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ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2007, Vol. 33, No. 1, pp. 99109. Pleiades Publishing, Inc.

., 2007. Original Russian Text N.E. Byramova, A.B. Tuzikov, T.V. Tyrtysh, N.V. Bovin, 2007, published in Bioorganicheskaya Khimiya, 2007, Vol. 33, No. 1, pp. 108118.

SYNTHETIC STUDIES

1,6-Anhydro-N-Acetyl-b-D-Glucosamine in Oligosaccharide Synthesis: II.1 The Synthesis of the Spacered Ley Tetrasaccharide
N. E. Byramova2, A. B. Tuzikov, T. V. Tyrtysh, and N. V. Bovin
ShemyakinOvchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia
Received April 03, 2006; in nal form, May 11, 2006

Abstract3-Aminopropyl glycoside of 3,2'-di-O--L-fucosyl-N-acetyllactosamine (Ley tetrasaccharide) was synthesized. The glycosyl donor, 2-O-acetyl-2,4,6-tri-O-benzoyl--D-galactopyranosyl bromide, was coupled with glycosyl acceptor, 1,6-anhydro-2-acetamido-2-deoxy--D-glucopyranose or its 3-O-acetyl derivative, to give the corresponding N-acetyllactosamine derivatives in 20 and 71% yields, respectively. The glycosyl donor was synthesized from 1,2-di-O-acetyl-3,4,6-triO-benzoyl-D-galactopyranose, which was obtained by the treatment of benzobromogalactose with sodium borohydride to yield 1,2-O-benzylidene derivative and subsequent removal of benzylidene group and acetylation. Acidic methanolysis of the disaccharide derivatives resulted in the selective removal of one or both acetyl groups to give the disaccharide acceptor bearing hydroxy groups at C3 of the glucosamine residue and C2 of the galactose residue. The introduction of fucose residues in these positions by the treatment with tetrabenzylfucopyranosyl bromide resulted in a tetrasaccharide derivative, which was converted into 3,2'-di-O--L-fucopuranosyl-1,6-anhydro-N-acetyllactosamine peracetate after substitution of acetyl groups for benzoyl and benzyl groups. Opening of the anhydro ring by acetolysis resulted in peracetate, which was then converted into the corresponding oxazoline derivative by two steps. Glycosydation of the oxazoline derivative with 3-triuoroacetamidopropan-1-ol and removal of O-acetyl and N-triuoroacetyl protective groups resulted in a free spacered Ley tetrasaccharide. Key words: 1,6-anhydro-GlcNAc, glycosylation, oligosaccharides, tumor-associated antigens DOI: 10.1134/S1068162007010128

INTRODUCTION Previously [1], we suggested 1,6-anhydro-GlcNAc as a synthon for the syntheses of oligosaccharides containing the N-acetyllactosamine unit (LacNAc, Gal14GlcNAc disaccharide). N-Acetyllactosamine substituted with the -L-fucose residue at 2 of the galactose moiety is a constituent of group-specic H (type 2) antigen; N-acetyllactosamine bearing the -L-fucose residue in O3 of the GlcNAc moiety is a constituent of Lex antigen, and, finally, N-acetyllactosamine substituted with two -L-fucose residues in both 2 of Gal and O3 of GlcNAc is a part of Ley antigen (see Scheme 1) [2, 3]. A number of communications report the synthesis of H (type 2), Lex, and Ley oligosaccharides [4]. For example, a synthesis of Ley 2(4-nitrophenyl)ethyl glycoside is described in [5] (see also communications cited therein). A synthesis of Ley from the derivative of galactose glycal is reported in [6]. In our laboratory, synthesis of 3-aminopropyl glycosides of H (type 2)
1 2

and Ley using another strategy, i.e., starting from the GlcNAc derivative with a spacer group, has also been carried out [7]. The following strategy for the synthesis is suggested [8, 9]. First, the lactosamine core (I) disaccharide bearing only acetyl and benzoyl protective groups in the corresponding positions is built up, and the hydroxy group at the reducing end is blocked by including into the 1,6-anhydro ring [(Ib), (Ic), and (Id)] (Scheme 2). The core (I) is constructed by condensation of glycosyl donor (II) with the corresponding acceptor (IIIa)
Gal1-4GlcNAc Gal1-4GlcNAc Gal1-4GlcNAc 3 2 2 Fuc1 H (type 1) Fuc1 Fuc1 Ley Gal1-4GlcNAc 3 Fuc1 Lex

For Part I, see [1]. Corresponding author, phone: +7 (495) 330-7492; e-mail: tuzikov@carb.ibch.ru.

Scheme 1.

99

100 OR O O R'O (I) (I) R = R' = H (Ib) R = R' = Ac (Ic) R = H, R' = Ac (Id) R = Ac, R' = H (Ie) R = Bz, R' = Ac (If) R = Ac, R' = Bz

BYRAMOVA et al. O BzO BzO OBz O NHAc BzO BzO AcO (II) Br H3C BnO (IV)
Scheme 2.

OBz O Br HO OR O

NHAc (IIIc) (III) R = H (IIIb) R = Bz (IIIc) R = Ac

O OBn

OBn

BzO BzO

OBz O BzO (V) Br

BzO BzO

OBz O O O BzO BzO RO OR (VII) R = H (II) R = Ac OBz O (II)

Ph (VI)
Scheme 3.

(IIIc), a derivative of 1,6-anhydro-GlcNAc. A mild acidic methanolysis [10] of the key disaccharide (I), which allows the selective removal of acetyl groups in the presence of benzoyl groups, results in deprotection of the hydroxy groups in the positions to be fucosylated. Mono- or bisfucosylation in these positions with donor (IV) results in the target oligosaccharides. The next step includes deprotection of the reducing end by opening of the 1,6-anhydro ring and subsequent treatment with the spacer alcohol. We herein report the synthesis of protected Ley tetrasaccharide (XVII) and its transformation to free aminopropyl glycoside (XVIII) convenient for further modications at the amino group. Neoglycoconjugates obtained on the basis of tetrasaccharide (XVIII) according to the method [11], have been used in a number of biological studies [1215], in particular, for obtaining monoclonal antibodies to Ley antigen [12]. RESULTS AND DISCUSSION The substituted galactose donor, 2--acetyl-3,4,6tri--benzoyl--D-galactopyranosyl bromide (II) also necessary for obtaining of the H (type 2) trisaccharide was synthesized starting from bezobromogalactose (V) according to a tree-step scheme. This scheme methodologically coincides with the scheme used by Kochet-

kov et al. [16, 17] for the synthesis of the L-rhamnose derivative with similar protective groups. Benzobromogalactose (V) was converted into tribenzoate of 1,2--benzylidene derivative (VI) as a mixture of R- and S-isomers by the treatment with sodium borohydride in acetonitrile in the presence of tetrabutylammonium iodide according to the method [18] (see Scheme 3). The removal of the benzylidene protective group with triuoroacetic acid resulted in diol (VII), which was then acetylated to diacetate (II), the precursor of bromide (II). Diacetate (IIa) can be synthesized according to the scheme (V) (VI) (VII) (II) without purification of intermediates [except for diol(VII)] and results in diacetate (II) in a high yield from the starting bromide (V). In the original variant of core (I) synthesis, diol (III) [1] was used as acceptor to provide the shortest route to Ley tetrasaccharide. The reaction of acceptor (III) with donor (II) in acetonitrile or in acetonitrile dichloromethane in the presence of mercury cyanide mercury bromide resulted in disaccharide (Ic) in approximately 3% yield. The use of nitromethanebenzene as a solvent at room temperature allowed an increase in the yield up to 10%. The proceeding of the reaction in reuxing nitromethanebenzene appeared to be the best variant of condensation conditions. The yield of the target -(14)disaccharide (Ic) was 20%.
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1,6-ANHYDRO-N-ACETYL--D-GLUCOSAMINE BzO BzO AcO HO OBz O O O NHAc O OBz BzO AcO BzO
Scheme 4.

