Highly efficient synthesis of β(1 → 4)-oligo- and -polysaccharides using a mutant cellulase

Article abstract of DOI:10.1021/ja9936520

This report describes an efficient chemoenzymatic synthesis of a variety of regioselectively modified β(1→4)-oligo, and -polysaccharides. This successful approach was based on: (i) the use of a 'glycosynthase' which is a Glu-197-Ala nucleophile mutant of the retaining cellulase endoglucanase I (Cel7B) from Humicola insolens and (ii) the rational design of modified acceptor and donor molecules through a careful examination of information given by the X-ray structures of wild type and mutated enzymes. The mutant was able to catalyze, in high yield, the regio- and stereoselective glycosylation of α-glycobiosyl fluorides both unsubstituted and modified with various mono- and disaccharide acceptors, as well as the polymerization of these donors through a single-step inverting mechanism.

Full text of DOI:10.1021/ja9936520

J. Am. Chem. Soc. 2000, 122, 5429-5437
5429
Highly Efficient Synthesis of â(1 f 4)-Oligo- and -Polysaccharides
Using a Mutant Cellulase
Se´bastien Fort,† Viviane Boyer,† Lionel Greffe,† Gideon J. Davies,‡ Olga Moroz,‡
Lars Christiansen,§ Martin Schu1lein,§ Sylvain Cottaz,† and Hugues Driguez*,†
Contribution from the Centre de Recherches sur les Macromole´cules Ve´ge´tales, CNRS, BP 53, 38041
Grenoble Cedex 9, France, Structural Biology Laboratory, Department of Chemistry, UniVersity of York,
Heslington, York, Y010 5DD, UK, and NoVo-Nordisk A/S, NoVo alle´, DK-2880 BagVaerd, Denmark
ReceiVed October 12, 1999
Abstract: This report describes an efficient chemoenzymatic synthesis of a variety of regioselectively modified
â(1f4)-oligo- and -polysaccharides. This successful approach was based on: (i) the use of a “glycosynthase”
which is a Glu-197-Ala nucleophile mutant of the retaining cellulase endoglucanase I (Cel7B) from Humicola
insolens and (ii) the rational design of modified acceptor and donor molecules through a careful examination
of information given by the X-ray structures of wild type and mutated enzymes. The mutant was able to
catalyze, in high yield, the regio- and stereoselective glycosylation of R-glycobiosyl fluorides both unsubstituted
and modified with various mono- and disaccharide acceptors, as well as the polymerization of these donors
through a single-step inverting mechanism.
Introduction
Very recently, a new and highly efficient approach based on
protein engineering of retaining â-glucosidase has been devel-
oped by Withers and co-workers.4 As expected, a glycoside
hydrolase in which the carboxylate nucleophile was replaced
by alanine was unable to form the glycosyl enzyme intermediate
and had no hydrolytic activity. However, a mimic of this
intermediate, an R-glycosyl fluoride, was recognized and used
for transglycosylation reactions, with the advantage that the
reaction products cannot be hydrolyzed and were accumulated
almost quantitatively. This original concept was also applied
successfully by Planas and Malet to an endo-glycosidase, the
1,3-1,4-â-glucanase from Bacillus licheniformis, which gave tri-
and tetrasaccharides in very high yield.5
To overcome the problems related to chemical syntheses of
cellooligosaccharides and cellulose having â(1f4)-glycosidic
linkages, Kobayashi and co-workers developed a transglyco-
sylation approach using â-cellobiosyl or â-lactosyl fluorides,
catalyzed by partially purified cellulases in hydroorganic
medium.1a-c,g We also applied the same methodology for the
preparation of hemithiocellodextrins6 and for the synthesis of a
bifunctionalized fluorogenic tetrasaccharide.3 In the most recent
example, the enzymatic condensation was achieved by a
recombinant endocellulase Cel7B (formerly called endogluca-
nase I) from the fungus Humicola insolens, an enzyme which
was known to have high transglycosylation activity.7 In a
mixture of acetonitrile and maleate buffer, condensation of
â-lactosyl fluoride onto a cellobiosyl acceptor gave the expected
tetrasaccharide in 60% yield. The same strategy was, however,
unsuccessful for the synthesis of higher oligomers, when
The use of enzymes is a theoretically straightforward ap-
proach for the formation of glycosidic bonds in oligo- and
polysaccharides, and the potential of glycoside hydrolases as
tools for the synthesis of glycosides has been realized in many
laboratories.1 These enzymes as well as the requisite donor
substrates (aryl glycosides, glycosyl fluorides, or disaccharides)
are readily available and inexpensive. Retaining glycosidases,
which hydrolyze glycosides through a glycosyl-enzyme inter-
mediate which is formed by nucleophilic attack of one catalytic
amino acid (the nucleophile) onto the anomeric carbon of the
donors, are known to be effective for oligosaccharide synthesis.
New oligosaccharides can be formed when a sugar competes
with water as acceptor during the deglycosylation step.2 The
transglycosylation catalyzed by endo-enzymes is always highly
regioselective, and under appropriate conditions (organic co-
solvent and pH) oligosaccharides are isolated in good yields
(up to 60%).3
† Centre de Recherches sur les Macromole´cules Ve´ge´tales.
‡ University of York.
§ Novo-Nordisk A/S.
(1) (a) Kobayashi, S.; Kashiwa, K.; Kawasaki, T.; Shoda, S-i. J. Am.
Chem. Soc. 1991, 113, 3079-3084. (b) Shoda, S-i.; Kawasaki, T.; Obata,
K.; Kobayashi, S. Carbohydr. Res. 1993, 249, 127-137. (c) Karthaus, O.;
Shoda, S-i.; Takano, H.; Obata, K.; Kobayashi, S. J. Chem. Soc., Perkin
Trans 1 1994, 1851-1857. (d) Singh, S.; Scigelova, M.; Crout, D. H. G.
Chem. Commun. 1996, 993-994. (e) Kobayashi, S.; Kiyosada, T.; Shoda,
S-i. J. Am. Chem. Soc. 1996, 118, 13113-13114. (f) Murata, T.; Tashiro,
A.; Itoh, T.; Usui, T. Biochim. Biophys. Acta 1997, 1335, 326-334. (g)
Okamoto, E.; Kiyosada, T.; Shoda, S-i.; Kobayashi, S. Cellulose 1997, 4,
161-172. (h) Vic, G.; Tran, C. H.; Schigelova, M.; Crout, D. H. G. Chem.
Commun. 1997, 169-170. (i) Viladot, J.-L.; Moreau, V.; Planas, A.;
Driguez, H. J. Chem. Soc., Perkin Trans 1 1997, 2383-2387. (j) Hrmova,
M.; Fincher, G. B.; Viladot, J. L.; Planas, A. Driguez, H. ibid. 1998, 3571-
3576. (k) Fujita, M.; Shoda, S-i; Kobayashi, S. J. Am. Chem. Soc. 1998,
120, 6411-6412. (l) Li, J.; Robertson, D. E.; Short, J. M., Wang, P. B.
Biorg. Med. Chem. 1999, 9, 35-38. (m) Macmanus, D. A.; Grabowska,
U.; Biggadike, K.; Bird, M. I.; Davies, S.; Vulfson, E. N.; Gallagher, T. J.
Chem. Soc., Perkin Trans 1 1999, 295-305.
(3) Armand, S.; Drouillard, S.; Schu¨lein, M.; Henrissat, B.; Driguez, H.
J. Biol. Chem. 1997, 272, 2709-2713.
(4) Mackenzie, L. F.; Wang, Q.; Warren, R. A. J.; Withers, S. G. J. Am.
Chem. Soc. 1998, 120, 5583-5584.
(5) Malet, C.; Planas, A. FEBS Lett. 1998, 440, 208-212.
(6) Moreau, V.; Driguez, H. J. Chem. Soc., Perkin Trans 1 1996, 525-
527.
(7) Schou, C.; Ramussen, G.; Kaltoft, M.-B.; Henrissat, B.; Schu¨lein,
M. Eur. J. Biochem. 1993, 217, 947-953.
(2) Sinnott, M. L. Chem. ReV. 1990, 90, 1171-1202.
10.1021/ja9936520 CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/23/2000

5430 J. Am. Chem. Soc., Vol. 122, No. 23, 2000
Fort et al.
Figure 1. Stereo representation of the observed electron density around the Glu197Ala mutation. The refined coordinates of the mutant structure
are shown in bold, and the overlapped wild-type coordinates, in faint lines. The two structures are almost identical except for the inclusion of two
solvent molecules in the space provided by the Glu197Ala mutation. The map shown is a maximum likelihood and σAweighted 2Fobs - Fcalc
synthesis contoured at 0.44 e/Å3.
cellotrioside was used as acceptor. This molecule competes with
lactosyl fluoride as a donor and a cellobiosyl-enzyme intermedi-
ate was formed which led to an untractable mixture of products
with both nonreducing end galactosyl or glucosyl units (un-
published results).
To overcome these problems we have developed a “glyco-
synthase” mutant of Cel7B. The Glu-197-Ala mutant has been
expressed and purified and its X-ray structure determined at
1.75 Å resolution. The Cel7B glycosynthase is a highly efficient
catalyst for the synthesis of novel â(1 f 4)-oligo- and
-polysaccharides.
