Stepwise synthesis of cellodextrins assisted by a mutant cellulase

Article abstract of DOI:10.1560/5NCY-L9K2-JND4-MB6Q

A 4¹¹-protected α-cellobiosyl fluoride is an efficient donor for the enzymatic stepwise condensations onto methyl β-D-glycoside acceptors. These reactions, catalyzed by the Ce17B E197A glycosynthase from Humicola insolens, gave cellodextrins from degree of polymerization 3 to 6 in high yields.

Full text of DOI:10.1560/5NCY-L9K2-JND4-MB6Q

Stepwise Synthesis of Cellodextrins Assisted by a Mutant Cellulase
a,
SÉBASTIEN FORT,a LARS CHRISTIANSEN,b MARTIN SCHÜLEIN,b SYLVAIN COTTAZ,a AND HUGUES DRIGUEZ *
aCentre de Recherches sur les Macromolécules Végétales, CNRS, BP 53, 38041 Grenoble cedex 9, France
bNovo-Nordisk a/s, Novo allé, DK-2880 Bagvaerd, Denmark
(Received 21 August 2000 and in revised form 19 December 2000)
Abstract. A 4II-protected α-cellobiosyl fluoride is an efficient donor for the
enzymatic stepwise condensations onto methyl β-D-glycoside acceptors. These reac-
tions, catalyzed by the Cel7B E197A glycosynthase from Humicola insolens, gave
cellodextrins from degree of polymerization 3 to 6 in high yields.
INTRODUCTION
tetraose,5 and allowed Nishimura and Nakatsubo to pre-
Cellulases (1,4-β-glucanases) are glycoside hydrolases pare higher oligomers up to an eicosamer.6,7 However,
able to cleave the β-1,4 linkages of cellulose and its due to their low overall yield, chemical methods have
oligomers. These enzymes have raised considerable in- been restricted to small-scale syntheses, and enzymatic
terest in the past decade especially for their environmen- alternatives have emerged. Transglycosylation and
tally-sound bio-applications in the food, paper, and tex- phosphorolytic reactions, catalyzed by cellulases8,9 and
tile industries.1 To develop new processes and cellulosic cellodextrin phosphorylase,10 respectively, were found
products, a clearer picture of structure–function rela- to be useful for the formation of 1,4-β-glycosidic link-
tionships of cellulases is a necessary prerequisite. For ages. The former approach was successfully applied for
this reason, very fundamental studies to understand the the synthesis of hemithiocellodextrins as cellulase in-
protein–substrate interactions have been undertaken re- hibitors11 and fluorogenic substrates12 for endoglu-
quiring a series of 1,4-β-oligosaccharides.
canase assays. However, synthesis of higher oligomers
Cellodextrins, with degree of polymerization (DP) than DP 4 by this method is not practical since the
between 2 and 7, form an important homologous family reaction product is rapidly hydrolyzed by the enzyme,
of water-soluble substrates for cellulase assays. Re- leading to an untractable mixture. Recently, thanks to
duced, radioactive labeled, or chromogenic cello-oli- protein engineering of retaining β-glycosidases, enzy-
gosaccharides have become very useful to follow fer- matic syntheses of oligosaccharides have been im-
mentation processes or to study steady-state kinetics of proved. As expected, retaining enzyme in which the
cellulolytic enzymes.2 The synthesis of cellodextrins carboxylate nucleophile is replaced by a non-carboxylic
has been investigated for many years and the acetolysis amino acid is unable to form the glycosyl enzyme inter-
or acid hydrolysis of cellulose constitute expeditious mediate and has no hydrolytic activity, but can still
ways of preparing low molecular weight oligomers in transfer a sugar residue from an α-glucosyl fluoride
spite of difficult isolation and purification.3,4 Chemical onto an acceptor molecule. This original concept devel-
approaches based on the selective blocking, deblocking, oped by Withers on the β-glucosidase from Agro-
and activation of D-glucose derivatives have also been bacterium sp13,14 was successfully applied to the 1,3-1,4-
undertaken by several groups. The trichloroacetimidate β-glucanase from Bacillus lichenfornis,15 the Cel7B cel-
methodology was used for the synthesis of cello-
This paper is dedicated to Professor R.U. Lemieux.