101 RO OR O O O O O O O OR (X) R = Bn, R' = Bz (XI) R = Bn, R' = H (XII) R = R' = H (XIII) R = R' = Ac
Scheme 5.

(VIII) (13) 7% R'O (I) + (IV) R'O

H3C OR'

OR O

NHAc

OH O O O NHAc

(IX) (14) ~1.4%

H3C RO

OR

Glycosylation in the O4 position was evident from the Overhauser effect in the 1H NMR spectra. To complete the conrmation of the formation of the -(14) linkage, the resulting derivative (Ic) was transformed to acetate (Ib). The signal of H3 of the GlcNAc residue, which appeared as a broadened singlet at 3.894 ppm in the 1H NMR spectrum of the starting hydroxy derivative (Ic), underwent the characteristic downeld shift to 4.995 in the case of 3--acetylated derivative (Ib). The 1H NMR spectrum of (Ib) totally coincided with that of the disaccharide obtained by condensation of bromide (II) with acceptor (IIc), 3-Oacetyl derivative of 1,6-anhydro-GlcNAc, reported in the previous communication [1]. Along with the target -(14)-disaccharide (Ic), we have also isolated from the mixture of diol (IIIa) galactosylation products (VIII) and (IX) in 7 and 1.4% yield, respectively (Scheme 4), which were identied as -(13) and -(14) isomers by 1H NMR spectra. Further, we carried out the condensation of donor (II) with 4-hydroxy derivative (IIIc) as the acceptor. Glycosylation under the conditions of the Helferich reaction proceeded as expected with a high efciency and resulted in disaccharide derivative (Ib) in 71% yield; it was identical to that obtained from disaccharide (Ic) by acetylation. Disaccharides (Ic) and (Ib) were subjected to selective acidic deacetylation [10]. We succeeded in conversion of derivative (Ic) bearing the only hydroxyl group at 2 of the galactose residue to the target diol (I) in 52% yield and returned 19% of the starting (Ic). An increase in the reaction time (more than 24 h at room temperature) raised the conversion of the starting (Ic), but resulted in appearance of side products. Deacetylation of disaccharide derivative (Ib) under the same conditions proceeded with the same effectiveness and resulted in diol (Ia) (55%) and starting diacetate (Ib) recovered (20%, data not shown in the Experimental section). Milder reaction conditions due to increase in the chloroform content in reaction mixture
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resulted in a decrease in the diol (Ia) yield to 40%, but allowed us to isolate disaccharide (Ic), the product of deacetylation at 3 of the glucosamine residue, in 31% yield. Moreover, disaccharide (Id), the product of deacetylation at 2 of the galactose residue (8%), and the starting diacetate (Ib) (4%) were also isolated. The results demonstrate that the acetyl group at O2 of the -galactopyranose residue of diacetate (Ib) is more stable than that at O3 of the glucosamine residue under the conditions of acidic methanolysis. This correlates well with the literature data on the increased stability of acetyl group at O2 of galactose -glycoside under the conditions of the Zemplen deacetylation [19]. Thus, a selective deacetylation of diacetate (Ib) enables the preparation of all the core disaccharide acceptors required for the syntheses of the corresponding oligosaccharides Ley, Lex, and H (type 2). The resulting diol (I) was condensed with fucosyl bromide (IV) [20] by the Lemieux method [21, 22] (Scheme 5). The product (X) was debenzoylated by sodium methylate in methanol to give triol (XI), which was isolated in 57% yield by column chromatography [from starting diol (I) in two steps].The 1H NMR spectrum of (XI) exhibited two doublets at 1.347 and 1.388 ppm with a coupling constant of 6.5 Hz corresponding to H6 atoms of fucose residues. The -conguration of the fucose residues was conrmed by the presence of two doublets at 5.346 and 5.414 ppm with J 3.6 and 3.1 Hz, respectively (see the Experimental section). The catalytic hydrogenolysis of benzyl groups in (XI) over Pd/C resulted in the free tetrasaccharide (XII) in 96% yield. Acetylation of (XII) with the acetic anhydride in pyridine resulted in the peracetate of 1,6-anhydro derivative of Ley tetrasaccharide (XIII) in 94% yield. The derivative (XIII) was acetolyzed by acetic anhydride in acetic acid in the presence of concentrated
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BYRAMOVA et al. O AcNH OH (XV) O N (XVI) O

AcNH OAc (XIV)

HO H 3C

CH3 OH O O OH OH O O OH O OH
Scheme 6.

O O(CH2)3NH2 AcNH

HO HO

O(CH2)3NHCOCF

H 3C HO

AcNH (XVIII) OH

(XVII)

H2SO4 to open the 1,6-anhydro ring. Substitution of triuoroacetic acid for sulfuric acid did not result in opening of the 1,6-anhydro ring. It should be noted that free tetrasaccharide (XII) can also be subjected to acetolysis, as it reacts similarly to its acetate (XIII). The attempt to acetolyze derivative (XI) with benzylated fucose residues resulted in almost complete cleavage of fucoside bond. Similar examples of the fucoside bond lability in perbensylated fucose were reported in [23, 24]. Therefore, the removal of benzyl groups before acetolysis is necessary in our case. After 17-h acetolysis, the starting anhydride (XIII) completely reacted and the reaction mixture contained peracetate (XIV) as a mixture of anomers with different Rf values and oxazoline (XVI) (Scheme 6). As the content of oxazoline (XVI) did not exceed 60% (visually, TLC), it was hydrolyzed to anomeric 1-OH derivatives (XV), which differed in Rf (TLC) like acetates. The resulting mixture of acetates (XIV) and 1-OH derivatives (XV) was treated with acetic anhydride in pyridine, to transform derivatives (XV) to the corresponding acetates (XIV). The mixture of anomers (XIV) was obtained in 89% yield, with the -anomer of the less chromatographic mobility prevailing. A comparison of the 1H NMR spectrum of the starting anhydro derivative (XIII) and the resulting peracetate (XIV) conrmed the transformation of the 41 unit of GlcNAc to the relaxed 41 conformation with axial H2H5. The signals of H1, H2, H6a, and H6b were signicantly (by 0.350.74 ppm) shifted downeld. The coupling constants for H2H5 from approximately 1 Hz in (XIII) rose up to 810 Hz for (XIV), which is characteristic of trans-diaxial protons. The transformation of (XIV) to the target spacered tetrasaccharide (XVIII) was achieved according to Scheme 6. The mixture of anomeric acetates (XIV) was selectively de-1--acetylated by hydrazine acetate in

DMF by the method [25] to give monohydroxy derivative (XV) as a mixture of anomers in 80% yield. The mixture of anomers (XV) was converted into oxazoline (XVI) with mesyl chloride in the presence of collidine [26]. The resulting oxazoline (XVI) was homogenous according to TLC data and was used at the next step without additional purication. The reaction of oxazoline (XVI) with spacered alcohol in toluene in the presence of anhydrous TsOH as described in [26] resulted in the target protected glycoside (XVII) in 81% yield. The signal of H1 of the GlcNAc unit in the 1H NMR spectrum of glycoside (XVII) was shifted upeld (5.95 4.63 ppm) as compared to that in the spectrum of the starting -anomer of acetate (XIV) because of the substitution of the O-alkyl residue for the O-acyl unit. The value of the coupling constant that characterized the -conguration (3.9 Hz) changed for 8.3 Hz, which corresponds to the -conguration. The removal of - and N-protective groups in (XVII) in the presence of ion-exchange resin (OH-) by the method [11] resulted in free aminopropyl glycoside (XVII). The structure of (XVIII) was conrmed by 1and 13C NMR spectra (see the Experimental section). EXPERIMENTAL NMR spectra [1D, the values of chemical The shifts ( ppm) relative to Me4Si and the values of coupling constants (J, Hz) are given) and 13C NMR (at a working frequency of 67.5 MHz, , ppm) of CDCl3 solutions were registered on WM-500 and WM-250 Bruker spectrometers. The values of optical rotation were measured on a Jasco DIP-360 polarimeter at 20. LC was carried out on silica gel 60 (Merck, 5553). TLC was carried out on precoated silica gel 60 (Merck) glass plates or aluminum sheets using the following developing systems: chloroformmethanol
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1H

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1,6-ANHYDRO-N-ACETYL--D-GLUCOSAMINE