) 59.87 Å, and â ) 103.81°. Synchrotron data collected from
a single crystal at 100 K have an overall Rmerge (Rmerge ) Σhkl
Σi|Ihkli - Ihkl |/ΣhklΣi Ihkl ) of 0.075, a mean I/σI of 19.7 and a
mean multiplicity of 4.9 observations/reflection. The data are
complete. In the outer resolution shell (1.81-1.75 Å) the
corresponding values are Rmerge 0.45, I/σI 3.3, multiplicity 4.8,
and are again 100% complete. Structure solution by molecular
replacement, using the native H. insolens Cel7B structure, was
trivial and maximum likelihood refinement proceeded smoothly.
The final model coordinates have a crystallographic R-factor
of 0.16, with an Rfree of 0.20. They consist of 3104 protein atoms
(residues 1 to 398), 595 water molecules (with temperature
factors e60 Å2) and 28 atoms resulting from single N-
acetylglucosamine residues N-linked to Asn 89 and Asn 247.
Crystal structures of Cel7B enzymes from H. insolens,8
Fusarium oxysporum,10 and Trichoderma reesei11 reveal their
close architectural similarity with an active site located in a long
(50 Å) and deep (20 Å) open groove with the active site
nucleophile located about two-thirds of the way along the cleft.
The structure of the Glu-197-Ala mutant is virtually identical
to the native H. insolens Cel7B structure; the only changes are
in the vicinity of the mutation where the solvent structure is
slightly altered (Figure 1). The mutation of Glu-197-Ala liberates
a volume of approximately 120 Å3 which is more than sufficient
to accommodate the axial fluoride of the â(1f4)-disaccharide
donor. Two solvent water molecules occupy this newly formed
cavity in the uncomplexed Cel7B glycosynthase structure. One
can also imagine that other “short” nucleophile mutations such
as glutamate to serine or cysteine would also generate enough
space to accommodate an axial fluoride.
Results and Discussion
Specific Mutagenesis of Cel7B from Humicola insolens.
Glu 197 has been identified in Cel7B from Humicola insolens
as the catalytic nucleophile.8 The Glu-197-Ala variant was made
by site directed mutagenesis (Chameleon, Stratagene), directly
in the expression plasmid pHW704, an Escherichia coli plasmid
in which the transcription of Cel7B is governed by an Aspergil-
lus R-amylase promoter and glucoamylase terminator. The Glu-
197-Ala modification was verified by complete sequencing of
the gene. The plasmid harboring pCE10 was transformed into
Aspergillus oryzae JaL228, together pToC104, encoding the
selectable marker amdS, encoding an acetamidase, and plated
with acetamide as the sole nitrogen source.9
The amdS transformants were screened for expression of the
inactive Cel7B variant by Ouchterlony method on fermentation
samples, applying a polyclonal Ab raised against wild-type
Cel7B. The highest expressing transformant was reisolated three
times, and stored as LaC2922. The transformed A. oryzae was
grown in a fermentor, and the extracellular expressed protein
was purified to homogeneity using cationic (S-Sepharose)
chromatography. The purified protein gave a single band in
SDS-PAGE of 50 kDa and had no activity on CMC.
The high degree of sequence similarity (57%) between the
H. insolens and F. oxysporum enzymes suggest that the same
interactions may occur in the active site of both enzymes. The
structure of Cel7B from F. oxysporum complexed to both a non-
hydrolyzable thiooligosaccharide substrate analogue and cello-
Crystal Structure of the Ala Mutant of Cel7B from H.
insolens. The Cel7B Glu-197-Ala mutant crystallizes in space
group P21 with cell-dimensions a ) 49.53 Å, b ) 73.98 Å, c
(10) (a) Sulzenbacher, G.; Driguez, H.; Henrissat, B.; Schu¨lein, M.;
Davies, G. J. Biochemistry 1996, 35, 15280-15287. (b) Sulzenbacher, G.;
Schu¨lein, M.; Davies, G. J. ibid. 1997, 36, 5902-5911
(11) Kleywegt, G. J.; Zou, J.-Y., Divine, C.; Davies, G. J.; Sinning, J.;
Sta¨hlberg, J.; Srisoduk, M., Teeri, T. T.; Jones, T. A. J. Mol. Biol. 1997,
272, 383-397.
(8) Mackenzie, L. F.; Sulzenbacher, G.; Divne, C.; Jones, T. A.; Wo¨ldike,
H.; Schu¨lein, M.; Withers, S. G.; Davies, G. J. Biochem J. 1998, 335, 409-
416.
(9) Kelly, J. M.; Hynes, M. EMBO J. 1985, 4, 475-479.

Synthesis of â(1 f 4)-Polysaccharides
J. Am. Chem. Soc., Vol. 122, No. 23, 2000 5431
Table 1. Obtention of â-(1f4)-Oligosaccharides by Enzymatic Condensation of Lactosyl Fluoride 1 on Mono- and Disaccharide Acceptors
2-8, 16e Using Cel7B Glu197Ala
biose has shown that the three hydroxyl groups of -1 subsite
sugar strongly interact with Asp 173, Gln 175, and Trp 347 (F.
oxysporum numbering). There are no interactions with the
protein involving the 6-OH in both the -2 and +1 glucosyl
residues. Indeed, the -2 subsite 6-OH is extremely solvent
exposed and frequently disordered.10 It was thus anticipated that
the mutated enzyme could not only be used as a “glycosynthase”
for the synthesis of natural oligo- and polysaccharides, but that
the lack of steric restriction on the C6-OH interactions could
be exploited for the synthesis of substituted â(1f4)-glucans.
Enzymatic Reaction Catalyzed by Ala Mutant of Cel7B
from H. insolens. The scope of the enzyme condensation
catalyzed by the Ala mutant of Cel7B from H. insolens was
first explored using R-lactosyl fluoride 112 as a donor in the
presence of a range of mono- and disaccharide acceptors 2-8,
16e (Table 1) in ammonium carbonate solution at pH 8.0 or in
phosphate buffer at pH 7.0. The transfer of the fluoride 1 on
â-D-mannopyranoside 413 and â-D-xylopyranoside 314 was
slightly less efficient than on the â-D-glucoside 215 but resulted
in higher yields than reported using a commercially available
enzyme in hydro-organic solvents.1c The present yields of
acetylated purified products 9-11 were respectively 83, 51, and
61%. These results confirm those of the X-ray structure of the
complexed wild type enzyme in which the 6-OH and 2-OH of
the glucosyl unit in subsite +1 were not involved in key polar
interactions. It must be emphasized that only â(1f4) linkages
were formed whatever monosaccharide acceptors were involved.
This conclusion arises from the lack of the characteristic signals
at C-2 or C-3 of 2- or 3-O-substituted glycosyl units in 13C
NMR of unprotected compounds obtained by de-O-acetylation
1
of 9 and 10, at 79-85 ppm.16 In the H NMR spectrum of the
tetraacetylated p-nitrophenyl 1-thio-â-D-mannoside, the triplet
signal at δ 5.26 was assigned to H-4.13 The complete interpreta-
1
tion of the H NMR spectrum of the trisaccharide 11 shows
that H-4 was shifted upfield to δ 3.86. On the other hand, the
position of C-4 at δ 65.46 for the acetylated mannoside was
missing in the 13C NMR spectrum of 11 and as expected for a
(12) Ju¨nnemann, J.; Thiem, J.; Pedersen, C. Carbohydr. Res. 1993, 249,
91-94.
(13) Blanc-Muesser, M.; Defaye, J.; Driguez, H. ibid. 1978, 67, 305-
(15) Fischer, E.; Helferich, B. Justus Liebigs Ann. Chem. 1911, 71, 68-
71.
328.
(14) Ballou, C. E.; Roseman, S.; Link, K. P. J. Am. Chem. Soc. 1951,
73, 1140-1144.
(16) Bock, K.; Pedersen, C.; Pedersen, H. AdV. Carbohydr. Chem.
Biochem. 1984, 42, 193-224.

5432 J. Am. Chem. Soc., Vol. 122, No. 23, 2000
Fort et al.
Scheme 1. Enzymatic Polymerization of Cellobiosyl Fluorides 16a-16d Using Cel7B Glu197Ala
4-O-substituted D-mannosyl residue was found at δ 73.60. This
is in contrast with the â(1f3) reaction product obtained when
xyloside was used with the Ala mutant of â-glucosidase from
data of the wild-type enzyme. The 6II-bromo-6II-deoxy-R-
cellobiosyl fluoride 16b, 6II-amino-6II-deoxy-R-cellobiosyl fluo-
ride 16c, and 6II-S-(R-D-xylopyranosyl)-6II-thio-R-cellobiosyl
fluoride 16d were selected.
4
Agrobacterium sp.
Several â(1f4)- and â(1f3)-disaccharides were also used
as acceptors. Methyl â-cellobioside 5,17 methyl 6-bromo-6-
deoxy-â-cellobioside 6,18 N-acetyl-chitobiose 7,19 and benzyl
â-laminaribioside 820 gave the expected tetrasaccharides 12-
15 in excellent yields (Table 1).21 Only N,NII-diacetyl-chitobiose,
a close analogue of 7 with an additional acetyl group which
should fit in +1 subsite, was found to be unable to act as an
acceptor molecule.