*Authortowhomcorrespondenceshouldbeaddressed. E-mail:
Hugues.Driguez@cermav.cnrs.fr
Israel Journal of Chemistry
Vol. 40
2000 pp. 217–221

218
lulase from Humicola insolens,16 a thermophilic glu-
cosidase from Solfolobus solfataricus,17 and a β-1,3-
glucanase from barley.18 In the 1,4-β-D-gluco-oligosac-
charide series, the common glycosidic donors used for
glycosidase-assisted syntheses are lactosyl or D-galacto-
syl fluorides, since the axial orientation of the hydroxyl
at C-4 prevents these substrates from being acceptors and
leads to a single-step condensation with an appropriate
acceptor. This method is, however, limited to the synthesis
of oligosaccharides via a single enzymatic reaction, as the
reaction product can be used neither as a novel donor nor
as an acceptor unless the terminal galactosyl residue is
selectively cleaved before the next enzymatic coupling.8
In this paper, we describe the chemical synthesis of
the selectively 4II-protected α-cellobiosyl fluoride 1 as a
versatile building block for iterative cellodextrin syn-
theses. This key derivative, easily prepared from
2I,II,3I,II-tetra-O-acetyl-4II,6II-O-benylidene-1I,6I-
anhydro-β-cellobiose19 (2), is efficiently condensed
step-by-step by the Cel7B E197A glycosynthase from
Humicola insolens leading to cellodextrins from DP 3 to 6.
To a solution of this compound (1.7 g, 3.45 mmol) in
CH2Cl2 (20 mL) were added 1-(benzoyloxy)-benzotriazole
(825 mg, 1 equiv) and Et3N (480 µL, 1 equiv). After stirring
the solution for 48 h at room temperature, trichloroacetyl
chloride (775 µL, 2 equiv) and Et3N (1 mL, 2 equiv) were
added. After 10 min, the reaction mixture was diluted with
CH2Cl2 and successively washed with H2O and saturated aque-
ous NaHCO3. The organic layer was dried, concentrated, and
purified by flash chromatography (EtOAc–petroleum ether
1:1 v/v) to give 3, which crystallized in EtOH (2.3 g, 90% over
the two last steps); mp 159–160 °C; [α]25D –30 (c 0.55, CHCl3);
13C NMR (CDCl3, 75 MHz) δ 169.8–160.6 (CO), 133.2 (Cq
arom.), 129.6–128.4 (CH arom.), 100.8, 98.8 (C-1I,II), 77.4,
73.7, 72.5, 72.1, 71.6, 71.5, 69.3, 68.5 (C-2I,II, C-3I,II, C-4I,II,
C-5I,II), 64.8, 62.0 (C-6I,II), 20.6–20.4 (CH3); 1H NMR (CDCl3,
300 MHz) δ 8.0–7.24 (m, 5H, H arom.), 5.39 (t, 2H, J = 9 Hz),
5.26 (t, 1H, J = 9.5 Hz), 5.17 (s, 1H), 5.05 (t, 1H, v = 8.5 Hz),
4.99 (t, 1H, J = 8 Hz), 4.55 (m, 3H), 4.36 (dd, 1H, J = 5 and
12.5 Hz), 4.09 (ddd, 1H, J = 2 and 5 and 7 Hz), 3.88 (d, 1H, J =
8 Hz), 3.72 (dd, 1H, J = 5.8 and 8 Hz), 3.50 (s, 1H), 1.98–1.94
(m, 12, CH3); Anal. Calcd for C29H31Cl3O16: C, 46.95; H, 4.21;
Cl, 14.34. Found: C, 47.51; H, 4.30; Cl, 14.45.
(2,3-Di-O-acetyl-6-O-benzoyl-4-O-trichloroacetyl-β-D-
glucopyranosyl)-(1→4)-1,2,3,6-tetra-O-acetyl-β-D-glucopy-
ranose (4). A catalytic amount of triethylsilyl triflate (40 µL)
was added, dropwise via a syringe, to a solution of compound
3 (1.02 g, 1.38 mmol) in acetic anhydride (25 mL) cooled at
0 °C. The mixture was stirred at 0 °C for 30 min, then saturated
aqueous NaHCO3 was added to quench the reaction, and the
products were extracted with CH2Cl2. The organic layer was
dried, concentrated, and co-evaporated 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, then at room temperature for an additional 1 h, the
reaction mixture was diluted with CH2Cl2, washed with ice-
cold H2O, ice-cold saturated aqueous NaHCO3 (3×), dried, and
concentrated. The resulting bromide and AgOAc (460 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, 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 6:4 v/v) gave compound 4 (995 mg, 85% over
EXPERIMENTAL
General
NMR spectra were recorded on a Brüker AC 300. Proton
chemical shifts (δ) are reported in ppm downfield from TMS.