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hexane, hexanechloroformisopropanol, tolueneacetone, ethyl acetatetoluene, and isopropanolethyl acetatewater. Carbohydrates were visualized by heating the plates after the treatment with 7% orthophosphoric acid; carbohydrates with the free amino group were visualized by soaking in a ninhydrin solution and subsequent heating. The solutions were dried by ltration through a cotton layer. Molecular sieves 4 (Fluka) were dried at 300 for 3 h. Tribenzylfucosyl bromide (IV) [20] was obtained from the corresponding thioglycoside as described in [27]. 2,3,4,6-Tetra--benzoyl-D-galactopyranosyl bromide (V) was prepared from penta--benzoyl--D-galactopyranose [28] by the procedure [16, 17]. 1,2--Benzylidene-3,4,6-tri--benzoyl-a-Dgalactopyranose (VI). Tetrabutylammonium iodide (3.99 g, 10.8 mmol) was added to a mixture of benzoylbromogalactose (V) (13 g, 19.7 mmol), anhydrous acetonitrile (50 ml) and sodium borohydride (1.06 g, 28 mmol); the mixture was stirred at room temperature for 48 h and then quenched carefully with an acetic acid solution (2.2 ml in 15 ml of water) at stirring. The transparent solution was diluted with chloroform (250 ml) and washed with water (3 200 ml). The chloroform solution was concentrated in a vacuum to give the mixture of S- and R-isomers (in 9 : 5 ratio according to the 1H NMR) [18] of benzylidene derivative (VI) as a syrup, which was used in the next step without purication; 1H NMR, S-isomer: 4.378 (1 H, dd, J6,6b 12, J6a,5 6, H6a), 4.477 (1 H, dd, J2,1 = J2,3 5, H2), 5.60 (1 H, dd, J3,2 6, J3,4 4, H3), 5.858 (1 H, dd, J4,3 = J4,5 3.54.0, H4), 4.574.61 (in m, H5), 5.916 (d, J2,1 5, H1), 5.988 (1 H, s, CHPh), 7.5 (m, Ar), 8.0 (m, Ar); R-isomer: 4.312 (1 H, dd, J6a,6b 14, J6a,5 6, H6), 4.574.61 (in m, H2, H5), 5.66 (1 H, dd, J3,2 7, J3,4 3.5, H3), 5.962 (1 H, m, J1,2 4, H4), 6.34 (1 H, s, CHPh), 7.5 (m, Ar), 8.0 (m, Ar). 3,4,6-Tri--benzoyl-a,b-D-galactopyranose VII. The product (VI) was dissolved in chloroform (75 ml), a mixture of TFA (40 ml) and water (2 ml) was added, and the mixture was kept for 2 h until the complete disappearance of the starting compound (VI) [(Rf 0.80, 2 : 8 ethyl acetatetoluene) and Rf 1.00 (9 : 1 : 1 chloroformmethanolhexane)] and formation of diol (VII) (Rf 0.00 (2 : 8 ethyl acetatetoluene) and 0.51 and 0.49 (the mixture of - and -anomers, 9 : 1 : 1 chloroform methanolhexane)]. The solution was washed with water (2 150 ml), saturated Na2CO3 (4 200 ml), ltered through a cotton layer, and concentrated. The residue was chromatographed in a gradient of methanol 0 3) in chloroform to give diol (VII) (a mixture of anomers) as white foam; yield 8.46 g (87% from the 20 bromide); [ ] D +50.5 (c 0.93; chloroform); 1H NMR, -anomer: 4.32 [1 H, ddd (m), J2,1 3, H5], 4.36 (1 H, dd, J6a,6b 11, J6a,5 6, H6a), 4.59 (1 H, dd, J6b,6a 11, J6b,5 7, H6b), 5.58 [1 H, dd (t), J1,2 = J1,OH 33.5, H1], 5.66 (1 H, dd, J3,2 10, J3,4 33.5, H3), 5.96 (1 H, dd, J4,5 11.5, J4,3 3.5, H4), 2.40 (1 H, br. d, JOH,1 5, 2-OH), 3.28 (1 H,
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br. s, 1-OH), 7.407.770 (m, Ar), 7.85 (m, Ar), 6.06.25 (m, Ar); -anomer: 4.12 [1 H, ddd (m), H5], 4.38 (1 H, dd, J6a,6b 11, J6a,5 23, H6a), 4.63 (1 H, dd, J6b,6a 11.5, J6b,5 6, H6b), 5.40 (1 H, dd, J3,2 10, J3,4 4, H3), 5.93 (1 H, dd, J4,5 11.5, J4,3 3.5, H4), 2.40 (1 H, br. d, JOH,1 5, 2-OH), 3.28 (1 H, br. s, 1-OH), 7.407.770 (m), 7.85 (m), 6.06.25 (m, Ar). 1,2-Di--acetyl-3,4,6-tri--benzoyl-a,b-Dgalactopyranose (IIa). Diol ((VII), 8.40 g) was acetylated with 2 (20 ml) in pyridine (40 ml) (48 h, 20). The reaction mixture was carefully decomposed with water (5 ml) at cooling with ice. After 40 min, the solution was diluted with CHCl3 (400 ml); washed with water, saturated NaHCO3, water, and 1 N HCl; ltered through a cotton layer; and concentrated to give 9.8 g (100%) of diacetate (II) as a mixture of anomers (/ ~ 3/2); 1H NMR, -anomer: 2.12 and 2.38 (3 H, 2 s, 2 Ac), 4.512 (1 H, dd, J6a,5 6.7, J6a,6b 11, H6a), 4.740 (1 H, dd, J6b,5 6, H6b), 4.845 [1 H, dd (t), H5], 5.855 (1 H, dd, J2,1 3.5, J2,3 11, H2), 6.215 (1 H, dd, J4,3 3, J4,5 1, H4), 5.938 (1 H, dd, H3), 6.725 (1 H, d, H1), 7.50 8.20 (m, Ar); -anomer: 2.13 and 2.35 (3 H, 2 s, 2 Ac), 4.565 (1 H, dd, J6a,5 6.7, J6a,6b 11, H6a), 4.70 (m, H6b), 4.845 [1 H, dd (t), J5,4 1, J5,6a J5,6b 5.56.0, H5], 5.646 (1 H, dd, J3,2 10.5, J3,4 3.5, H3), 5.818 (1 H, dd, J2,1 8, H2), 6.08 (1 H, d, H1), 6.138 (1 H, dd, H4), 7.50 8.20 (m, Ar). 2--Acetyl-3,4,6-tri--benzoyl-a-D-galactopyranosyl bromide (II). A solution of (IIa) (4.05 g, 7.02 mmol) was dissolved in a mixture of chloroform (40 ml) and AcOH (20 ml) and cooled to 0; AcBr (9 ml, 122 mmol) and then a solution of water (1.26 ml, 70 mmol) in AcOH (5 ml) were added. Cooling was removed, and the reaction mixture was kept for 3 h at room temperature, monitoring the process with TLC (1 : 9 ethyl acetatetoluene) for the starting acetate, Rf 0.47, and for the bromide, Rf 0.71. The reaction mixture was poured on ice and diluted with chloroform. The organic layer was twice washed with saturated NaHCO3, dried, and evaporated to dryness to give 4.18 g (quantitative) of chromatographically individual bromide (II) as light-yellow foam, which was immediately used in the synthesis of (Ic); 1H NMR: 2.07 (3 H, s, An), 4.44 (1 H, dd, J6a,5 6, J6a,6b 11.5, H6a), 4.61 (1 H, dd, J6b,5 6.8, H6b), 4.86 (1 H, ddd, J5.4 1, H5), 5.42 (1 H, dd, J2,1 4, J2,3 10.5, H2), 5.86 (1 H, dd, J3,4 3.3, H3), 6.07 (1 H, dd, H4), 6.87 (1 H, d, H1), 8.087.32 (m, Ar). 1,6-Anhydro-2-acetamido-4--(2--acetyl-3,4,6tri--benzoyl-b-D-galactopyranosyl)-2-deoxy-b-Dglucopyranose (Ic). Diol (IIIa) (1.42 g, 7 mmol), mercury cyanide (1.77 g, 7 mmol), mercury bromide (0.25 g, 0.7 mmol), and powdered molecular sieves 4(5 g) were stirred in a mixture of nitromethane (50 ml) and anhydrous benzene (5 ml) at room temperature for 2 h. The mixture was heated at a moderate reux (~80) and a solution of bromide (II) [obtained from 7.02 mmol of diacetate (II)] in anhydrous benNo. 1 2007