These results demonstrate that the acceptor subsites of Glu-
197-Ala mutant of Cel7B from H. insolens may accommodate
various mono- and disaccharides structurally different from the
natural cellobiosyl unit. As already observed and expected for
an endoglucanase, disaccharides are better acceptors than
monosaccharides for transglycosylation reaction.5
Scheme 2 outlines the synthesis of these compounds. The
synthesis began with the mild and selective bromination of the
free primary hydroxyl group of 1,6-anhydro-â-cellobiose (18a)23
by a method described for the bromination of methyl R-D-
glucoside.24 Thus 18a reacted with carbon tetrabromide and
triphenylphosphine in pyridine for 2 h at 50 °C. After addition
of acetic anhydride, the expected bromide 18b was isolated in
54% yield. Acetolysis of the 1,6-anhydro ring was performed
by treatment of 18b with acetic anhydride in the presence of
triethylsilyl triflate. The anomeric mixture of acetates was
converted via the corresponding R-bromide, followed by the
action of silver acetate in acetic acid-acetic anhydride mixture
into the hepta-O-acetyl-6II-bromo-6II-deoxy-â-cellobiose 19, the
precursor for the synthesis of the corresponding fluoride 20.
The fluorination was achieved in 90% yield using commercially
available pyridine-hydrogen fluoride reagent, as already de-
scribed in the maltose and cellobiose series.12 The substituted
R-cellobiosyl fluoride 20 was de-O-acetylated with sodium
methoxide in methanol at 0 °C to give the corresponding
unprotected pure compound 16b in quantitative yield. This
fluoride 20 was the key compound for other modified R-cel-
lobiosyl fluorides. Treatment with sodium azide in N,N-
dimethylformamide generated the 6II-azido-6II-deoxy derivative
21 in 88% yield.25 After de-O-acetylation with sodium meth-
oxide in methanol at 0 °C and hydrogenation in ethanol using
Pd/C as catalyst, the fluoride 16c was obtained (90% yield over
the two steps). The fluoride derivative 16d was also prepared
from 20, since the synthesis of 1,6-thiooligosaccharides is easy
and effective by displacement of a 6-halide derivative with a
1-thiolate glycose as a donor.26 The â-D-xylosyl chloride 2227
was treated with the tetrabutylammonium salt of triphenyl-
methanethiol in toluene28 to produce stereoselectively the R-thio-
In a second set of experiments, the polymerization reaction
of cellobiosyl fluoride 16a and various derivatives 16b-16e
catalyzed by the Glu-197-Ala mutant of Cel7B was investigated.
(Scheme 1). Mild de-O-acetylation of the known acetylated
R-cellobiosyl fluoride12 gave the corresponding compound 16a
which was incubated with enzyme, in phosphate buffer (0.1M,
pH 7.0) at 40 °C for 24 h. As the reaction proceeded a white
crystalline precipitate was formed.The structure of the insoluble
material 17a (Scheme 1) was investigated by electron and X-ray
1
diffraction analysis, together with H- and 13C NMR in 0.2 M
sodium hydroxide (NaOD) at room temperature and in dimethyl
sulfoxide (CD3)2SO at 353 K, respectively. The spectra are
identical to those of low molecular weight cellulose II obtained
by us using phosphorolytic synthesis.22
Polymerization reactions were then attempted using R-cel-
lobiosyl fluorides modified at C-6II as suggested from the X-ray
(17) Newth, F. H.; Nicholas, S. D.; Smith, F.; Wiggins, L. F. J. Chem.
Soc 1949, 2550-2553.
(18) Takeo, K.; Fukatsu, T.; Yasato, T. Carbohydr. Res. 1982, 107, 71-
90.
(23) Fujimaki, I.; Ichikawa, Y.; Kuzuhara, H. ibid. 1982, 101, 148-
151.
(19) Drouillard, S.; Armand, S.; Davies, G. J.; Vorgias, C. E.; Henrissat,
B. Biochem. J. 1997, 328, 945-949.
(20) Obtained by deacetylation of acetylated benzyl â-laminaribioside
prepared as described in Thiem J.; Horst, K. Chem. Ber. 1979, 112, 1046-
1056.
(21) To ensure that only â(1f4) linkages were formed during the reaction
catalyzed by the “glycosynthase”, all the new isolated oligo- and polysac-
charides were subjected to enzymatic hydrolysis by the wild-type enzyme
Cel7B from H. insolens and their hydrolysis products were characterized
by TLC using autenthic samples.
(22) Samain, E.; Lancelon-Pin, C.; Ferigo, F.; Moreau, V.; Chanzy, H.;
Heyraud, A.; Driguez, H. Carbohydr. Res. 1995, 271, 217-226.
(24) Anisuzzaman, A. K. M.; Whistler, R. L. ibid. 1978, 61, 511-518.
(25) As already reported in the glucose series, under these conditions,
no â-D-glycosyl azide was detected (Horneman A. M.; Lundt, I. J.
Carbohydr. Chem. 1995, 14, 1-8).
(26) (a) Hasegawa, A.; Terada, T.; Ogawa, H.; Kiso, M. J. Carbohydr.
Chem. 1992, 11, 319-331. (b) Bennet, S.; von Itzstein, M.; Kiefel, M. J.
Carbohydr. Res. 1994, 259, 293-299.
(27) Holland, C. V.; Horton, D.; Jewell, J. S. J. Org. Chem. 1967, 32,
1818-1821.
(28) Blanc-Muesser, M.; Vigne, L.; Driguez, H. Tetrahedron Lett. 1990,
3869-3870.

Synthesis of â(1 f 4)-Polysaccharides
J. Am. Chem. Soc., Vol. 122, No. 23, 2000 5433
Scheme 2. Syntheses of Cellobiosyl Fluorides 16b-16ea
a (a) PPh3, CBr4, pyridine, 0 to 50 °C, 2 h, then Ac2O, 3 h. (b) (1) TESTf, AC2O, 0 °C, 30 min, aq. NaHCO3, 30 min; (2) HBr-AcOH, CH2Cl2,
0 °C, 30 min then rt, 1 h; (3) AgOAc, Ac2O, AcOH, rt, 12 h; (c) HF-pyridine, 0 °C, 1.5 h; (d) (1) NaOMe, MeOH, 0 °C, 4 h; (2) H+ resin; (e)
NaN3, DMF, 80 °C, 12 h; (f) (1) d; (2) 10% Pd/C, H2, EtOH, 2 h; (g) Bu4NSTr, toluene, rt, 24h; (h) (1) PhHgOAc, MeOH-CH2Cl2, rt, 1 h; (2)
H2S, Ac2O, pyridine, CH2Cl2, rt, 12h; (i) DTE, cysteamine, HMPA, rt, 7h.
glycoside 23a (54% yield). Well-known two-step procedures28
led to 23b from 23a in 89% yield. Selective de-S-acetylation
and activation, using cysteamine in HMPA in the presence of
DTE,29 and reaction with glycosyl acceptor 20 produced the
desired trisaccharide fluoride 24 in 68% yield, but 20 was
recovered in 30% yield. De-O-acetylation as already described,
afforded 16d in quantitative yield. This compound is the thio-
analogue fluoride of the trisaccharide R-D-Xylp-(1f6)-â-D-
Glcp-(1f4)-D-Glc, one of the building blocks of xyloglucan, a
widely distributed polymer which interacts intimately with
cellulose in primary cell walls of higher plants,30 and is a
substrate for wild-type Cel7B.31
Conclusions
This work clearly shows the benefit of a multi-disciplinary
approach for the rational design of new tools in organic
synthesis. The 1.75 Å X-ray structure of the Glu-197-Ala mutant
reveals its structural integrity with no change in the position of
critical substrate-binding residues such as Trp 347 and Trp 356.
The catalytic acid/base, Glu 202, (functioning as a Brønsted
base for the enzymatic condensation) is found in an unchanged,
and hence catalytically viable, position. Initial modeling, using
the wild-type H. insolens Cel7B structure and the thio-
oligosaccharide and cellobiose complexes of the closely related
Cel7B structures from F. oxysporum correctly indicated that
the -2 subsite would tolerate substantial substitutions, particu-
larly at the C6 position, which has been successfully exploited
for the synthesis of new glucans.
In the presence of a catalytic amount (10 µM) of the Glu-
197-Ala mutant of Cel7B, the fluorides 16b-16d were incu-
bated in buffers at 40 °C for 24 h. Water insoluble polymers
17b and 17d and the soluble one 17c (at pH 7.0) were
characterized by NMR, MS, or GPC and enzymatic hydrolysis.21
Experimental Section
An attempt at polymerization of 16e, readily obtained from
16b via the acetylated dibromo compound 25 (Scheme 2), failed.
However, this molecule was an acceptor when incubated in the
presence of lactosyl fluoride 1, and the expected tetrasaccharide
26 (Table 1) was isolated in 80% yield.
