrile/water, 95:5, 9:1, 85:15, 8:2, and 75:
25 v/v) was used to elute the undecasaccharide 32 (12.3 mg, 7.2
µmol) in 48% yield: MALDI-TOF-MS m/z ) 1733 [M + Na]+;
ES-HRMS m/z calcd for C63H106O53Na [M + Na]+ 1733.5497,
â-D-Glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-
glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-glucopy-
ranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-[R-D-xylopyranosyl-
(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-
â-D-glucopyranosyl-(1f4)-[R-D-xylopyranosyl-(1f6)]-â-D-
glucopyranosyl-(1f4)-D-glucopyranose (GXXGXXXG 27). A
mixture of deacetylated fluoride (15.0 mg, 13.7 µmol) obtained
from 17 as described for the preparation of 26 and heptasaccharide
2 (16.0 mg, 15.1 µmol, 1.1 equiv) in phosphate buffer (0.1 m, pH
7, 1.5 mL) was incubated with H. insolens Cel7B E197A glyco-
synthase (3 mg). The solution was placed in a rotative shaker for
12 h at 37 °C, and then the solution was diluted with acetonitrile/
water (1:1 v/v, 10 mL), silica gel was added, and the suspension
was evaporated. The powder was deposited at the top of a silica
gel column, and a gradient (acetonitrile then acetonitrile/water, 95:
5160 J. Org. Chem., Vol. 71, No. 14, 2006

ActiVe-Site Mapping of Xyloglucan endo-Transglycosylase
found 1733.5411; LC/ES-MS analysis gave m/z 1882 [M(ABEE)
+ Na]+; tR ) 16.33 min.
constant at I ) 0.5 M with added KCl. Donor (1 mM) and acceptor
(7 mM) were dissolved and enzyme added to a final concentration
of 0.5-2 µM. Reactions were performed in a final volume of 100
µL in a thermostated bath at 30 °C. Aliquots (20 µL) were
withdrawn at different time intervals and mixed with 20 µL of
ManANTS 2 mM in water as internal reference, the mixture was
heated at 100 °C for 10 min in a sealed tube, and finally samples
were analyzed by HPCE.35
Capillary electrophoresis was performed on a system equipped
with a diode array UV-vis detector, using a fused silica capillary,
72 cm effective length, 50 µm internal diameter with an extended
light path bubble (150 µm) in the detection window. Samples
(ANTS-labeled oligosaccharide standards or enzymatic reaction
mixtures) were loaded into the capillary under hydrodynamic
injection mode at 40 mbar pressure during 6 s. Electrophoretic
separations were performed at -30 kV and constant temperature
of 30 °C under inverted EOF conditions (anodic detection) using
50 mM phosphoric acid adjusted to pH 2.5 with triethylamine as
running buffer. Electropherograms were recorded at 270 nm (20
nm slit).
R-D-Xylopyranosyl-(1f6)-â-D-glucopyranosyl-(1f4)-[R-D-xy-
lopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-xylopyrano-
syl-(1f6)]-â-D-glucopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-
[R-D-xylopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-D-
glucopyranose (XXXGXG 33). A mixture of deacetylated fluoride
(21.0 mg, 15.1 µmol) obtained from 18 as described for the
preparation of 31 and trisaccharide 30 (7.9 mg, 16.6 µmol, 1.1
equiv) in phosphate buffer (0.1 m, pH 7, 1.5 mL) was incubated
with H. insolens Cel7B E197A glycosynthase (3 mg). The solution
was placed in a rotative shaker for 20 h at 37 °C, and then H.
insolens Cel7B E197A glycosynthase (0.5 mg) was added again.
