Israel Journal of Chemistry Vol. 40 2000 pp. 217–221

*Author to whom correspondence should be addressed. E-mail:Hugues.Driguez@cermav.cnrs.fr

Stepwise Synthesis of Cellodextrins Assisted by a Mutant Cellulase

SÉBASTIEN FORT,a LARS CHRISTIANSEN,b MARTIN SCHÜLEIN,b SYLVAIN COTTAZ,a AND HUGUES DRIGUEZa,*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 theenzymatic stepwise condensations onto methyl β-D-glycoside acceptors. These reac-tions, catalyzed by the Cel7B E197A glycosynthase from Humicola insolens, gavecellodextrins from degree of polymerization 3 to 6 in high yields.

INTRODUCTIONCellulases (1,4-β-glucanases) are glycoside hydrolasesable to cleave the β-1,4 linkages of cellulose and itsoligomers. These enzymes have raised considerable in-terest in the past decade especially for their environmen-tally-sound bio-applications in the food, paper, and tex-tile industries.1 To develop new processes and cellulosicproducts, a clearer picture of structure–function rela-tionships of cellulases is a necessary prerequisite. Forthis reason, very fundamental studies to understand theprotein–substrate interactions have been undertaken re-quiring a series of 1,4-β-oligosaccharides.

Cellodextrins, with degree of polymerization (DP)between 2 and 7, form an important homologous familyof water-soluble substrates for cellulase assays. Re-duced, radioactive labeled, or chromogenic cello-oli-gosaccharides have become very useful to follow fer-mentation processes or to study steady-state kinetics ofcellulolytic enzymes.2 The synthesis of cellodextrinshas been investigated for many years and the acetolysisor acid hydrolysis of cellulose constitute expeditiousways of preparing low molecular weight oligomers inspite of difficult isolation and purification.3,4 Chemicalapproaches based on the selective blocking, deblocking,and activation of D-glucose derivatives have also beenundertaken by several groups. The trichloroacetimidatemethodology was used for the synthesis of cello-

tetraose,5 and allowed Nishimura and Nakatsubo to pre-pare higher oligomers up to an eicosamer.6,7 However,due to their low overall yield, chemical methods havebeen restricted to small-scale syntheses, and enzymaticalternatives have emerged. Transglycosylation andphosphorolytic reactions, catalyzed by cellulases8,9 andcellodextrin phosphorylase,10 respectively, were foundto be useful for the formation of 1,4-β-glycosidic link-ages. The former approach was successfully applied forthe synthesis of hemithiocellodextrins as cellulase in-hibitors11 and fluorogenic substrates12 for endoglu-canase assays. However, synthesis of higher oligomersthan DP 4 by this method is not practical since thereaction product is rapidly hydrolyzed by the enzyme,leading to an untractable mixture. Recently, thanks toprotein engineering of retaining β-glycosidases, enzy-matic syntheses of oligosaccharides have been im-proved. As expected, retaining enzyme in which thecarboxylate nucleophile is replaced by a non-carboxylicamino acid is unable to form the glycosyl enzyme inter-mediate and has no hydrolytic activity, but can stilltransfer a sugar residue from an α-glucosyl fluorideonto an acceptor molecule. This original concept devel-oped by Withers on the β-glucosidase from Agro-bacterium sp13,14 was successfully applied to the 1,3-1,4-β-glucanase from Bacillus lichenfornis,15 the Cel7B cel-

This paper is dedicated to Professor R.U. Lemieux.

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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 forglycosidase-assisted syntheses are lactosyl or D-galacto-syl fluorides, since the axial orientation of the hydroxylat C-4 prevents these substrates from being acceptors andleads to a single-step condensation with an appropriateacceptor. This method is, however, limited to the synthesisof oligosaccharides via a single enzymatic reaction, as thereaction product can be used neither as a novel donor noras an acceptor unless the terminal galactosyl residue isselectively cleaved before the next enzymatic coupling.8

In this paper, we describe the chemical synthesis ofthe selectively 4II-protected α-cellobiosyl fluoride 1 as aversatile building block for iterative cellodextrin syn-theses. This key derivative, easily prepared from2I,II,3I,II-tetra-O-acetyl-4II,6II-O-benylidene-1I,6I-anhydro-β-cellobiose19 (2), is efficiently condensedstep-by-step by the Cel7B E197A glycosynthase fromHumicola insolens leading to cellodextrins from DP 3 to 6.

EXPERIMENTAL

GeneralNMR 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 internalreference of solvent. Low-resolution mass spectra (MS) wererecorded 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 Centrald’Analyses du CNRS (Vernaison).

Evolution of reactions was monitored by analytical thin-layer chromatography using silica gel 60 F254 precoatedplates (E. Merck, Darmstadt).

All reactions in organic medium were carried out underargon using freshly distilled solvents. After workup, organicphases were dried over anhydrous Na2SO4.