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zene (30 ml) was added for 0.5 h at a vigorous stirring. The resulting mixture was heated for another 2 h and kept for 16 h at room temperature. The mixture was ltered; the sieves were washed with chloroform (2 10 ml) and 1 : 1 chloroformmethanol (2 10 ml); the combined ltrate and organic washings were concentrated; and the residue was dissolved in chloroform (200 ml). The organic layer was washed with water (2 75 ml), 1 N sodium iodide (75 ml), and water (100 ml); dried; and evaporated to dryness to give 4.64 g of the residue, which was chromatographed in a gradient of methanol (0 2%) in chloroform. Fractions containing disaccharide (Ic) (Rf 0.45, 9 : 1 : 1 chloroform methanolhexane) were combined, concentrated and dried to give 1.78 g of the product containing the admixtures of three more compounds (obviously stereo- and regioisomeric disaccharides). The resulting mixture was additionally fractured by LC on silica gel (100 g) in a gradient of isopropanol (0 12%) in 2 : 1 hexanechloroform; yield 1.02 g (20%) of pure disaccharide (Ic); Rf 0.45 (4 : 2 : 1 hexanechloroform isopropanol). [ ] D 24 (c 1, chloroform); 1H NMR: 2.040 and 2.085 (2 3 H, 2 s, NAc, OAc), 3.659 (1 H, br. s, II, GlcNAc), 3.735 [1 H, dd (br. s), J4,3 1, J4,5 1, H4, GlcNAc], 3.765 (1 H, dd, J6a,6b7.2, J6a,5 5.5, H6a, GlcNAc), 3.894 [1 H, dd (br. s), J3,2 1, H3, GlcNAc], 4.175 [1 H, ddd (br. d), J2, NH 10, J2,1 1, H2, GlcNAc], 4.247 [1 H, ddd (br. t), J5,4 1, J5,6a 6.2, J5,6b 6.6, H5, Gal], 4.297 [1 H, dd (d), J6b,5 1, H6b, GlcNAc], 4.375 (1 H, dd, J6a,6b 11.5, H6a, Gal), 4.528 [1 H, ddd (br. d), H5, GlcNAc], 4.613 (1 H, dd, H6b, Gal), 4.742 (1 H, d, J1,2 7.2, H1, Gal), 5.341 [1 H, d (br. s), H1, GlcNAc], 5.467 (1 H, dd, J2,3 10.2, H2, Gal), 5.504 (1 H, dd, J3,4 ~ 3, H3, Gal), 5.932 [1 H, dd (d), H4, Gal], 6.348 (1 H, d, NH), 7.286, 7.393, and 7.458 [3 2 H, ttt, m-H(Bz)], 7.479, 7.524, and 7.545 [3 1 H, ttt, para-H(Bz)], 7.802, 7.971, and 8.033 [3 2 , ddd, o-H(Bz)]. We have also isolated 1,6-anhydro-2-acetamido-3--(2--acetyl-3,4,6tri--benzoyl-b-D-galactopyranosyl)-2-deoxy-b-Dglucopyranose (VIII): 360 mg (7%), Rf 0.36 (4 : 2 : 1 hexanechloroformisopropanol); 1H NMR: 1.960 and 2.039 (2 3 H, s, 2OAc), 2.892 [1 H, dd (br. s), H4, GlcNAc]; 3.707 (1 H, dd, J6a,6b 7.4, J6a,5 6.1, H6a, GlcNAc), 3.774 [1 H, dd (br. s), H3, GlcNAc], 3.902 [1 H, dd (br. s), H4, GlcNAc], 3.971 [1 H, ddd (br. d), J2,HN, 9.4, H2, Gal], 4.131 [1 H, dd (br. d), I6b, GlcNAc], 4.323 [1 H, ddd (dd), J5,6a 6.5, J5,6b 6.2, H5, Gal]; 4.371 (1 H, dd, J6a,6b 11, H6a, Gal), 4.516 [1 H, ddd (br. d), H5, GlcNAc], 4.645 (1 H, dd, H6b, Gal); 5.005 (1 H, d, J1,2 7.9, Gal), 5.353 [1 H, d (br. s), H1, GlcNAc], 5.517 (2 H, m, H2, H3, Gal); 5.960 [1 H, dd (br. d), J4,3 < 1.5, H4, Gal], 6.240 (1 H, d, NH, GlcNAc), 7.305, 7.409, and 7.466 [3 2 H, ddd, Jm,o 8, Jm,p 7.7, metha-H(Bz)], 7.490, 7.545, and 7.605 [3 1 H, ttt, para-H(Bz)], 7.833, 7.994, and 8.048 [3 2 H, ddd, ortho-H(Bz)];
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1,6-anhydro-2-acetamido-4--(2--acetyl-3,4,6tri--benzoyl-a-D-galactopyranosyl)-2-deoxy-b-Dglucopyranose (IX): 73 mg (1.4%); Rf 0.52 (hexane chloroformisopropanol 4 : 2 : 1); 1H NMR: 1.974 and 2.068 (2 3 H, s, 2OAc), 3.487 (1 H, dd, J6a,6b 7.4, J6a,5 5.7, H6a, GlcNAc), 3.681 [1 H, dd (br. s), H3, GlcNAc], 3.888 [1 H, dd (br. s), H4, GlcNAc], 4.056 [1 H, ddd (br. d), J2,HN 9.4, H2, GlcNAc], 5.400 [1 H, d (br. s), H1, GlcNAc], 5.423 (1 H, d, J1,2 3.6, H1, Gal), 5.523 (1 H, dd, J2,3 10.8, H2, Gal), 5.810 (1 H, dd, J3,4 3.3, H3, Gal), 5.942 (1 H, dd (br. d), H4, Gal), 6.329 (1 H, d, NH, GlcNAc); 1,6-Anhydro-2-acetamido-3--acetyl-4--(2-acetyl-3,4,6-tri--benzoyl-b-D-galactopyranosyl)2-deoxy-b-D-glucopyranose (Ib). (A) Mercury cyanide (835 mg, 3.3 mmol) and mercury bromide (119 mg, 0.33 mmol) were added to a solution of acceptor (IIIc) (540 mg, 2.2 mmol) in a mixture of anhydrous nitromethane (7 ml) and anhydrous benzene; the mixture was stirred with freshly calcined molecular sieves 4(3 g) for 3 h. A solution of bromide (II) [prepared from 1.9 g (3.3 mmol) of diacetate (II) in anhydrous benzene (10 ml)] was added to the reaction mixture together with molecular sieves 4 (2 g). The mixture was stirred at room temperature for 16 h. Then, mercury cyanide (279 mg, 1.1 mmol), mercury bromide (40 mg, 0.1 mmol), and bromide (II) (1.1 mmol) were added. The mixture was stirred at room temperature for 16 h, diluted with chloroform, and ltered off; the sieves were washed with chloroform on lter; and the ltrate was concentrated. The residue was dissolved in chloroform (30 ml); washed with 2 N sodium iodide, water, saturated NaHCO3, and water; ltered through a cotton layer; and concentrated. The residue was chromatographed on silica gel (30 g) in a gradient of isopropanol (0 3%) in 2 : 1 hexane chloroform to give 1.192 g (71%) of disaccharide (Ib); 20 [ ] D 24 (c 1, chloroform); 1H NMR: 1.991, 2.012, and 2.068 (3 3 H, 3 s, NAc, 2 OAc), 3.735 [1 H, dd (br. s), J4.3 1, J4.5 1, H4, GlcNAc], 3.792 (1 H, dd, J6a,6b 7.5, J6a,5 5.5, H6a, GlcNAc), 4.059 [1 H, dd (d), J6b,5 1, H6b, GlcNAc], 4.073 [1 H, ddd (br. d), J2,NH 10, J2.1 1, J2,3 1, H2, GlcNAc], 4.266 [1 H, ddd (t), J5,4 1, J5,6 = J5,6b 7, H5, Gal], 4.401 (1 H, dd, J6a,6b 11.5, H6a, Gal), 4.517 [1 H, ddd (br. d), H5-GlcNAc], 4.576 (1 H, dd, H6b, Gal), 4.800 (1 H, d, J1,2 7.3, H1, Gal), 4.995 [1 H, dd (br. s), H3-GlcNAc], 5.322 [1 H, d (br. s), H1-GlcNAc], 5.463 (1 H, dd, J3,2 10.2, J3,4 ~ 3, H3, Gal), 5.509 (1 H, dd, H2, Gal), 5.916 [1 H, dd (d), H4, Gal], 6.288 (1 H, d, NH-GlcNAc), 7.289, 7.387, and 7.479 [3 2 H, ttt, metha-H(Bz)], 7.480, 7.520, and 7.611 [3 1 H, ttt, para-H(Bz)], 7.805, 7.958, and 8.049 [3 2 H, ddd, o-H(Bz)]. (B) Acetylation of monohydroxy derivative (Ic) with acetic anhydride in pyridine resulted in (Ib) in quantitative yield. According to the 1H NMR, the