General. NMR spectra were recorded on a Bruker AC 300, Bruker
Avance 400 or Varian Unity 500. Proton chemical shifts (δ) are reported
in ppm downfield from TMS. Coupling constants (J) are in hertz (Hz)
with singlet (s), doublet (d), doublet of doublet (dd), triplet (t), multiplet
(m), broad (b). Carbon chemical shifts (δ) are reported in ppm with
internal reference of solvent. Complete assignment of the oligomers
11 and 15 was performed using a combination of COSY or TOCSY
1D, HMQC, and HMBC experiments. One bond 13C-1H correlations
were obtained from HMQC data, and the position of the glycosidic
linkages was determined using HMBC data. High-resolution mass
spectra (HRMS) were recorded on VG ZAB and low-resolution (MS)
on a Nermag R-1010C spectrometers. Multidetection steric exclusion
(29) Blanc-Muesser, M.; Driguez, H. J. Chem. Soc., Perkin Trans 1 1988,
3345-3351.
(30) Perrin, R. M.; De Rocher, A. E.; Bar-Peled, M.; Zeng, W.;
Norambuena, L.; Orellana, A.; Raikhel, N. V.; Keegstra, K. Science 1999,
284, 1976-1978.
(31) Vargas-Rechia, C.; Reicher, F.; Sierakowski, M. R.; Heyraud, A.;
Driguez, H.; Lienart, Y. Plant Physiol. 1998, 116, 1013-1021.

5434 J. Am. Chem. Soc., Vol. 122, No. 23, 2000
Fort et al.
chromatography was performed on 150 C Waters chromatograph
equipped with a multiangle DAWN DSP-F light scattering detector
(laser wavelength 632.8 nm), an ambient flow cell for aqueous solution
K5, and the ASTRA 4 software (Wyatt technology Corporation, Santa
Barbara, CA). Optical rotations were measured with a Perkin-Elmer
341 polarimeter. Melting points were measured on a Bu¨chi 535
apparatus. Microanalyses were performed by the “Laboratoire Central
d’analyses du CNRS” (Vernaison).
Evolution of reactions was monitored by analytical thin-layer
chromatography using silica gel 60 F254 precoated plates (E. Merck,
Darmstadt).
Di-N,NII-acetyl chitobiose was commercially available. All reactions
in organic medium were carried out under argon using freshly distilled
solvents. After workup, organic phases were dried over anhydrous Na2-
SO4.
The reaction mixture was gently shaken in an oven at 40 °C. After 48
h, the solution was filtered on C18 Cartridge Sep-Pak Plus (Waters)
and then concentrated to dryness and acetylated (acetic anhydride-
pyridine 1:1 v/v, 10 mL). After 12 h of stirring at 25 °C, the reaction
was quenched by adding MeOH at 0 °C and concentrated in vacuo.
The residue was dissolved in CH2Cl2 and washed with 20% aqueous
KHSO4 and saturated aqueous NaHCO3. The organic layers were
concentrated and purified by flash chromatography (EtOAc-petroleum
ether 6:4 v/v) to generate the title trisaccharide 9 (105 mg, 83%): [R]25
D
-31.5 (c 1.33, CHCl3); 13C NMR (CDCl3, 75 MHz) δ 169.6-169.2
(CO), 136.6 (Cq arom), 128.3-127.6 (C arom), 101.0 (C-1I), 100.3
(C-1II), 99.0 (C-1III), 76.3, 75.9 (C-4I, II), 72.9, 72.6, 72.4, 71.8, 71.6,
II,
II,
70.8, 70.65, 70.6 (C-2I, II, C-3I, III, C-5I, III, CH2Ph), 69.0 (C-2III),
66.5 (C-4III), 62.2, 61.7, 60.7 (C-6I, II, III), 20.7-20.5 (CH3); 1Η ΝΜR
(CDCl3, 300 MHz) δ 7.28-7.22 (m, 5H, H arom), 5.30 (d, 1H, J3, 4
)
3.3 Hz, H-4III), 5.13-4.76 (m, 7H, H-2I, II, III, H-3I, II, III, CH2Ph), 4.58-
4.31 (m, 6H, H-1I, II, III, CH2Ph, H-6I, IIa), 4.05 (m, 4H, H-6I, IIb, H-6IIIa,
H-6IIIb), 3.84-3.71 (m, 3H, H-4I, II, H-5III), 3.57-3.50 (m, 2H, H-5I,
II), 2.11-1.93 (m, 30H, CH3); FABMS: m/z 1038 [M + Na]+; Anal.
Calcd for C45H58O26: C,53.25; H,5.76. Found: C, 52.48; H, 5.89.
Benzyl (2,3,4,6-Tetra-O-acetyl-â-D-galactopyranosyl)-(1f4)-
(2,3,6-tri-O-acetyl-â-D-glucopyranosyl)-(1f4)-2,3-di-O-acetyl-â-D-
xylopyranoside (10). Benzyl xyloside 3 (22 mg,0.092 mmol) and then
Cel7B Glu197Ala (0.5 mg) were added to a solution of lactosyl fluoride
1 (31.5 mg, 1 equiv) in phosphate buffer (0.1M, pH 7.0, 1 mL). The
reaction mixture was gently shaken in an oven at 40 °C. After 48 h,
the solution was filtered on C18 Cartridge Sep-Pak Plus (Waters), and
then concentrated to dryness and acetylated (acetic anhydride-pyridine
1:1 v/v, 10 mL). After 12 h of stirring at 25 °C, the reaction mixture
was quenched by adding MeOH at 0 °C and concentrated in vacuo.
The residue was dissolved in CH2Cl2 and washed with 20% aqueous
KHSO4 and saturated aqueous NaHCO3. The organic layers were
concentrated and purified by flash chromatography (EtOAc-petroleum
ether 6:4 v/v) to generate the title trisaccharide 10 (26.5 mg, 51%):
X-ray Structure Determination. The Glu-197-Ala mutant of Cel7B
was purified as described for the CE8 double mutant.32 Crystals were
grown by the hanging-drop vapor-phase diffusion technique in 4 µL
hanging-drops. The protein concentration was 20 mg‚mL-1 in 20 mM
Tris-HCl buffer with 20-30% (w/v) poly(ethylene glycol) 4000 as
precipitant. 5% (v/v) DMSO was included to help prevent the formation
of disordered arrays of plates. X-ray data to 1.75 Å resolution were
collected on the EMBL BW7A beam line (λ ) 0.9998 Å) at the DORIS
storage ring in Hamburg from a single crystal in a stream of N2 gas at
100 K. A 300 mm Mar-Research Imaging Plate Scanner was used as
detector. Data processing and reduction were carried out with the HKL
program suite,33 and all further computing used the CCP4 suite of
programs34 unless otherwise stated.
The structure was solved by molecular replacement using the native
H. insolens Cel7B structure as the search model (PDB code 2A39).
The program AMORE35 was used in conjunction with data in the
resolution range 20-4 Å and an outer radius of Patterson integration
of 30 Å. Structure solution revealed just one significant solution. For
refinement, 5% of the observations were immediately set aside for cross
validation analysis36 and were used to monitor various refinement
strategies such as geometric and temperature-factor restraint values,
the insertion of solvent water and as the basis for the maximum
likelihood refinement using REFMAC.37 Manual corrections of the
model using the X-FIT routines of the program QUANTA (Molecular
Simulations Inc., San Diego, CA) were interspersed with cycles of
maximum-likelihood based refinement. Water molecules were added
in an automated manner using ARP38 and verified manually prior to
coordinate deposition. Hydrogen scattering from “riding” hydrogen
atoms was included and justified by a decrease in the Rcryst and Rfree of
1.2%/1.2%, respectively. Coordinates have been deposited with the
Protein Databank.39
[R]25 -49.0 (c 1.0, CHCl3); 13C NMR (CDCl3, 75 MHz) δ 169.9-
D
169.0 (CO), 136.9 (Cq arom), 128.4-127.5 (C arom), 101.0, 100.0,
II,
99.5 (C-1I, III), 76.0, 75.4 (C-4I, II), 72.8, 72.7, 71.73, 71.68, 70.9,
70.8, 70.7, 70.4 (C-2I, II, C-3I, II, III, C-5II, III, CH2Ph), 69.0 (C-2III), 66.6
(C-4III), 62.6, 62.0, 60.8 (C-5I, C-6II, III), 20.7-20.4(CH3), Anal. Calcd
for C42H54O24: C, 53.50; H, 5.77. Found: C, 53.68; H, 5.88.
p-Nitrophenyl (2,3,4,6-Tetra-O-acetyl-â-D-galactopyranosyl)-
(1f4)-(2,3,6-tri-O-acetyl-â-D-glucopyranosyl)-(1f4)-2,3,6-tri-O-
acetyl-1-thio-â-D-mannopyranoside (11). Lactosyl fluoride 1 (21.7
mg, 0.063 mmol) and p-nitrophenyl 1-thio-â-D-mannopyranoside 4
(22.6 mg, 1 equiv) in ammonium carbonate solution (0.1M, pH 8.0, 1
mL) were incubated with Cel7B Glu197Ala (0.6 mg) at 40 °C for 48
h. The condensation product which precipitated in the reaction medium
was isolated by centrifugation and removal of the supernatant, washed
with H2O, dried in vacuo over sodium pentoxide at 40 °C for 24 h,
and then acetylated and purified as described for compound 9, to give
Benzyl (2,3,4,6-Tetra-O-acetyl-â-D-galactopyranosyl)-(1f4)-
(2,3,6-tri-O-acetyl-â-D-glucopyranosyl)-(1f4)-2,3,6-tri-O-acetyl-â-
D-glucopyranoside (9). Benzyl glucoside 2 (34 mg, 0.125 mmol) and
then Cel7B Glu197Ala (0.45 mg) were added to a solution of lactosyl
fluoride 1 (43 mg, 1 equiv) in phosphate buffer (0.1M, pH 7.0, 1 mL).