After 12 h at 37 °C, recombinant â-glucosidase/galactosidase (Agro-
bacterium sp., 0.2 mg) was added. The reaction mixture was kept
at 37 °C for 24 h, and another quantity of â-glucosidase/galac-
tosidase (0.2 mg) was added. After 20 h at 37 °C, the solution was
diluted with acetonitrile/water (1:1 v/v, 10 mL), silica gel was
added, and the suspension was evaporated. The powder was depos-
ited at the top of a silica gel column and a gradient (acetonitrile then
acetonitrile/water, 95:5, 9:1, 85:15, 8:2 and 75:25 v/v) was used to
elute the decasaccharide 33 (14.4 mg, 9.5 µmol) in 63% yield:
MALDI-TOF-MS m/z ) 1541 [M + Na]+; ES-HRMS m/z calcd
for C56H94O47Na [M + Na]+ 1541.4863, found 1541.4841; LC/
ES-MS analysis gave m/z 1690 [M(ABEE) + Na]+; tR ) 16.49 min.
R-D-Xylopyranosyl-(1f6)-â-D-glucopyranosyl-(1f4)-[R-D-xy-
lopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-xylopyrano-
syl-(1f6)]-â-D-glucopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-
[R-D-xylopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-[R-D-
xylopyranosyl-(1f6)]-â-D-glucopyranosyl-(1f4)-D-
glucopyranose (XXXGXXG 34). A mixture of deacetylated
fluoride (21.0 mg, 15.1 µmol) obtained from 18 as described for
the preparation of 31 and pentasaccharide 1 (12.8 mg, 16.6 µmol,
1.1 equiv) in phosphate buffer (0.1 m, pH 7, 1.5 mL) was incubated
with H. insolens Cel7B E197A glycosynthase (3 mg). The solution
was placed in a rotative shaker for 20 h at 37 °C, and then H.
insolens Cel7B E197A glycosynthase (1 mg) was added again. After
12 h at 37 °C, recombinant â-glucosidase/galactosidase (Agrobac-
terium sp., 0.2 mg) was added. After 48 h at 37 °C, the solution
was diluted with acetonitrile/water (1:1 v/v, 10 mL), silica gel was
added, and the suspension was evaporated. The powder was
deposited at the top of a silica gel column, and a gradient
(acetonitrile then acetonitrile/water, 95:5, 9:1, 85:15, 8:2, 75:25 and
7:3 v/v) was used to elute the dodecasaccharide 34 (17.7 mg, 9.8
µmol) in 65% yield: MALDI-TOF-MS m/z ) 1835 [M + Na]+;
ES-HRMS m/z calcd for C67H112O56Na [M + Na]+: 1835.5814,
found 1835.5816; LC/ES-MS analysis gave m/z 1984 [M(ABEE)
+ Na]+; tR ) 16.25 min.
Product concentrations from the electropherograms were deter-
mined from relative peak areas using ManANTS as internal
reference. Initial rates in product formation (V0 in mM/min) were
calculated as the slope of the linear region of the time course (0-
20 min) corresponding to < 10% substrate to product conversion.
All kinetics were done in duplicate, and standard deviations in V0/
[E] were e 5%.
Acknowledgment. This work was supported in part by EU
contract QLK5-CT-2001-00443. We thank Prof. S. G. Withers
(Department of Chemistry, University of British Columbia,
Vancouver, Canada) for the generous gift of â-glucosidase/
galactosidase from Agrobacterium sp. We also thank Dr. K.
Piens (School of Biotechnology, Royal Institute of Technology,
Stockholm, Sweden) for preparation of Populus xyloglucan
endo-transglycosylase 16A and for helpful discussions. We
thank Ste´phanie Boullanger for her skillful technical assistance.
Dr. L. Greffe (School of Biotechnology, Royal Institute of
Technology, Stockholm, Sweden) is also acknowledged for his
contribution to this work.
Supporting Information Available: 1H NMR data for com-
pounds 9, 10, 13, 14, and 17; 1H and 13C NMR spectra for
compounds 7-10, 13, 14, 16-18, 26; quantitative HPLC-ESMS
for ABEE derivatives of compounds 15, 20-27, 31-34.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Enzyme Kinetics. All enzymatic reactions were done in 50 mM
citrate-50 mM phosphate buffer, pH 5.5. Ionic strength was kept
JO0525682
J. Org. Chem, Vol. 71, No. 14, 2006 5161