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resulting compound was identical to diacetate (Ib) obtained as described above. 1,6-Anhydro-2-acetamido-4--(3,4,6-tri--benzoyl-b-D-galactopyranosyl)-2-deoxy-b-D-glucopyranose (Ia). (A) By deacetylation of monoacetate (Ic). A solution of HCl in methanol prepared by a dropwise addition of acetyl chloride (0.5 ml, 7 mmol) to methanol (8 ml) at 0 was added to a solution of monoacetate (Ic) (1.02 g, 1.417 mmol) in chloroform (2 ml). The mixture was kept at room temperature for 24 h, diluted with chloroform, and washed with saturated NaHCO3. The aqueous washings were extracted with chloroform. The combined organic extracts were washed with water, dried, and concentrated. The residue (980 mg) was subjected to column chromatography in a gradient of isopropanol (4 15%) in 2 : 1 hexanechloroform; yields: 500 mg (52%) of diol (I), Rf 0.25 (4 : 2 : 1 hexanechloroformisopropanol); [ ] D 14.9 (c 1, chloroform) and 190 mg (19%) of the starting (Ic), Rf 0.49 (4 : 2 : 1 hexanechloroformisopropanol). 1H NMR of (Ia): 1.905 (3 H, s, NAc), 3.522 (1 H, br. s, OH), 3.650 (1 H, dd, J6a,6b7.5, J6a,5 5.5, H6a, GlcNAc); 3.687 (1 H, br. s, OH'), 3.857 [1 H, dd (br. s), J4,3 1, J4,5 1, J3,2 1, H4, GlcNAc], 3.945 [1 H, dd (br. s), J2,NH 9.5, J2,1 1, H3, GlcNAc], 4.004 [1 H, ddd (br. d), J2,NH 9.5, J2,1 1, H2, GlcNAc], 4.103 (1 H, dd, J2,1 7.3, J2,3 10, H2, Gal), 4.192 [1 H, dd (d), J6b,5 1, H6bGlcNAc], 4.254 [1 H, ddd (t), J5,4 1, J5,6a = J5,6b 6.5, H5, Gal], 4.343 (1 H, dd, J6a,6b H1, H6a, Gal), 4.545 (1 H, dd, H6b, Gal), 4.679 [1 H, ddd (br. d), H5GlcNAc], 4.800 (1 H, d, H1, Gal), 5.383 [1 H, d (br. s), H1-GlcNAc], 5.397 (1 H, dd, J3,4 ~ 3.5, H3, Gal), 5.858 [1 H, dd (d), H4, Gal], 6.480 (1 H, d, NH-GlcNAc), 7.246, 7.370, and 7.439 [3 2 H, ttt, metha-H(Bz)], 7.445, 7.504, and 7.586 [3 , ttt, para-H(Bz)], 7.802, 7.945, and 8.017 [3 2 , 3 d, ortho-H(Bz)]. (B) By deacetylation of diacetate (Ib). A solution of HCl in methanol [prepared from acetyl chloride (2.4 ml, 34 mmol) and methanol (30 ml) at 0] was added to a solution of diacetate (Ib) (1.07 g, 1.4 mmol) in a mixture of chloroform (20 ml) and methanol (10 ml). The mixture was kept at room temperature for 20 h until disappearance (TLC) of the starting (Ib) (Rf 0.39, 9 : 1 : 6 chloroformmethanolhexane). The reaction mixture was quenched with anhydrous sodium acetate and evaporated to dryness. The residue was dissolved in chloroform (30 ml); the organic layer was washed with water (10 ml), saturated NaHCO3, and water; dried with anhydrous sodium sulfate; and concentrated. The residue was chromatographed in a gradient of isopropanol (0 10%) in 2 : 1 chloroform hexane to give (in the order of elution): the starting diacetate (Ib); yield 40 mg (4%); 1,6-anhydro-2-acetamido-4--(2--acetyl-3,4,6tri--benzoyl-b-D-galactopyranosyl)-2-deoxy-b-Dglucopyranose (Ic); yield 311 mg (31%), Rf 0.32
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(9 : 1 : 6 chloroformmethanolhexane); [ ] D 24 (c 1, chloroform); 1H NMR: 2.040 and 2.085 (2 3 H, 2 s, NAc, OAc), 3.659 (1 H, br. s, OH, GlcNAc), 3.735 [1 H, dd (br. s), J4,3 1, J4,5 1, H4, GlcNAc], 3.765 (1 H, dd, J6a,6b7.2, J6a,5 5.5, H6a, GlcNAc), 3.894 [1 H, dd (br. s), J3,2 1, H3, GlcNAc], 4.175 [1 H, ddd (br. d), J2,NH 10, J2.1 1, H2, GlcNAc], 4.247 [1 H, ddd (br. t), J5,4 1, J5,6a 6.2, J5,6b 6.6, H5, Gal], 4.297 [1 H, dd (d), J6b,5 1, H6b, GlcNAc], 4.375 (1 H, dd, J6a,6b 11.5, H6a, Gal), 4.528 [1 H, ddd (br. d), H5, GlcNAc), 4.613 (1 H, dd, H6b, Gal], 4.742 (1 H, d, J1,2 7.2, H1, Gal), 5.341 [1 H, d (br. s), H1, GlcNAc], 5.467 (1 H, dd, J2,3 10.2, I2, Gal), 5.504 (1 H, dd, J3,4 ~ 3, H3, Gal), 5.932 [1 H, dd (d), H4, Gal], 6.348 (1 H, d, NH, GlcNAc), 7.286, 7.393, and 7.458 [3 2 H, ttt, m-H(Bz)], 7.479, 7.524, and 7.545 [3 1 H, ttt, p-H(Bz)], 7.802, 7.971, and 8.033 [3 2 H, ddd, o-H(Bz)]; 1,6-anhydro-2-acetamido-4--(3,4,6-tri--benzoyl-b-D-galactopyranosyl)-3--acetyl-2-deoxy-bD-glucopyranose (Id); yield 77 mg (8%), Rf 0.28 (9 : 1 : 6 chloroformmethanolhexane), [ ] D 15.5 (c, chloroform); 1H NMR: 1.936 and 2.016 (2 3 H, 2 s, NAc, OAc), 3.766 [1 H, dd (br. s), J4,3 1, J4,5 1, H4, GlcNAc], 3.766 (1 H, dd, J6a,6b7.8, J6a,5 5.3, H6a, GlcNAc), 4.010 [1 H, ddd (br. d), J2,NH 8.8, J2,1 1, J2,3 1, I2, GlcNAc], 4.010 [1 H, dd (d), J6b,5 1, H6b, GlcNAc), 4.200 (1 H, dd, J2,1 7.8, J2,3 10, H2, Gal], 4.414 [1 H, ddd (br. q), J5,4 1, J5,6a 5.1, J5,6b 7.8, H5, Gal], 4.470 (1 H, dd, J6a,6b 11.5, H6a, Gal), 4.520 (1 H, dd, H6b, Gal), 4.701 [1 H, ddd (br. d), H5, GlcNAc], 4.950 (1 H, d, H1, Gal), 5.164 [1 H, dd (br. s), H3, GlcNAc], 5.429 (1 H, dd, J3,4 3.5, H3, Gal), 5.439 [1 H, d (br. s), H1, GlcNAc], 5.898 [1 H, dd (d), H4, Gal], 6.456 (1 H, d, NH, GlcNAc), 7.311, 7.412, and 7.498 [3 2 H, ttt, m-H(Bz)], 7.505, 7.543, 7.646 [3 1 H, ttt, para-H(Bz)], 7.852, 7.970, and 8.063 [3 2 H, ddd, o-H(Bz)]; and the target disaccharide diol (Ia), yield 385 mg (40%), identical to diol (Ia) obtained from (Ic) as described above. 