11 (41.0 mg, 61%): [R]25 -53 (c 0.58, CHCl3); 13C NMR (CDCl3,
D
75 MHz) δ 169.6-168.9 (CO), 146.6, 143.1 (Cq arom), 129.3, 123.8
(C arom), 101.0 (C-1III), 100.0 (C-1II), 83.4 (C-1I), 77.1 (C-5I), 75.8
(C-4II), 73.6 (C-4I), 72.74 (C-3II), 72.69 (C-5II), 71.9 (C-2II), 71.5 (C-
3I), 70.8 (C-3III), 70.6 (C-5III), 70.4 (C-2I), 69.0 (C-2III), 66.5 (C-4III),
62.7 (C-6I), 62.1 (C-6II), 60.6 (C-6III), 20.7-20.3 (CH3); 1Η ΝΜR
(CDCl3, 300 MHz) δ 8.09 et 7.52 (2d, 4H, J ) 9.1 Hz, H arom), 5.60
(d, 1H, J2, 3 ) 2.9 Hz, H-2I), 5.30 (d, 1H, J3, 4 ) 2.9 Hz, H-4III), 5.13
(32) Davies, G. J.; Lewis, R. J.; Ducros, V.; Borchert, T.; Schu¨lein, M.
J. Biotechnol. 1997, 57, 91-100.
(33) (a) Otwinowski, Z. Oscillation data reduction program. In Data
Collection and Processing: proceedings of the CCP4 study weekend;
Sawyer, L., Isaacs, N., Bailey, S., Eds.; Science and Engineering Research
Council: Daresbury, UK, 1993. (b) Otwinowski, Z.; Minor, W. Processing
of X-ray diffraction data collected in oscillation mode. In Methods in
Enzymology: Macromolecular Crystallography, part A; Carter, C. W. J.,
Sweet, R. M., Eds.; Academic Press: London & New York, 1997; Vol.
276, pp 307-326.
(t, 1H, J2, ) 9.1 Hz, H-3II), 5.13-5.03 (m, 3H, H-1I, H-2III, H-3I),
3
4.91 (dd, 1H, J2, ) 10.6 Hz, H-3III), 4.78 (dd, 1H, J1, ) 7.6 Hz,
3
2
H-2II), 4.54 (d, 1H, J1, 2 ) 8 Hz, H-1II), 4.46-4.43 (m and d, 2 H, J1,
2 ) 7.6 Hz, H-1III, H-6Ia), 4.35 (dd, 1H, J5, 6 ) 1.8 Hz, Ja, b ) 12 Hz,
H-6IIa), 4.18-4.04 (m, 4H, H-6Ib, H-6IIb, H-6IIIa, H-6IIIb), 3.87-3.73
(m, 4H, H-4I, II, H-5I, III), 3.56 (m, 1H, H-5II), 2.16-1.93 (m, 30H, CH3);
ΗRFABMS calcd for C44H55NO27S (M + Na)+ 889.2590, found
889.2595.
(34) Collaborative Computational Project Number 4, 1994. The CCP4
suite: programs for protein crystallography. Acta Crystallogr. 1994, D50,
760-763.
(35) Navaza, J. Acta Crystallogr. 1994, A50, 157-163.
(36) Bru¨nger, A. T. Nature 1992, 355, 472-475.
(37) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr.
1997, D 53, 240-255.
Methyl â-D-Galactopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-
â-D-glucopyranosyl -(1f4)-â-D-glucopyranoside (12). Lactosyl fluo-
ride 1 (30 mg, 0.087 mmol) and methyl â-cellobioside 5 (31.0 mg, 1
equiv) in phosphate buffer (0.1M, pH 7.0, 1 mL) were incubated with
Cel7B Glu197Ala (0.5 mg) at 40 °C. After 24 h, the solution was
(38) Lamzin, V. S.; Wilson, K. S. Acta Crystallogr. 1993, D49, 129-
147.
(39) Bernstein, F. C.; Koetzle, T. F.; Williams, G. J. B.; Meyer, E. T.,
Jr.; Brice, M. D.; Rodgers, J. R.; Kennard, O.; Shimanouchi, T.; Tasumi,
M. J. Mol. Biol. 1977, 112, 535-542

Synthesis of â(1 f 4)-Polysaccharides
J. Am. Chem. Soc., Vol. 122, No. 23, 2000 5435
filtered on C18 Cartridge Sep-Pak Plus (Waters) and then freeze-dried.
The expected tetrasaccharide 121b was obtained in quantitative yield
(59 mg): [R]25D -1 (c 0.32, H2O); 13C NMR (D2O, 75 MHz) δ 103.9,
103.7, 103.1 (2C) (C-1I, II, III, IV), 79.4, 79.1, 79.0 (C-4I, II, III), 76.2, 75.6,
75.1, 74.9, 74.8, 73.7, 73.3, 71.7 (C-2I, II, III, IV, C-3I, II, III, IV, C-5I, II, III,
IV), 69.3 (C-4IV), 61.8-60.8 (C-6I, II, III, IV), 58.0 (OCH3); FABMS: m/z
703 [M + Na]+.
Methyl â-D-Galactopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-
â-D-glucopyranosyl -(1f4)-6-bromo-6-deoxy-â-D-glucopyranoside
(13). Lactosyl fluoride 1 (9.0 mg, 0.026 mmol) and methyl 6-bromo-
6-deoxy-â-cellobioside 6 (11.0 mg, 1 equiv) in phosphate buffer (0.1M,
pH 7.0, 1 mL) were incubated with Cel7B Glu197Ala (0.6 mg) at 40
°C. After 7 h, the solution was filtered on C18 Cartridge Sep-Pak Plus
(Waters) and then freeze-dried. The expected tetrasaccharide 13 was
2,3,4-Tri-O-acetyl-6-bromo-6-deoxy-â-D-glucopyranosyl-(1f4)-
1,2,3,6-tetra-O-acetyl-â-D-glucopyranose (19). A catalytic amount of
triethylsilyl triflate (20 µL) was added, dropwise via a syringe, to a
solution of compound 18b (1.5 g, 2.5 mmol) in acetic anhydride (20
mL) cooled at 0 °C. The mixture was stirred at 0 °C for 30 min, and
then saturated aqueous NaHCO3 was added to quench the reaction,
and the products were extracted with CH2Cl2. The organic layers were
dried, concentrated and coevaporated with toluene. HBr (30% w/v in
AcOH, 5 mL) was added to the crude anomeric mixture in CH2Cl2 (10
mL) at 0 °C. After stirring at 0 °C for 30 min and then at room
temperature for an additional 1 h, the reaction mixture was diluted with
CH2Cl2, washed with ice-cold H2O and ice-cold saturated aqueous
NaHCO3 (3×), dried, and concentrated. The resulting bromide and
AgOAc (840 mg, 2 equiv) in a mixture of Ac2O-AcOH (20 mL, 1:1
v/v) were stirred in the dark overnight at room temperature. The mixture
was diluted with CH2Cl2 and filtered through a Celite bed, and the
filtrate was washed with saturated aqueous NaHCO3 (3×), dried, and
concentrated. Filtration through silica gel (EtOAc-petroleum ether 7:3
v/v) gave compound 19 (1.53 g, 88% three steps) which was crystallized
in EtOH, mp 156 °C, [R]25D -2.5 (c 0.81, CHCl3); 13C NMR (CDCl3,
75 MHz) δ 169.3-166.2 (CO), 99.8 (C-1II), 91.5 (C-1I), 75.2 (C-4I),
73.4, 73.3, 72.5, 71.9, 71.4, 70.5, 70.4 (C-2I, II, C-3I, II, C-5I, II, C-4II),
61.5 (C-6I), 30.2 (C-6II), 20.9-20.3 (CH3); 1Η ΝΜR (CDCl3, 300 MHz)
obtained in quantitative yield (19.3 mg): [R]25D +3 (c 0.93, H2O); 13
C
NMR (D2O, 75 MHz) δ 103.9, 103.7, 103.2, 103.1 (C-1I, II, III, IV), 80.9
(C-4I), 79.0, 78.97 (C-4II, III), 76.2, 75.6, 74.9, 74.8, 74.7, 73.76, 73.66,
73.6, 73.3, 71.8 (C-2I, II, III, IV, C-3I, II, III, IV, C-5I, II, III, IV), 69.4 (C-4IV),
III,
61.8, 60.8, 60.7 (C-6II,
IV), 58.2 (OCH3), 33.2 (C-6I); ΗRFABMS
calcd for C25H43BrO20 (M + Na)+ 765.1429, found 765.1435.