Coupling constants (J) are in hertz (Hz) with singlet (s), dou-
blet (d), doublet of doublet (dd), triplet (t), multiplet (m).
Carbon chemical shifts (δ) are reported in ppm with internal
reference of solvent. Low-resolution mass spectra (MS) were
recorded on a Nermag R-1010C spectrometer. Optical rota-
tions were measured with a Perkin-Elmer 341 polarimeter.
Melting points were measured on a Büchi 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).
All reactions in organic medium were carried out under
argon using freshly distilled solvents. After workup, organic
phases were dried over anhydrous Na2SO4.
3 steps) which crystallized in EtOH; mp 195 °C; [α]25 +1 (c
D
The Cel7B E197A glycosynthase was prepared as already
0.71, CHCl3); 13C NMR (CDCl3, 75 MHz) δ 170.0–165.8
(CO), 133.5 (Cq arom.), 129.6–128.6 (CH arom.), 100.7 (C-
1II), 91.5 (C-1I), 76.0, 73.4, 72.2, 72.0, 71.6, 71.3, 70.3 (C-2I,II,
C-3I,II, C-4I,II, C-5I,II), 61.8, 61.3 (C-6I,II), 20.7–20.3 (CH3); 1H
NMR (CDCl3, 300 MHz) δ 8.03–7.45 (m, 5H, H arom.), 5.62
(d, 1H, J1,2 = 8 Hz, H-1I), 5.33–5.16 (m, 3H), 5.02–4.93 (m,
2H), 4.59–4.39 (m, 4H), 4.06 (dd, 1H, J = 4.5 and 12 Hz), 3.93
(m, 1H), 3.83–3.70 (m, 2H), 2.07–1.86 (m, 18H, CH3);
FABMS: m/z 865 [M + Na]+; Anal. Calcd for C33H37Cl3O19: C,
46.96; H, 4.42; Cl, 12.60. Found: C, 47.21; H, 4.36; Cl, 12.49.
described.16
(2,3-Di-O-acetyl-6-O-benzoyl-4-O-trichloroacetyl-β-D-
glucopyranosyl)-(1→4)-2,3-di-O-acetyl-1,6-anhydro-β-D-
glucopyranose (3). To a solution of 2I,II,3I,II-tetra-O-acetyl-
4II,6II-O-benzylidene-1I,6I-anhydro-β-cellobiose19 (2, 2.75 g,
4.74 mmol) in CH2Cl2 (30 mL) was added a TFA–H2O mixture
(9:1 v/v, 3 mL). After 15 min at room temperature, the solution
was diluted with CH2Cl2, then washed with saturated aqueous
NaHCO3. The organic layer was dried, concentrated, and fil-
tered through silica gel (CH2Cl2–MeOH 95:5 v/v). The ex-
(2,3-Di-O-acetyl-6-O-benzoyl-4-O-trichloroacetyl-β-D-
pected tetra-O-acetyl-1,6-anhydro-β-cellobiose was obtained glucopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-α-D -
(1.7 g, 73%).
glucopyranosyl fluoride (5). A solution of compound 4
Israel Journal of Chemistry
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2000

219
(750 mg, 0.9 mmol) in HF–pyridine (5 mL, 7:3 v/v ) was mmol) was de-O-acylated in methanol (100 mL) at 0 °C by
stirred at 0 °C for 1 h in a plastic vial. The mixture was diluted successive additions of sodium methoxide (1 M, 500 µL ) over
with CH2Cl2 and poured into ice-cold aqueous NH3 (3 M), the 30 h. The mixture was neutralized with Amberlite IR 120 (H+)
organic layer was washed with saturated aqueous NaHCO3 resin, the resin was removed by filtration and the filtrate was
(3×), dried, and concentrated. Flash chromatography (EtOAc– concentrated. The free fluoride 1 was dissolved with the mini-
petroleum ether 1:1 v/v) gave compound 5, which crystallized mum of H2O, washed with Et2O to remove the methyl ben-
in EtOH (675 mg, 94%); mp 134 °C; [α]25 +31 (c 0.73, zoate, and freeze dried (580 mg, 89%); FABMS: m/z 451 [M +
D
CHCl3); 13C NMR (CDCl3, 75 MHz) δ 170.0–160.5 (CO), Na]+.