1,6-Anhydro-2-acetamido-3--(2,3,4-tri--benzyl-a-L-fucopyranosyl)-4--[2--(2,3,4-tri--benzyl-a-L-fucopyranosyl)-3,4,6-tri--benzoyl-b-Dgalactopyranosyl]-2-deoxy-b-D-glucopyranose (X). A suspension of diol (Ia) (500 mg, 0.738 mmol), tetraethylammonium bromide (1.06 g, 5.04 mmol), and molecular sieves 4 (1 g) in anhydrous methylene chloride (20 ml) was stirred at 20 for 2 h. A solution of diisopropylethylamine (1.06 ml, 6.17 mmol) in DMF (5 ml) and then a solution of fucosyl bromide (IV) obtained from the corresponding thioglycoside (2.41 g, 5.04 mmol) and Br2 (287 l, 5.56 mmol) in dichloromethane (5 ml) were added. The reaction mixture was stirred at room temperature for 48 h and ltered; and the residue was washed on lter with chloroform
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(5 5 ml). The ltrate was washed with 1 N HCl, water, saturated NaHCO3, and water; dried; and evaporated to dryness. The residue (the brownish-red syrup) was chromatographed on silica gel (25 g) in a gradient of ethyl acetate (12 20%) in toluene. The fractions containing (X) (Rf 0.58, 1 : 2 ethyl acetatetoluene) were combined and evaporated to dryness to yield 1.2 g of syrup, which was then debenzoylated without further purication. 1,6-Anhydro-2-acetamido-3--(2,3,4-tri--benzyl-a-L-fucopyranosyl)-4--[2--(2,3,4-tri--benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl]-2deoxy-b-D-glucopyranose (XI). A 2 N solution of sodium methylate in methanol (2.5 ml) was added to a solution of (X) (1.2 g) in anhydrous methanol (25 ml). The mixture was kept for 17 h at room temperature, quenched with acetic acid (75 l), and concentrated. The residue was chromatographed on silica gel (25 g) in a gradient of isopropanol (0 12%) in 1 : 2 chloroformhexane to give 504 mg (57%) of triol (XI); Rf 0.47 (4 : 2 : 1 chloroformhexaneisopropanol), [ ] D 72 (c 3, chloroform). 1H NMR (CD3OD): 1.347 and 1.388 (2 3 H, dd, J6,5 6.5, H6, Fuc1 and H6, Fuc2), 2.044 (3 H, s, OAc), 3.703 (1 H, dd, J5,6a 6.3, J5,6b 5.9, H5, Gal), 3.855 (1 H, dd, J6,6b 7.2, J6a,5 6.3, H6a, GlcNAc), 3.904 (4 H, m, H4, GlcNAc, H6a, Gal, H6b, Gal, H4, Fuc1), 3.970 [1 H, dd (br. d), H4, Fuc1], 3.992 (2 H, m, H2, Gal, H3, Gal), 4.007 [1 H, dd (br. s), H3, GlcNAc], 4.052 [1 H, dd (br. d), J4,3 2, H4, Gal], 4.094 (1 H, dd, J3,2 10.3, J3,4 2.8, H3, Fuc1), 4.165 (1 H, dd, J2,3 10.3, J2,1 3.6, H2, Fuc1), 4.175 (2 H, m, H2, Fuc2, H3, Fuc2), 4.189 [1 H, dq (br. d), H5, Fuc1], 4.197 [1 H, ddd (br. s), H2, GlcNAc], 4.313 (1 H, dd, J6b,5 ~ 1, H6b, GlcNAc), 4.467 [1 H, dq (br q), H5, Fuc2], 4.743 (1 H, d, J1,.2 7, H1, Gal), 4.787 and 4.796 (2 1 H, dd, J 11.2, CH-Ar), 4.803 [1 H, ddd (br. d), H5, GlcNAc], 4.841 (1 H, d, J 11.7, CH-Ar), 4.925.02 (7 H, m, CHAr), 5.090 and 5.105 (2 1 H, dd, J 11.2, CH-Ar), 5.343 (1 H, d, J1,2 3.6, H1, Fuc1), 5.414 (1 H, d, J1,2 3.1, H1, Fuc2), 5.465 (1 H, d (br. s), H1, GlcNAc), 7.44 7.61 (30 H, m, Ar). 1,6-Anhydro-2-acetamido-3--a-L-fucopyranosyl-4--[2--a-L-fucopyranosyl-b-D-galactopyranosyl]-2-deoxy-b-D-glucopyranose (XII). Palladium on carbon (10%, 10 mg) was added to a solution of triol (XI) (450 mg, 0.376 mmol) in a mixture of methanol (24 ml) and ethyl acetate (6 ml). The reaction mixture was subjected to hydrogenolysis at 20 and atmospheric pressure under stirring for 20 h. The reaction was monitored by TLC [Rf of the starting triol (XI) is 0.82 (4 : 3 : 2 isopropanolethyl acetatewater), Rf of the target (XII) is 0.25 (4 : 3 : 2 isopropanolethyl acetatewater)]. The suspension was ltered, the precipitate was washed on lter with methanol, and the ltrate was evaporated to dryness to give 236 mg (96%) of tetrasaccharide (XII) as white downy semicrystalline
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powder; [ ] D 157 (c 1, water); 1H NMR (D2O): 1.242 and 1.255 (2 3 H, dd, J6,5 6.5, H6, Fuc1, H6, Fuc2), 2.059 (3 H, s, Ac), 3.704 [1 H, ddd (br dq), I5, Gal], 3.709 (1 H, dd, J2,1 7.8, J2,3 10, H2, Gal), 3.757 (2 H, m, H6a, Gal, H6b, Gal), 3.788 (1 H, dd, J2,1 4, J2,3 10.4, H2, Fuc1), 3.84 and 3.89 (5 H and 3 H, mm, H4, GlcNAc, H6, GlcNAc, H3, Gal, H3, Fuc1, H4, Fuc1, H2, Fuc2, H3, Fuc2, H4, Fuc2), 3.930 [1 H, dd (br s), H3, GlcNAc], 3.966 [1 H, dd (br d), J4,3 3.5, H4, Gal], 4.063 [1 H, dd (br s), H2, GlcNAc], 4.129 [1 H, dq (br q), H5, Fuc1], 4.222 [1 H, dd (br d), J6b,6a 7.8, H6b, GlcNAc], 4.377 [1 H, dq (br q), H5, Fuc2], 4.680 (1 H, d, J1,2 7.8, H1, Gal), 4.796 [1 H, ddd (br d), J5,6 5.5, I5, GlcNAc], 5.062 (1 H, d, J1,2 4, H1, Fuc1), 5.329 (1 H, d, J1,2 3.8, H1, Fuc2), 5.537 [1 H, d (br. d), I1, GlcNAc]. 