2-Acetamido-1,3,6-tri-O-acetyl-2-deoxy-(2,3,4,6-tetra-O-acetyl-â-
D-galactopyranosyl)-(1f4)-(2,3,6-tri-O-acetyl-â-D-glucopyranosyl)-
(1f4)-(2-acetamido-3,6-di-O-acetyl-2-deoxy-â-D-glucopyranosyl)-
(1f4)-r,â-D-glucopyranose (14). Lactosyl fluoride 1 (10.0 mg, 0.029
mmol) and N-acetyl-chitobiose 7 (11.1 mg, 1 equiv) in ammonium
carbonate solution (0.1M, pH 8.0, 1 mL) were incubated with Cel7B
Glu197Ala (0.6 mg) at 40 °C for 24 h. Then the solution was
evaporated, dried in vacuo over sodium pentoxide at 40 °C for 24 h,
acetylated as described for compound 9 and purified by flash column
chromatography using a mixture CH2Cl2-MeOH (95:5 v/v) as eluent.
The tetrasaccharide 14 was obtained in 65% yield (23.6 mg): 13C NMR
(CDCl3, 75 MHz) δ 170.0-169.0 (CO), 101.8, 101.0, 101.3 (C-1II, III,
IV), 92.3, 90.3 (C-1I R,â), 77.1, 75.9, 75.7, 72.7, 71.8, 70.8, 70.6, 69.0,
66.5, 62.2, 62.0, 61.4, 60.6, 53.7, 51.5, 51.0 (C-2I, II, III, IV to C-5 I, II, III,
IV), 20.8-20.5 (CH3); ΗRFABMS calcd for C52H72O33N2 (M + Na)+
1275.3915, found 1275.3914.
δ 5.57 (d, 1H, J1, ) 8.2 Hz, H-1I), 5.20-4.78 (m, 5H, H-2I, H-2II,
2
H-3I, H-3II, H-4II), 4.47 (d, 1H, J1, ) 8 Hz, H-1II), 4.39 (dd, 1H, J5,
2
6 ) 1.6 Hz, Ja, b ) 12.2 Hz, H-6Ia), 4.02 (dd, 1H, J5, 6 ) 4.6 Hz, H-6Ib),
3.80 (t, 1H, J2, 3 ) J3, 4 ) 9 Hz, H-4I), 3.62-3.56 (m, 2H, H-5I, H-5II),
3.40 (dd, 1H, J5, 6 ) 2.6 Hz, Ja, b ) 11.5 Hz, H-6IIa), 3.25 (dd, 1H, J5,
6 ) 7 Hz, H-6IIb), 2.10-1.95 (m, 21H, CH3); DCIMS: m/z 718 [M +
H + NH3]+, 641 [M - OAc]; Anal. Calcd for C26H35BrO17: C,44.65;
H,5.04; Br, 11.42. Found: C, 44.64; H, 5.13; Br,. 11.07.
2,3,4-Tri-O-acetyl-6-bromo-6-deoxy-â-D-glucopyranosyl-(1f4)-
2,3,6-tri-O-acetyl-r-D-glucopyranosyl fluoride (20). A solution of
compound 19 (1.4 g, 2.0 mmol) in HF-pyridine (10 mL, 7:3 v/v) was
stirred at 0 °C for 1.5 h in a plastic vial. The mixture was diluted with
CH2Cl2 and poured into ice-cold aqueous NH3 (3 M); the organic layer
was washed with saturated aqueous NaHCO3 (3×), dried, and
concentrated. Crystallization from EtOH- Et2O gave pure 20 (1.2 g,
90%): mp 177-178 °C; [R]25D +40 (c 1.1, CHCl3); 13C NMR (CDCl3,
Benzyl â-D-Galactopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-
â-D-glucopyranosyl-(1f3)-â-D-glucopyranoside (15). Lactosyl fluo-
ride 1 (12.0 mg, 0.035 mmol) and benzyl laminaribioside 8 (15.0 mg,
1 equiv) in phosphate buffer (0.1M, pH 7.0, 1 mL) were incubated
with Cel7B Glu197Ala (0.6 mg) at 40 °C. After 6 h, the solution was
filtered on C18 Cartridge Sep-Pak Plus (Waters) and then freeze-dried.
The expected tetrasaccharide 15 was obtained in quantitative yield (26.0
75 MHz) δ 170.0-168.7 (CO), 103.6 (d, JC, ) 230 Hz; C-1I), 99.8
F
II
(C-1II), 77.1, 74.7, 73.5, 72.6, 71.5, 70.6, 70.2, 68.3 (C-2I, II, C-3I,
,
C-4I, , C-5I, II), 60.9 (C-6I), 30.2 (C-6II), 20.8-20.4 (CH3); Η ΝΜR
II
1
(CDCl3, 300 MHz) δ 5.62 (dd, 1H, J1, 2 ) 2.7 Hz, J1, F ) 53 Hz, H-1I),
mg): [R]25 -16 (c 0.5, H2O); 13C NMR (D2O, 125 MHz) δ 137.4
D
5.42 (t, 1H, J2, ) J3,4 ) 9.8 Hz, H-3I), 5.10 (t, 1H, J2,3 ) J3,4 ) 9.8
3
(Cq arom), 129.5-129.3 (C arom), 103.82 (C-1IV), 103.47 (C-1II),
103.26 (C-1III), 101.86 (C-1I), 85.25 (C-3I), 78.98 (C-4III), 78.85 (C-
4II), 76.50 (C-5I), 75.76 (C-5III), 75.70 (C-5II), 75.12 (C-3III), 74.98 (C-
3II), 74.12 (C-2II), 73.80 (C-2I), 73.78 (C-2III), 72.3 (CH2Ph), 71.80 (C-
2IV), 69.45 (C-4IV), 69.10 (C-4I), 62.82 (C-6IV), 61.59 (C-6I), 60.89 (C-
6III), 60.74 (C-6II); ΗRFABMS calcd for C31H48O21 (M + Na)+
779.2586, found 779.2589.
Hz, H-3II), 4.89 (m, 3H, H-2I, H-2II, H-4II), 4.50 (m, 2H, H-1II, H-6Ia),
4.10 (m, 2H, H-5I, H-6Ib), 3.83 (t, 1H, J4,5 ) 9.8 Hz, H-4I), 3.63 (m,
1H, H-5II), 3.42 (dd, 1H, J5,6 ) 2.4 Hz, Ja,b) 11.5 Hz, H-6IIa), 3.26
(dd, 1H, J5,6 ) 7.3 Hz, H-6IIb), 2.10-1.93 (m,18H, CH3); FABMS:
m/z 683 [M + Na]+; Anal. Calcd for C24H32BrFO15: C,43.71; H,4.89;
Br, 12.12. Found: C, 43.87; H, 5.02; Br,. 12.13.
2,3,4-Tri-O-acetyl-6-azido-6-deoxy-â-D-glucopyranosyl-(1f4)-
2,3,6-tri-O-acetyl-r-D-glucopyranosyl fluoride (21). Compound 20
(282 mg, 0.43 mmol) was dissolved in N,N-dimethylformamide (20
mL) which contained sodium azide (298 mg, 11 equiv).The mixture
was heated at 80 °C for 12 h and then concentrated in vacuo. The
residue was dissolved in CH2Cl2, and the organic layer was washed
with H2O and dried and concentrated. Filtration through silica gel
(EtOAc-petroleum ether 1:1 v/v) gave compound 21 (235 mg, 88%)
which was crystallized in EtOH, mp 155-156 °C, [R]25D +28 (c 0.51,
CHCl3); 13C NMR (CDCl3, 75 MHz) δ 169.9-168.7 (CO), 103.5 (d,
JC,F ) 230 Hz; C-1I), 99.8 (C-1II), 74.3, 72.8, 72.5, 71.5, 70.63, 70.58,
70.4, 70.1, 69.2, 68.6 (C-2I, II, C-3I, II, C-4I, II, C-5I, II), 61.0 (C-6I), 50.8
2,3,4-Tri-O-acetyl-6-bromo-6-deoxy-â-D-glucopyranosyl-(1f4)-
2,3-di-O-acetyl-1,6-anhydro-â-D-glucopyranose (18b). To a solution
of 1,6-anhydro-â-cellobiose 18a (281 mg, 0.87 mmol) in pyridine (20
mL) at 0 °C was added triphenylphosphine (911 mg, 4 equiv) and
tetrabromomethane (578 mg, 2 equiv). After stirring the solution for
30 min at 0 °C and 2 h at 50 °C, acetic anhydride (10 mL) was added.
After an additional 3 h, the reaction mixture was worked up as described
for compound 9 and purified by flash chromatography (EtOAc-
petroleum ether 8:2 v/v) to give 18b (280 mg, 54%) which was
crystallized in MeOH, mp 140-141 °C, [R]25D -34.5 (c 0.73, CHCl3);
13C NMR (CDCl3, 75 MHz) 100δ 169.4-168.9 (CO), 0.3, 98.9 (C-1I,
II), 76.9, 73.7, 73.2, 72.7, 71.3, 70.6, 69.6, 68.5 (C-2I, II, C-3I, II, C-4I, II
,
1
(C-6II), 20.6-20.3 (CH3); Η ΝΜR (CDCl3, 300 MHz) δ 5.62 (dd,
C-5I, II), 64.9 (C-6I), 30.6 (C-6II), 20.9-20.5 (CH3); 1Η ΝΜR (CDCl3,
300 MHz) δ 5.41 (s, 1H, H-1I), 5.22-5.03 (m, 2H, H-3I, H-3II), 5.03-
4.96 (m, 2H, H-2II, H4II), 4.90 (d, 1H, J1, 2 ) 8.2 Hz, H-1II), 4.56 (m,
2H, H-2I, H-5I), 3.93 (d, 1H, Ja, b ) 7.7 Hz, H-6Ia), 3.77 (m, 2H, H-5II,
H-6Ib), 3.53 (s, 1H, H-4I), 3.44 (dd, 1H, J5, 6 ) 2.7 Hz, Ja, b ) 11.5 Hz,
H-6IIa), 3.33 (dd, 1H, J5, 6 ) 6.6 Hz, H-6IIb), 2.12-1.96 (m, 15H, CH3);
DCIMS: m/z 616 [M + H + NH3]+; Anal. Calcd for C22H29BrO14:
C,44.23; H,4.89; Br, 13.38. Found: C, 43.95; H, 5.10; Br,. 13.26.