133.6 (Cq arom.), 129.6–128.9 (CH arom.), 103.5 (d, JC,F
=
230 Hz, C-1I), 100.6 (C-1II), 75.4, 72.2, 72.0, 71.6, 71.3, 70.5,
70.3, 70.0, 68.5 (C-2I,II, C-3I,II, C-4I,II, C-5I,II), 61.9, 60.8 (C-
General procedure for the enzymatic reactions
THP fluoride 1 (0.23 mmol) and acceptors (1 equiv) in
carbonate/bicarbonate buffer (0.1 M, pH 10, 5 mL) were incu-
bated with Cel7B E197A (1.5 mg) for 15 h at 40 °C. The
solution was diluted with H2O to make the reaction product
soluble and 1 M HCl (1–2 drops) was added. After 5 min at pH
1–2, the solution was neutralized with Et3N, 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
at 0 °C by addition of MeOH and concentrated. The residue
was dissolved in CH2Cl2 and washed with 20% aqueous
KHSO4 and saturated aqueous NaHCO3. The organic layers
were dried, concentrated, and purified by flash chromatogra-
phy (EtOAc–petroleum ether 7:3 v/v) to give methyl β-
cellotrioside 10a (78%), methyl β-cellotetraoside 11a (90%),
methyl β-cellopentaoside 12 (83%) or methyl β-
cellohexaoside 13 (60%). The NMR and mass spectra were in
accordance with those recently described in the literature.4
1
6I,II), 20.7–20.3 (CH3); H NMR (CDCl3, 300 MHz) δ 7.60–
7.43 (m, 5H, H arom.), 5.65 (dd, 1H, J1,2 = 3 Hz, J1,F = 53 Hz,
H-1I), 5.45-5.25 (m, 3H), 4.97 (t, 1H, J = 9 Hz), 4.80 (dd, 1H,
J = 3 and 10 Hz), 4.63–4.52 (m, 3H including H-1II), 4.38 (dd,
1H, J = 4.5 and 12.5 Hz), 4.12–4.04 (m, 2H), 3.95 (m, 1H),
3.80 (t, 1H, J = 10 Hz), 2.08–1.87 (m, 15H, CH3); Anal. Calcd
for C31H34Cl3FO17: C, 46.31; H, 4.26; Cl, 13.23; F, 2.36.
Found: C, 46.46; H, 4.02; Cl, 13.22; F, 2.22.
(2,3-Di-O-acetyl-6-O-benzoyl-β-D-glucopyranosyl)-
(1→4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl fluoride (6). To
a solution of fluoride 5 (1.19 g, 1.48 mmol) in DMF (5 mL) at
50 °C, hydrazine acetate (136 mg, 1 equiv) was added every 5
min until the total transformation of the starting material. The
solution was then diluted with EtOAc, washed with saturated
aqueous NaCl, dried, concentrated, and purified by flash chro-
matography (EtOAc–petroleum ether 7:3 v/v) to give pure
compound 6 (855 mg, 88%); [α]25 +18 (c 0.87, CHCl3);
Methyl (2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-
(1→4)-(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-2,3,6-
tri-O-acetyl-β-D-glucopyranoside (10a). 13C NMR (CDCl3, 75
MHz) δ 170.3–168.9 (CO), 101.2, 100.6, 100.3 (C-1I–III), 76.3,
D
13C NMR (CDCl3, 75 MHz) δ 170.0–166.8 (CO), 133.5 (Cq
arom.), 129.6–128.5 (CH arom.), 103.5 (d, JC,F = 230 Hz,
C-1I), 100.7 (C-1II), 75.4, 74.2, 71.5, 70.7, 70.6, 70.4, 70.0,
68.7, 68.4 (C-2I,II, C-3I,II, C-4I,II, C-5I,II), 63.1, 61.0 (C-6I,II),
20.6–20.4 (CH3); 1H NMR (CDCl3, 300 MHz) δ 8.01–7.42 (m,
5H, H arom.), 5.62 (d, 1H, J1,2 = 2.5 Hz, J1,F = 53 Hz, H-1I),
5.41 (t, 1H, J = 9.5 Hz), 5.0 (t, 1H, J = 9 Hz), 4.87–4.81 (m,
1H), 4.77 (dd, 1H, J = 3 and 10 Hz), 4.65 (dd, 1H, J = 3.7 and
12 Hz), 4.53 (m, 3H), 4.14–4.02 (m, 3H), 3.79 (t, 1H, J = 10
Hz), 3.65 (m, 1H), 2.06–1.94 (m, 15H, CH3); DCIMS: m/z 676
[M + H + NH3]+; Anal. Calcd for C29H35FO16: C, 52.89; H,
5.36; F, 2.88. Found: C, 53.15; H, 5.52; F, 2.68.