The Cel7B E197A glycosynthase was prepared as alreadydescribed.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 solutionwas diluted with CH2Cl2, then washed with saturated aqueousNaHCO3. The organic layer was dried, concentrated, and fil-tered through silica gel (CH2Cl2–MeOH 95:5 v/v). The ex-pected tetra-O-acetyl-1,6-anhydro-β-cellobiose was obtained(1.7 g, 73%).

To a solution of this compound (1.7 g, 3.45 mmol) inCH2Cl2 (20 mL) were added 1-(benzoyloxy)-benzotriazole(825 mg, 1 equiv) and Et3N (480 µL, 1 equiv). After stirringthe solution for 48 h at room temperature, trichloroacetylchloride (775 µL, 2 equiv) and Et3N (1 mL, 2 equiv) wereadded. After 10 min, the reaction mixture was diluted withCH2Cl2 and successively washed with H2O and saturated aque-ous NaHCO3. The organic layer was dried, concentrated, andpurified by flash chromatography (EtOAc–petroleum ether1:1 v/v) to give 3, which crystallized in EtOH (2.3 g, 90% overthe two last steps); mp 159–160 °C; [α]25

D –30 (c 0.55, CHCl3);13C NMR (CDCl3, 75 MHz) δ 169.8–160.6 (CO), 133.2 (Cqarom.), 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 and12.5 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 compound3 (1.02 g, 1.38 mmol) in acetic anhydride (25 mL) cooled at0 °C. The mixture was stirred at 0 °C for 30 min, then saturatedaqueous NaHCO3 was added to quench the reaction, and theproducts were extracted with CH2Cl2. The organic layer wasdried, concentrated, and co-evaporated with toluene. HBr(30% w/v in AcOH, 5 mL) was added to the crude anomericmixture in CH2Cl2 (10 mL) at 0 °C. After stirring at 0 °C for30 min, then at room temperature for an additional 1 h, thereaction mixture was diluted with CH2Cl2, washed with ice-cold H2O, ice-cold saturated aqueous NaHCO3 (3×), dried, andconcentrated. The resulting bromide and AgOAc (460 mg, 2equiv) in a mixture of Ac2O–AcOH (20 mL, 1:1 v/v) werestirred in the dark overnight at room temperature. The mixturewas diluted with CH2Cl2, filtered through a Celite bed, and thefiltrate 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% over3 steps) which crystallized in EtOH; mp 195 °C; [α]25

D +1 (c0.71, 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); 1HNMR (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.

(2,3-Di-O-acetyl-6-O-benzoyl-4-O-trichloroacetyl-β-D-g l u c o p y r a n o s y l ) - ( 1 →4 ) - 2 , 3 , 6 - t r i - O - a c e t y l - α - D -glucopyranosyl fluoride (5). A solution of compound 4

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(750 mg, 0.9 mmol) in HF–pyridine (5 mL, 7:3 v/v ) wasstirred at 0 °C for 1 h in a plastic vial. The mixture was dilutedwith CH2Cl2 and poured into ice-cold aqueous NH3 (3 M), theorganic layer was washed with saturated aqueous NaHCO3

(3×), dried, and concentrated. Flash chromatography (EtOAc–petroleum ether 1:1 v/v) gave compound 5, which crystallizedin EtOH (675 mg, 94%); mp 134 °C; [α]25

D +31 (c 0.73,CHCl3); 13C NMR (CDCl3, 75 MHz) δ 170.0–160.5 (CO),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-6I,II), 20.7–20.3 (CH3); 1H 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. Calcdfor 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). Toa solution of fluoride 5 (1.19 g, 1.48 mmol) in DMF (5 mL) at50 °C, hydrazine acetate (136 mg, 1 equiv) was added every 5min until the total transformation of the starting material. Thesolution was then diluted with EtOAc, washed with saturatedaqueous NaCl, dried, concentrated, and purified by flash chro-matography (EtOAc–petroleum ether 7:3 v/v) to give purecompound 6 (855 mg, 88%); [α]25

D +18 (c 0.87, CHCl3);13C NMR (CDCl3, 75 MHz) δ 170.0–166.8 (CO), 133.5 (Cqarom.), 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 and12 Hz), 4.53 (m, 3H), 4.14–4.02 (m, 3H), 3.79 (t, 1H, J = 10Hz), 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.

(2,3-Di-O-acetyl-6-O-benzoyl-4-O-tetrahydropyranyl-β-D -glucopyranosyl ) - (1→4)-2 ,3 ,6- tr i -O -acety l -α -D -glucopyranosyl fluoride (7). To a solution of compound 6(1.02 g, 1.56 mmol) in CH2Cl2 (10 mL) were added freshlydistilled dihydropyran (680 µL, 5 equiv) and camphorsulfonicacid (24 mg, 0.07 equiv). After 1 h at room temperature, thesolution was diluted with CH2Cl2 and successively washedwith H2O and saturated aqueous NaHCO3. The organic layerwas dried, concentrated and purified by flash chromatography(EtOAc–petroleum ether 1:1 v/v) to give compound 7, whichcrystallized in EtOH (1.13 g, 98%); mp 140 °C; [α]25

D +25 (c1.05, CHCl3); FABMS: m/z 765 [M + Na]+; Anal. Calcd forC34H43FO17: C, 54.98; H, 5.84; F, 2.56. Found: C, 55.11; H,6.15; F, 2.46.