1H, J1,2 ) 2.4 Hz, J1,F ) 53 Hz, H-1I), 5.44 (t, 1H, J2,3 ) J3,4 ) 9.8
Hz, H-3I), 5.13 (t, 1H, J2,3 ) J3,4 ) 9.3 Hz, H-3II), 4.95-4.80 (m, 3H,
H-2I, H-2II, H-4II), 4.57-4.50 (m, 2H, H-1II, H-6Ia), 4.10 (m, 2H, H-5I,
H-6Ib), 3.87 (t, 1H, J4,5 ) 9.3 Hz, H-4I), 3.60 (m, 1H, H-5II), 3.33 (m,
2H, H6IIa, H-6IIb), 2.10-1.95 (m,18H, CH3); DCIMS: m/z 639 [M +
H + NH3]+; Anal. Calcd for C24H32FN3O15: C,46.38; H,5.19; F, 3.06,
N, 6.76. Found: C, 46.67; H, 5.37; F, 2.87; N, 6.59.

5436 J. Am. Chem. Soc., Vol. 122, No. 23, 2000
Fort et al.
Triphenylmethyl 2,3,4-Tri-O-acetyl-1-S-r-D-xylopyranoside (23a).
A solution of triphenylmethanethiol (8 g, 29 mmol) and N-tetrabutyl-
ammonium hydroxide (37 mL, 25% in MeOH, 29 mmol) in a mixture
MeOH-toluene (60 mL, 1:1 v/v) was evaporated to dryness then
coevaporated with toluene (30 mL, 3×). The residual thiolate was
dissolved in toluene (75 mL), and then tri-O-acetyl-â-D-xylopyranosyl
chloride 22 (7.94 g, 27 mmol) was added. The solution was stirred
under argon for 24 h and then concentrated. The residue was purified
by flash-chromatography (EtOAc-petroleum ether 1:3 v/v), to give
after recrystallization in Et2O-petroleum ether 23a (8.44 g, 54%): mp
O-acetylated by treatment with sodium methoxide (1 M, 50 µL) in
methanol (10 mL) for 4 h at 0 °C. The mixture was neutralized with
Amberlite IR 120 (H+) resin, the resin was removed by filtration, and
the filtrate concentrated. Freeze-dried compounds 16a or 16b were
obtained in quantitative yield.
Compound (16a):13C NMR (D2O, 75 MHz) δ 107.8 (d, JC,F ) 223
Hz; C-1I), 103.3 (C-1II), 78.1, 76.8, 76.3, 74.0, 73.7, 71.8, 71.5, 70.3
1
(C-2I, II, C-3I, II, C-4I, II, C-5I, II), 61.4, 60.2 (C-6I, II); Η ΝΜR (D2O,
300 MHz) δ 5.58 (dd, 1H, J1,2 ) 2.7 Hz, J1,F ) 53.3 Hz, H-1I), 4.40
(d, 1H, J1,2 ) 7.9 Hz, H-1II), 3.8-3.2 (m, 12H)
93-95 °C; [R]25 +117 (c 1, CHCl3); 13C NMR (75 MHz) δ 169.4,
Compound (16b): FABMS: m/z 429 [M + Na]+.
D
169.2 and 169.0 (CO), 144.2, 129.6, 127.6 and 126.8 (C arom), 81.8
(C-1), 69.8, 68.5 and 67.8 (C-2, C-3, C-4), 62.5 (C-5), 20.4 and 20.5
(CH3); 1H NMR (300 MHz, CDCl3) δ 7.20-7.38 (m, 15 H, 3 H arom),
5.11 (t, 1 H, J2,3 ) J3,4 ) 7 Hz, H-3), 4.74 (dd, 1 H, J1,2 4.1 Hz, H-2),
4.69 (m, 1 H, H-4), 4.60 (d, 1 H, H-1), 3.99 (dd, 1 H, J4,5ax 7.15 Hz,
6-Amino-6-deoxy-â-D-glucopyranosyl-(1f4)-r-D-glucopyrano-
syl fluoride (16c). Acetylated 6II-azido-6II-deoxy-R-cellobiosyl fluoride
21 (100 mg, 0.16 mmol) was de-O-acetylated as described above, giving
the free fluoride which was used in the next step without further
characterization. This compound (20 mg, 0.054 mmol) in EtOH (5 mL)
was hydrogenolyzed on 10% Pd/C (20 mg) for 12 h under atmospheric
pressure. The catalyst was filtered off, the filtrate was concentrated,
and the residue was freeze-dried to give 16c (16.8 mg, 90%) which
was characterized by its DCIMS spectrum m/z 344 [M + H + NH3]+.
R-D-Xylopyranosyl-(1f6)-â-D-glucopyranosyl)-(1f4)-r-D-glu-
copyranosyl fluoride (16d). Compound 24 was de-O-acetylated as
already described to give 16d in quantitative yield: FABMS: m/z 513
[M + Na]+.
J5ax,5eq 12.4 Hz H-5ax), 3.57 (dd, 1 H, J4,5eq 4.5 Hz, H-5eq), 2.09, 2.01
and 1.92 (3 s, 9 H, 3 CH3); Anal. Calcd for C30H30O7S: C, 67.39; H,
5.65; S, 5.99. Found: C, 67.60; H, 5.47; S, 5.80.
S-Acetyl-2,3,4-tri-O-acetyl-r-D-xylopyranose (23b). Phenylmer-
cury(II) acetate (6.57 g, 19.49 mmol) was added to a solution of
compound 23a (9 g, 16.86 mmol) in a mixture of MeOH-CH2Cl2 (300
mL, 2:1 v/v). The reaction mixture was stirred for 1 h at room
temperature and then concentrated in vacuo. The residue, coevaporated
with toluene (50 mL), was dissolved in CH2Cl2 (270 mL), and then
pyridine (36 mL) and Ac2O (18 mL) were added. Hydrogen sulfide
was bubbled into the solution for 15 min, and the resulting cloudy
mixture was stirred overnight at room temperature. The precipitated
mercuric salts were removed by filtration through a bed of Celite, and
the resulting clear filtrate was concentrated. Flash-column chromatog-
raphy (EtOAc-petroleum ether 1:3 v/v) of the residue gave compound
23b which crystallized in Et2O-petroleum ether (5 g, 89%): mp 70
°C; [R]25D +63 (c 1, CHCl3); 13C NMR (75 MHz) δ 191.5 (SCOCH3),
169.0, 169.2 and 169.3 (3 OCOCH3), 78.0 (C-1), 69.4, 68.8, 67.7 (C-
2, C-3, C-4), 62.9 (C-5), 30.9 (SCOCH3), 20.1 and 20.3 (3 OCOCH3);
1H NMR (300 MHz, CDCl3) δ 5.85 (d, 1 H, J1,2 4.1 Hz, H-1), 4.95 (m,
2 H, H-2, H-3), 4.75 (m, 1 H, H-4), 3.80 (dd, 1 H, J4,5eq 5.1 Hz, J5ax,5eq
12 Hz, H-5eq), 3.50 (dd, 1 H, J4,5ax 8.9 Hz, H-5ax), 2.25 (s, 3 H, SAc),
1.85, 1.87 and 1.88 (3 s, 9 H, CH3); Anal. Calcd for C13H18O8S: C,
46.69; H, 5.42; S, 9.59. Found: C, 46.72; H, 5.51; S, 9.34.