76.0, 72.7, 72.5, 72.4, 71.8, 71.7, 71.5, 67.7 (C-2I–III, C-3I–III
,
C-4I–III, C-5I–III), 62.0, 61.6, 61.4 (C-6I–III), 56.7 (OCH3), 20.6–
20.3 (CH3); DCIMS: m/z 956 [M + H+ NH3]+.
Methyl (2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-
(1→4)-(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-
(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-2,3,6-tri-O-
acetyl-β-D-glucopyranoside (11a). 13C NMR (CDCl3, 75
MHz) δ 169.5–168.9 (CO), 101.3, 100.6, 100.3 (C-1I–IV), 76.3,
76.1, 76.0, 72.8, 72.6, 72.3, 71.9, 71.8, 71.6, 71.5 67.7 (C-2I–IV
,
C-3I–IV, C-4I–IV, C-5I–IV), 62.0, 61.7, 61.4 (C-6I–IV), 56.8
(2,3-Di-O-acetyl-6-O-benzoyl-4-O-tetrahydropyranyl-β-
D -glucopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-α-D -
glucopyranosyl fluoride (7). To a solution of compound 6
(1.02 g, 1.56 mmol) in CH2Cl2 (10 mL) were added freshly
distilled dihydropyran (680 µL, 5 equiv) and camphorsulfonic
acid (24 mg, 0.07 equiv). After 1 h at room temperature, the
solution was diluted with CH2Cl2 and successively washed
with H2O and saturated aqueous NaHCO3. The organic layer
was dried, concentrated and purified by flash chromatography
(EtOAc–petroleum ether 1:1 v/v) to give compound 7, which
crystallized in EtOH (1.13 g, 98%); mp 140 °C; [α]25D +25 (c
1.05, CHCl3); FABMS: m/z 765 [M + Na]+; Anal. Calcd for
C34H43FO17: C, 54.98; H, 5.84; F, 2.56. Found: C, 55.11; H,
6.15; F, 2.46.
(OCH3), 20.9–20.4 (CH3); FABMS: m/z 1250 [M + Na]+
Methyl β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-
(1→4)-β-D-glucopyranoside (10b) and Methyl β-D-gluco-
pyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-gluco-
pyranosyl-(1→4)-β-D-glucopyranoside (11b). They were ob-
tained by Zemplèn de-O-acetylation of compounds 10a and
11a with 1 M sodium methoxide (1% v/v in MeOH), neutral-
ization with Amberlite IR 120 (H+) resin, and concentration.
Methyl (2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-
(1→4)-(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-
(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-
acetyl-β-D-glucopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-
glucopyranoside (12). 13C NMR (CDCl3, 75 MHz) δ 169.5–
169.3 (CO), 101.7, 101.0, 100.7 (C-1I–V), 76.7, 76.4, 76.3,
73.1, 73.09, 72.99, 72.9, 72.8, 72.7, 72.3, 72.16, 72.06, 71.87,
(4-O-Tetrahydropyranyl-β-D-glucopyranosyl)-(1→4)-α-
D-glucopyranosyl fluoride (1). The fluoride 7 (1.13 g, 1.5
Fort et al. / Enzymatic Synthesis of Cellodextrins

220
Scheme 1. a. TFA/H2O (9/1), CH2Cl2, 73%; b. 1-(benzoyloxy)-benzotriazole, Et3N, CH2Cl2 then TCACl, 90%; c. TESOTf,
Ac2O; d. HBr then AgOAc, Ac2O/AcOH, 85% ( 2 steps); e. HF/pyridine, 94%; f. NH2NH2-AcOH, DMF, 88%; g. dihydropyran,
CH2Cl2, CSA, 98%; h. MeONa/MeOH, 89%.