(4-O-Tetrahydropyranyl-β-D-glucopyranosyl)-(1→4)-α-D-glucopyranosyl fluoride (1). The fluoride 7 (1.13 g, 1.5

mmol) was de-O-acylated in methanol (100 mL) at 0 °C bysuccessive additions of sodium methoxide (1 M, 500 µL ) over30 h. The mixture was neutralized with Amberlite IR 120 (H+)resin, the resin was removed by filtration and the filtrate wasconcentrated. The free fluoride 1 was dissolved with the mini-mum of H2O, washed with Et2O to remove the methyl ben-zoate, and freeze dried (580 mg, 89%); FABMS: m/z 451 [M +Na]+.

General procedure for the enzymatic reactionsTHP 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. Thesolution was diluted with H2O to make the reaction productsoluble and 1 M HCl (1–2 drops) was added. After 5 min at pH1–2, the solution was neutralized with Et3N, concentrated todryness, and acetylated (acetic anhydride–pyridine 1:1 v/v, 10mL). After 12 h of stirring at 25 °C, the reaction was quenchedat 0 °C by addition of MeOH and concentrated. The residuewas dissolved in CH2Cl2 and washed with 20% aqueousKHSO4 and saturated aqueous NaHCO3. The organic layerswere 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 inaccordance with those recently described in the literature.4

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, 75MHz) δ 170.3–168.9 (CO), 101.2, 100.6, 100.3 (C-1I–III), 76.3,76.0, 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, 75MHz) 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(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 and11a 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,

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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%.

Scheme 2. a. Cel7B E197A, carbonate/bicarbonate buffer; b. 1M HCl then concentration; c. Ac2O/pyridine; d. MeONa/MeOH.

(C-2I–VI, C-3I–VI, C-4I–VI, C-5I–VI), 62.3, 62.1, 61.8 (C-6I–VI), 57.2(OCH3), 21.0–20.7 (CH3); FABMS: m/z 1825 [M + Na]+.

RESULTS AND DISCUSSION

For the synthesis of compound 1 (Scheme 1), we had tocircumvent the problem of selective protection of adisaccharide and to find a protecting group compatiblewith de-O-acylation basic conditions and strong acidicconditions employed during the fluorination step. Thesynthesis began with the acidic hydrolysis of the ben-

68.1 (C-2I–V, C-3I–V, C-4I–V, C-5I–V), 62.3, 62.1, 61.8 (C-6I–V),57.2 (OCH3), 21.0–20.7 (CH3); FABMS: m/z 1537 [M + Na]+.

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-g l u c o p y r a n o s y l ) - ( 1→ 4 ) - 2 , 3 , 6 - t r i - O - a c e t y l - 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

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zylidene group20 of 2, followed by the selectivebenzoylation21 of the free primary hydroxyl group of thetetra-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-anhydroring was performed by treatment of 3 with acetic anhy-dride in the presence of trimethylsilyl triflate.22 Theanomeric mixture of acetates was converted via thecorresponding α-bromide, followed by the action ofsilver acetate in acetic acid–acetic anhydride23 into thehexa-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 ofthe 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 aftermild de-O-acylation in 89% yield.

The THP-cellobiosyl fluoride 1 was used as the keydonor molecule for the stepwise enzymatic synthesis ofcellodextrins assisted by the Cel7B E197A glyco-synthase (Scheme 2). Methyl β-D-glucoside 8 andmethyl β-cellobioside 926 respectively, were used asacceptors for the synthesis of DP 3 and 4. The methylβ-cellotrioside 10a and methyl β-cellotetraoside 11awere respectively isolated in 78% and 90% yield, aftercoupling of 1 onto 8 and 9 in carbonate/bicarbonatebuffer. Isolation was done after in situ deprotection ofthe THP group by addition of 1 M HCl, followed byacetylation. De-O-acetylation of 10a and 11a gave 10band 11b, which on coupling with 1 afforded the expectedmethyl β-cellopentaoside 12 and methyl β-cello-hexaoside 13 in 83% and 60% yield, respectively.

The 4II-THP-α-cellobiosyl fluoride 1 available on agram scale has proven to be a versatile building blockfor glycosynthase-assisted synthesis. We have demon-strated that this compound, condensed step by step bythe Cel7B E197A mutant, affords an efficient access tocellodextrins of controlled size.

Acknowledgment. This work was supported in part by CNRSand the European Union (BIO4-CT97-2303)

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