6-Bromo-6-deoxy-â-D-glucopyranosyl-(1f4)-6-bromo-6-deoxy-â-
r-D-glucopyranosyl fluoride (16e). Compound 16b (58.5 mg, 0.144
mmol) was dissolved in pyridine (5 mL) and treated as described for
the synthesis of 18b, and then the reaction mixture was acetylated under
standard conditions. Workup and flash chromatography (EtOAc-
petroleum ether 4:6 v/v) provided pure 25 in 23% yield: [R]25 +40
D
(c 0.79, CHCl3); 13C NMR (CDCl3, 75 MHz) δ 169.2-168.6 (CO),
103.6 (d, JC,F ) 229 Hz, C-1I), 99.7 (C-1II), 76.0 (C-4I), 73.3, 72.7,
71.6, 70.7, 70.6, 70.5, 68.2 (C-2I, II, C-3I, II, C-5I, II, C-4II), 31.5, 30.2
II
(C-6I, ), 21.3-20.4 (CH3); H NMR (CDCl3, 300 MHz) δ 5.69 (dd,
1
1H, J1,2 ) 2.7 Hz, J1,F ) 53 Hz, H-1I), 5.47 (t, 1H, J2, ) J3,4 ) 10 Hz,
H-3I), 5.16 (t, 1H, J2, ) J3, ) 9.3 Hz, H-3II), 5.01-4.88 (m, 3H, H-2,
H-2II, H-4II), 4.74 (d, 1H, J1,2 ) 8 Hz, H-1II), 4.05 (m, 1H, H-5I), 3.95
(t, 1H, J4,5 ) 9.3 Hz, H-4I), 3.67 (m, 3H, H-5II, H-6Ia, H-6Ib), 3.46
(dd, 1H, J5,6 ) 2.5 Hz, Ja,b ) 11.3 Hz, H-6IIa), 3.31 (dd, 1H, J5, 6 ) 6.7
Hz, H-6IIb), 2.10-1.97 (m, 15H, CH3); FABMS: m/z 703 [M + Na]+.
Unreacted compound 20 was recovered in 68% yield.
(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-glucopyranosyl fluo-
ride (24). Compound 23b (153 mg,0.45 mmol) was added to a solution
of bromide 20 (100 mg,0.3 equiv) in HMPA (2.5 mL) with dithio-
erythritol (23 mg, 1 equiv) and cysteamine (20 mg, 1.7 equiv). The
reaction mixture was stirred for 7 h at room temperature and then
precipitated in ice-cold water (10 mL). After filtration through a bed
of Celite, the precipitate was washed with H2O and then dissolved in
CH2Cl2. The organic phase was washed with H2O, dried, concentrated,
and purified by flash chromatography (EtOAc-petroleum ether 1:1
v/v) to give 24 (86 mg, 68%): [R]25D +80 (c 1.73, CHCl3); 13C NMR
(CDCl3, 75 MHz) δ 169.7-169.3 (CO), 103.5 (d, JC, F ) 230 Hz, C-1),
99.8 (C-1II), 82.4 (C-1III), 74.5, 74.4, 72.6, 71.6, 70.8, 70.6, 70.5, 70.2,
69.3, 68.8, 68.7 (C-2I, II, III, C-3I, II, III, C-4I, II, III, C-5I, II), 61.1, 59.4 (C-
6I, C-5III), 31.0 (C-6II), 20.7-20.4 (CH3); 1Η ΝΜR (CDCl3, 300 MHz)
Standard de-O-acetylation of compound 25 gave the corresponding
derivative 16e.
Enzymatic synthesis of modified celluloses. Cellobiosyl fluoride
16a (100 mg) in phosphate buffer (0.1M, pH 7.0, 6 mL) was incubated
with Cel7B Glu197Ala (2.5 mg) at 40 °C. After 24 h, the resulting
suspension was recovered by centrifugation and the solid was washed
with H2O (3×). The wet precipitate was used for electron microscopy
or X-ray analyses as already described.22 When dried under reduced
pressure at 40 °C in the presence of phosporous pentoxide, a white
1
powder of 17a was obtained (81 mg) with 13C and H NMR spectra
22
identical to those already reported.
6II-Bromo-6II-deoxy-R-cellobiosyl fluoride 16b (34 mg) in phosphate
buffer (0.1M, pH 7.0, 1.5 mL) was incubated with Cel7B Glu197Ala
(1.5 mg) at 40 °C for 24 h. A precipitate appeared (23 mg) and the
supernatant was freeze-dried and the residue was purified using steric
exclusion chromatography on a Biogel P2 column with water as eluent.
FABMS spectra with m/z 815 [M + Na]+and m/z 1203 [M + Na]+
allowed the characterization of corresponding tetra- and hexasaccharides
17b respectively. The precipitate was assumed to consist of higher
oligomers of 17b.
δ 5.64 (dd, 1H, J1,2 ) 2.4 Hz, J1,F ) 53 Hz, H-1I), 5.58 (d, 1H, J1,2
)
5.5 Hz, H-1III), 5.45 (t, 1H, J2,3 ) J3,4 ) 9.5 Hz, H-3I), 5.26 (t, 1H, J2,3
) J3,4 ) 9.2 Hz, H-3III), 5.10 (t, 1H, J2,3 ) J3,4 ) 9.3 Hz, H-3II), 4.92-
4.78 (m, 5H, H-2I, II, III, H-4II, III), 4.48 (m, 2H, J1,2 ) 8 Hz, H-1I, H-6Ia),
4.07 (m, 2H, H-5I, H-6Ib), 3.88 (m, 2H, H-4I, H-5IIIa), 3.76 (dd, 1H,
J4,5 ) 5.6 Hz, Ja,b ) 11.3 Hz, H-5IIIb), 3.53 (m, 1H, H-5II), 2.72 (dd,
1H, J5,6 ) 2.9 Hz, Ja,b ) 14 Hz, H-6IIa), 2.54 (dd, 1H, J5,6 ) 7.3 Hz,
H-6IIb), 2.1-1.93 (m, 27H, CH3); DCIMS: m/z 888 [M + H + NH3]+;
ΗRFABMS calcd for C35H47FO22S (M + Na)+ 893.2161, found
893.2156.
6II-Amino-6II-deoxy-R-cellobiosyl fluoride 16c (60 mg) in carbonate
solution (0.1M, pH 8.0, 2 mL) was incubated with Cel7B Glu197Ala
(1.2 mg) at 40 °C for 36 h. The solution was freeze-dried, a sample (2
mg‚mL-1) was filtered through Satorius membranes (0.45 and 0.2 µm)
and analyzed at 30 °C by steric exclusion chromatography on
Synchropak CATSEC 100 and 1000 columns using solution of AcOH
Unreacted fluoride 20 was also recovered (30 mg, 30%).
Preparation of the potential donors 16a - 16e. â-D-Glucopy-
ranosyl-(1f4)-r-D-glucopyranosyl fluoride (16a) and 6-Bromo-6-
deoxy-â-D-glucopyranosyl-(1f4)-r-D-glucopyranosyl fluoride (16b):
Hepta-O-acetyl-R-cellobiosyl fluoride or 2,3,6,2II,3II,4II-hexa-O-acetyl-
6II-bromo-6II-deoxy-R-cellobiosyl fluoride (20), 100 mg each were de-
(0.3M) containing AcONa (0.2 M)) with a flow rate of 0.2 mL‚min-1
.
The dn/dc value of 0.185 mL‚g-1 found for chitosan under the same
conditions was applied.40 The weight average molecular weight of 17c
is 5.000 ( 1.000.

Synthesis of â(1 f 4)-Polysaccharides
J. Am. Chem. Soc., Vol. 122, No. 23, 2000 5437
6II-Thioxylosyl -R-cellobiosyl fluoride 16d (25.5 mg) in phosphate
buffer (0.1 M, pH 7.0, 1 mL) was incubated with Cel7B Glu197Ala
(0.5 mg) at 40 °C for 24 h. Insoluble 17d was obtained (18.4 mg) after
centrifugation of the incubation mixture and H2O washings as already
Acknowledgment. This work was funded, in part, by CNRS,
the European Union (BIO4-CT97-2303), the Biotechnology and
Biological Sciences Research Council, the University of York
and Novo-Nordisk A/S. G.J.D. is a Royal Society University
Research Fellow. We thank Dr. C. Bosso, R. Vuong, C. Gey,
J. Mazet, and P. Colin-Morel for their assistance during the char-
acterization of the various compounds. This paper is dedicated
to Professor Pierre Sinay¨ on the occasion of his 62nd birthday.
1
described. Η ΝΜR (NaOD, 300 MHz) δ 5.13 (d, 1H, J1,2 ) 4.4 Hz,
H-1III), 4.3 (m, 2H, H-1I, II), 3.74-2.6 (m, 17H).
6,6II-Dibromo-R-cellobiosyl fluoride 16e ( 7.0 mg, 0.0015 mmol)
was found unchanged under the above conditions. In the presence of
lactosyl fluoride 1 (1 eq), incubation, acetylation, workup and purifica-
tion as described for the synthesis of 9 gave the expected tetrasaccharide
26 (15 mg, 80%): [R]25D +1.5 (c 0.45, CHCl3); 13C NMR (CDCl3, 75
MHz) δ 170.0-169.5 (CO), 103.7 (d, JC,F ) 222 Hz, C-1I), 100.9,
100.0, 99.7 (C-1II, III, IV), 77.1, 76.1, 75.7, 73.6, 72.9, 72.8, 72.4, 71.8,
71.7, 70.8, 70.6, 69.0, 68.3, 66.5 (C-2I, II, III, IV to C-5 I, II, III, IV), 62.1,
60.7 (C-6III, IV), 31.5, 31.0 (C-6I, II), 21.0-20.5 (CH3); ΗRFABMS calcd.
for C46H61Br2FO29 [M + Na]+: 1279.1547; found: 1279.1544.
Supporting Information Available: Coordinates for the
structures described in this paper have been deposited with the
Protein Data Bank with accession code 1DYM. Data for
determination of weight average molecular weight for 17c
(PDF). An X-ray crystallographic file in CIF format. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(40) Brugnerotto, J.; Rinaudo, M., personal communication.
JA9936520