68.1 (C-2I–V, C-3I–V, C-4I–V, C-5I–V), 62.3, 62.1, 61.8 (C-6I–V), (C-2I–VI, C-3I–VI, C-4I–VI, C-5I–VI), 62.3, 62.1, 61.8 (C-6I–VI), 57.2
57.2 (OCH3), 21.0–20.7 (CH3); FABMS: m/z 1537 [M + Na]+. (OCH3), 21.0–20.7 (CH3); FABMS: m/z 1825 [M + Na]+.
Methyl (2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-
RESULTS AND DISCUSSION
(1→4)-(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-
For the synthesis of compound 1 (Scheme 1), we had to
(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-
circumvent the problem of selective protection of a
disaccharide and to find a protecting group compatible
with de-O-acylation basic conditions and strong acidic
conditions employed during the fluorination step. The
synthesis began with the acidic hydrolysis of the ben-
acetyl-β-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-β-D-
glucopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D -
glucopyranoside (13). 13C NMR (CDCl3, 75 MHz) δ 170.4–
169.5 (CO), 101.7, 101.0, 100.7 (C-1I–VI), 76.7, 76.4, 76.3,
73.2, 73.1, 73.0, 72.9, 72.8, 72.3, 72.2, 72.1, 71.9, 68.1
Scheme 2. a. Cel7B E197A, carbonate/bicarbonate buffer; b. 1M HCl then concentration; c. Ac2O/pyridine; d. MeONa/MeOH.
Israel Journal of Chemistry 40 2000

221
zylidene group20 of 2, followed by the selective
benzoylation21 of the free primary hydroxyl group of the
tetra-O-acetyl-1I,6I -anhydro-β-cellobiose and in situ in-
troduction of a trichloroacetyl (TCA) group at its posi-
tion 4II. The expected compound 3 was isolated in 66%
yield over three steps. Acetolysis of the 1,6-anhydro
ring was performed by treatment of 3 with acetic anhy-
dride in the presence of trimethylsilyl triflate.22 The
anomeric mixture of acetates was converted via the
corresponding α-bromide, followed by the action of
silver acetate in acetic acid–acetic anhydride23 into the
hexa-O-acetyl-6II-O-benzoyl-4II-O-trichloroacetyl-β-
cellobiose 4 in 85% yield. Treatment of 4 with a hydro-
gen fluoride–pyridine mixture (7/3 v/v)24 led to the α-
cellobiosyl fluoride 5 in 94% yield. Selective removal of
the TCA group with hydrazine acetate25 afforded 6,
which could be converted in 98% yield into the tetra-
hydropyranyl (THP)-protected cellobiosyl fluoride 7.
The expected compound 1 was finally obtained after
mild de-O-acylation in 89% yield.
DC, 1998.
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The THP-cellobiosyl fluoride 1 was used as the key
donor molecule for the stepwise enzymatic synthesis of
cellodextrins assisted by the Cel7B E197A glyco-
synthase (Scheme 2). Methyl β-D-glucoside 8 and
methyl β-cellobioside 926 respectively, were used as
acceptors for the synthesis of DP 3 and 4. The methyl
β-cellotrioside 10a and methyl β-cellotetraoside 11a
were respectively isolated in 78% and 90% yield, after
coupling of 1 onto 8 and 9 in carbonate/bicarbonate
buffer. Isolation was done after in situ deprotection of
the THP group by addition of 1 M HCl, followed by
acetylation. De-O-acetylation of 10a and 11a gave 10b
and 11b, which on coupling with 1 afforded the expected
methyl β-cellopentaoside 12 and methyl β-cello-
hexaoside 13 in 83% and 60% yield, respectively.
The 4II-THP-α-cellobiosyl fluoride 1 available on a
gram scale has proven to be a versatile building block
for glycosynthase-assisted synthesis. We have demon-
strated that this compound, condensed step by step by
the Cel7B E197A mutant, affords an efficient access to
cellodextrins of controlled size.
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Acknowledgment. This work was supported in part by CNRS
and the European Union (BIO4-CT97-2303)
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