1,6-Anhydro-2-acetamido-4--[2--(2,3,4-tri-acetyl-L-a-fucopyranosyl)-3,4,6-tri--acetyl-b-Dgalactopyranosyl]-3--(2,3,4-tri--acetyl-a-L-fucopyranosyl)-2-deoxyb-D-glucopyranose (XIII). Acetic anhydride (5 ml) was added to a solution of (XII) (216 mg, 0.328 mmol) in pyridine (5 ml); the mixture was kept for 48 h at room temperature and then was cooled to 0; methanol (7 ml) was added dropwise to quench the excess of acetic anhydride; and methyl acetate was removed in a vacuum. The residue was dissolved in chloroform (30 ml); washed with 1 N HCl, water, and saturated NaHCO3; dried; and evaporated to dryness. The residue was puried by LC on silica (25 g) in a gradient of ethyl acetate (30 80%) in toluene to give 319 mg (94%) of peracetate (XIII); Rf 0.33 (3 : 1 ethyl acetatetoluene), Rf 0.42 (4 : 2 : 1 hexanechloroformisopropanol); [ ] D 127 ( 1, chloroform); 1H NMR: 1.171 and 1.198 (2 3 H, dd, J6,5 = J6,5 6.5, H6, Fuc1, H6, Fuc2), 1.988, 2.000, 2.008, 2.020, 2.039, 2.067, 2.110, 2.143, 2.162, and 2.165 (10 3 H, 10 s, 10 An), 3.725 [1 H, dd (br. s), H4, GlcNAc], 3.808 (1 H, dd, J6a,6b 7.2, J6a,5 6.1, H6, GlcNAc), 3.886 (1 H, dd, J2,1 7.7, J2,3 10, H2, Gal), 3.892 [1 H, ddd (br. dd), H5, Gal], 3.931 [1 H, dd (br s), H3, GlcNAc], 4.005 [1 H, ddd (br d), J2,HN 9.6, H2, GlcNAc], 4.006 (1 H, dd, J6a,6b 11.4, J6a,5 6.7, H6a, Gal), 4.114. [1 H, dq (br. q), H5, Fuc1], 4.161 (1 H, dd, J6b,5 6.4, H6b, Gal), 4.190 [1 H, dd (br. d), H6b, GlcNAc], 4.583 (1 H, d, H1, Gal), 4.585 [1 H, ddd (br. d), H5, GlcNAc], 4.620 [1 H, dq (br q), H5, Fuc2], 5.029 (1 H, dd, J2,1 3.7, J2,3 11, H2, Fuc1), 5.049 (1 H, dd, J3,4 3.5, H3, Gal), 5.092 (1 H, dd, J2,1 3.9, J2,3 10.8, H2, Fuc2), 5.272 (1 H, dd, J3,4 3.3, H3, Fuc2), 5.299 [1 H, dd (br. d), H4, Fuc1], 5.302 [1 H, d (br. s), H1, GlcNAc], 5.321 [1 H, dd (br. s), H4, Gal], 5.328 [1 H, dd (br. s), H4, Fuc2], 5.352 (1 H, dd, J3,4 3.3, H3, Fuc1), 5.431 (1 H, d, H1, Fuc2), 5.470 (1 H, d, H1, Fuc1), 6.013 (1 H, d, NH, GlcNAc). 2-Acetamido-1,6-di--acetyl-3--(2,3,4-tri-acetyl-a-L-fucopyranosyl)-4--[2--(2,3,4-tri-acetyl-a-L-fucopyranosyl)-3,4,6-tri--acetyl-b-Dgalactopyranosyl]-2-deoxy-a,b-D-glucopyranose
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(XIV). A 10 vol % solution of concentrated H2SO4 in acetic acid (0.5 ml) was added to a solution of anhydro derivative (XIII) (289 mg, 0.279 mmol) in acetic acid (5 ml) and acetic anhydride (5 ml); the mixture was kept at room temperature for 40 h. According to TLC, the mixture contained peracetate (XIV) as the mixture of - and -anomers and oxazoline (XVI) with Rf 0.44, 0.37, and 0.58, respectively (4 : 2 : 1 hexanechloroformisopropanol). Ground anhydrous NaOAc (0.5 g) and then pyridine (25 ml) were added to the reaction mixture; the mixture was cooled to 0; and water (15 ml) was added carefully portionwise. The mixture was kept for 1 h at room temperature and diluted with chloroform. The chloroform layer was washed with 1 N HCl, water, saturated NaHCO3, and water; dried; and evaporated to dryness. The residue, a mixture of anomeric acetates (XIV) and the product of oxazoline hydrolysis as anomeric derivatives (XV) with Rf 0.31 and 0.21 (4 : 2 : 1 hexanechloroformisopropanol) according to TLC, was subjected to additional acetylation with acetic anhydride (4 ml) in pyridine (4 ml). After 16 h, the mixture was cooled to 0 and quenched with methanol as described above for obtaining acetate (XIII). The solution was concentrated; the residue was diluted with chloroform; extracted with 1 N HCl, water, saturated NaHCO3, and water; dried; and evaporated to dryness. The residue (370 mg) was chromatographed on a silica gel column (25 g) in a gradient of isopropanol (4 13%) in 2 : 1 hexanechloroform to give 284 mg (89%) of acetate (XIV) as a mixture of anomers. The sample of the mixture was rechromatographed for analytical purposes to give the minor -anomer, Rf 0.44 (4 : 2 : 1 hexanechloroformisopropanol), [ ] D 109 ( 1, chloroform) and the major -anomer, Rf 0.37 (4 : 2 : 1 hexanechloroformisopropanol), [ ] D 80 ( 1, chloroform); 1H NMR: 1.194 (6 H, dd, J6,5 6.6, H6, Fuc1, H6, Fuc2), 1.962, 1.964, 1.970, 1.992, 2.018, 2.093, 2.112, 2.148, 2.157, 2.162, 2.166, and 2.167 (12 3 H, 12 s, 12 Ac), 3.736 (1 H, dd, J2,1 8, J2,3 10, H2, Gal), 3.858 [1 H, ddd (br dd), H5, Gal], 3.882 (1 H, dd, J3,2 10.3, J3,4 8.3, H3, GlcNAc), 4.010 (2 H, m, H4, H5, GlcNAc), 4.262 (1 H, dd, J6,6b 12.2, J6a,5 4.5, H6a, GlcNAc), 4.294 (1 H, dd, J6a,6b 11.6, J6a,5 7.6, H6a, Gal), 4.456 [1 H, dq (br q), H5, Fuc1], 4.473 (1 H, d, J1,2 8, H1, Gal), 4.494 (1 H, dd, J6b,5 6.5, H6b, Gal), 4.542 (1 H, dd, J6b,5 1.5, H6b, GlcNAc), 4.607 (1 H, ddd, J2,1 3.9, J2,NH 10.4, H2, GlcNAc), 4.957 [1 H, dq (br q), H5, Fuc2], 5.018 (1 H, dd, J3,4 3.7, H3, Gal), 5.031 (1 H, dd, J2,1 3.9, J2,3 11, H2, Fuc1), 5.057 (1 H, dd, J2,1 3.9, J2,3 11, H2, Fuc2), 5.147 (1 H, dd, J3,4 3.3, H3, Fuc1), 5.196 (1 H, dd, J3,4 3.3, H3, Fuc2), 5.261 (1 H, d, NH, GlcNAc), 5.308 [1 H, dd (br d), H4, Fuc1], 5.342 (1 H, d, H1, Fuc1), 5.358 [1 H, dd (br d), H4, Gal], 5.374 (1 H, d, H1, Fuc2), 5.387 [1 H, dd (br. d), H4, Fuc2], 5.954 (1 H, d, H1, GlcNAc).
20 20

2-Acetamido-6--acetyl-3--(2,3,4-tri--acetyla-L-fucopyranosyl)-4--[2--(2,3,4-tri--acetyla-L-fucopyranosyl)-3,4,6-tri--acetyl-b-D-galactopyranosyl]-2-deoxy-a,b-D-glucopyranose (XV). Hydrazine acetate (100 mg) was added to a solution of acetate (XIV) (218 mg, 192 mol) in acetonitrile (10 ml); the mixture was stirred at room temperature for 40 h and then concentrated. The residue was diluted with chloroform; washed with 0.5 N HCl, saturated NaHCO3, and water; dried; and evaporated to dryness. The residue was chromatographed on silica gel in a gradient of isopropanol (4 9%) in chloroform to give 167 mg (79%) of monohydroxy derivative (XV) as a mixture of anomers (Rf 0.31 and 0.21, 4 : 2 : 1 hexane chloroformisopropanol); [ ] D 111 (c 1, chloroform, 1.5 h) and 15 mg (7%) of the starting (XIV). 3-Triuoroacetamidopropyl 2-acetamido-6-acetyl-3--(2,3,4-tri--acetyl-a-L-fucopyranosyl)4--[2--(2,3,4-tri--acetyl-a-L-fucopyranosyl)3,4,6-tri--acetyl-b-D-galactopyranosyl]-2-deoxyb-D-glucopyranoside (XVII). Collidine (200 l, 1.52 mmol) and then mesyl chloride (60 l, 0.77 mmol) were added to a solution of (XV) (156 mg, 142 mol) in anhydrous methylene chloride (4.5 ml). After 5 h, the solution containing oxazoline (XVI) (Rf 0.58, 4 : 2 : 1 hexanechloroformisopropanol) as the only product was diluted with chloroform, washed with saturated NaHCO3 and water, dried, concentrated, and dried in a vacuum (1 mm Hg) to give (XVI). It was dissolved in anhydrous benzene (7 ml), 3-triuoroacetamidopropanol (1 ml, 8.19 mmol) was added, and then a solution of anhydrous TsOH in anhydrous toluene was added at vigorous stirring. The addition was controlled by measuring pH of the sample of the reaction mixture with wet indicator paper, so that pH maintained within 34. The reaction mixture was stirred for 3 h at room temperature; diluted with chloroform; washed with 1 N HCl, water, saturated NaHCO3, and water; dried; concentrated, and dried in a vacuum. The residue was chromatographed on a silica gel column (15 g) in a gradient of isopropanol (5 12%) in 2 : 1 hexanechloroform 20 to give 144 mg (81%) of glycoside (XVII), [ ] D 111 (c 2, chloroform), Rf 0.32 (4 : 2 : 1 hexanechloroform isopropanol) and 0.47 (4 : 2 :1 hexanechloroformethanol); 1H NMR: 1.196 and 1.203 (6 H, dd, J6,5 6.5, H6, Fuc1, H6, Fuc2), 1.967, two 1.971, 1.987, 2.007, 2.072, 2.121, 2.147, two 2.152, and 2.1156 (11 3 H, 11 s, 11 An), 3.290 (1 H, ddd, HNH), 3.590 (4 H, m, H'NH, OCH and 2), 3.528 (1 H, ddd, J5,6a 2.2, J5,6b 5, I5, GlcNAc), 3.640 (1 H, ddd, J2,1 8.3, J2,3 9.5, J2,NH 9, I2, GlcNAc), 3.764 (1 H, dd, J2,1 8, J2,3 10, H2, Gal), 3.863 [1 H, ddd (br. dd), J5,6a 7.5, J5,6b 6, H5, Gal], 3.894 (1 H, dd, J4,3 9.5, J4,5 9.5, H4, GlcNAc), 3.908 (ddd, JOCH,OCH 10, OCH), 4.043 (1 H, dd, H3, GlcNAc), 4.233 (1 H, dd, J6,6b 12.5, H6a, GlcNAc), 4.265 (1 H, dd, J6a,6b 11.5, H6a, Gal), 4.422 [1 H, dq (br. q), H5, Fuc2], 4.500
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(1 H, dd, H6b, Gal), 4.518 (1 H, d, H1, Gal), 4.630 (1 H, d, H1, GlcNAc), 4.696 (1 H, dd, H6b, GlcNAc), 4.926 [1 H, dq (br. q), H5, Fuc1], 5.007 (1 H, dd, J2,1 3.5, J2,3 11, H2, Fuc2), 5.010 (1 H, dd, J2,1 3.5, J2,3 11, H2, Fuc1), 5.028 (1 H, dd, J3,4 3.5, H3, Gal), 5.138 (1 H, dd, J3,4 3.5, H3, Fuc2), 5.194 (1 H, dd, J3,4 3.5, H3, Fuc1), 5.317 [1 H, dd (br. d), H4, Fuc2], 5.350 [1 H, dd (br. d), H4, Gal], 5.350 (1 H, d, H1, Fuc2), 5.368 [1 H, dd (br. d), H4, Fuc1], 5.426 (1 H, d, H1, Fuc1), 5.763 (1 H, d, NH, GlcNAc), 7.337 [1 H, dd (br. t), N HCOCF3). 13 NMR: 171.40, 171.27, 170.82, 170.72, 170.66, 170.43, 170.17, 169.94, and 169.88 [13C=O (Ac)], 100.65 (C1, Gal), 10.46 (C1, GlcN), 96.35 (C1, Fuc2), 95.80 (C1, Fuc1), 74.38, 74.29, 73.47, 73.34, 72.80, 71.41, 71.04, 68.27, 68.04, 67.82, 67.46, 67.20, and 66.94 (unidentied signals), 65.13 and 64.14 (C5, Fuc1, C5, Fuc2), 61.93 (C6, Gal), 60.76 (C6, GlcN), 57.20 (C2 GlcN), 37.36 ( CN), 28.67 (C C C), 23.49 (3 (AcN)), 20.972-0.66 (11 C 3 (AcO)], 15.99 and 15.77 (C6, Fuc1, C6, Fuc2). 3-Aminopropyl 2-acetamido-2-deoxy-3--(a-Lfucopyranosyl)-4--[2--(a-L-fucopyranosyl)-3,4,6b-D-galactopyranosyl]-b-D-glucopyranoside (XVIII). Ion-exchange resin Amberlist A-26 (OH) (2 ml) was added to a solution of glycoside (XVII) (61.6 mg, 49.3 mol) in a mixture of methanol (8 ml) and water (0.3 ml); the mixture was stirred for 20 h at room temperature and ltered; the resin was washed on lter with methanol and 2 : 1 methanolwater; and the ltrate was concentrated and dried in a vacuum to give 34.8 mg (96%) of aminopropyl glycoside (XVIII). Rf 0.49 (3 : 1 : 1 : 0.3 ethanolwaterpyridineacetic acid); 20 [ ] D 118 (c 1.5, water); 1H NMR (D2O): 1.232 and 1.264 (6 H, dd, J6,5 6.7, H6, Fuc1, H6, Fuc2), 1.831 (2 H, m, CCH2C) 2.885 (2 H, t, 2N), 2.034 (3 H, s, NHAc), 3.660 (1 H, m, OCH), 3.983 (1 H, m, OCH), 4.252 [1 H, dq (br q), H5, Fuc2], 4.518 (1 H, d, J1,2 7.7, H1, Gal), 4.630 (1 H, d, J1,2 8.8, I1, GlcNAc), 4.864 [1 H, dq (br q), H5, Fuc1], 5.101 (1 H, d, J1,2 4, H1, Fuc1), 5.273 (1 H, d, J1,2 3, H1, Fuc2); 13C NMR (D2O): 102.21 (N1, GlcN), 101.47 (C1, Gal), 100.66 (C1, Fuc2), 99.84 (C1, Fuc1), 77.57 (C2, Gal), 76.79 (C3, GlcN), 76.10 (C5, GlcN, C5, Gal), 74.78 (C3, Gal), 74.54 (C4, GlcN), 73.15 (C4, Fuc2), 72.92 (C4, Fuc1), 70.95 (C3, Fuc2), 70.39 (C3, Fuc1), 69.96 (C4, Gal), 69.48 (C2 Fuc1, C2 Fuc2), 68.90 (OC), 68.05 (C5, Fuc1, C5, Fuc2), 62.67 (C6, Gal), 61.13 (C6, GlcN), 38.77 (NC), 30.57 (CCC), 23.40 (CCON), 18.62 (C6, Fuc1, C6, Fuc2). ACKNOWLEDGMENTS We are grateful to A.S. Shashkov (Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences) and I.V. Maslennikov (ShemyakinOvchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sci-

ences) for registration of NMR spectra and to G.V. Pazynina for participation in the synthesis of the starting 1,6-anhydro-GlcNAc. REFERENCES
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