WO2001042264A1

WO2001042264A1 - Method for the synthesis of sialylated oligosaccharide donors

Info

Publication number: WO2001042264A1
Application number: PCT/CA2000/001487
Authority: WO
Inventors: Seema Mehta; Michel Gilbert; Warren W. Wakarchuk; Dennis M. Whitfield
Original assignee: National Research Council Of Canada
Priority date: 1999-12-10
Filing date: 2000-12-08
Publication date: 2001-06-14
Also published as: AU2136201A

Abstract

A method for the synthesis of aryl thioglycosides comprising a sialylated residue of β-D-galactose is disclosed. The method consists of preparing by a chemical synthesis a non-sialylated aryl thioglycoside, and enzymatically sialylating the latter with a sialic acid in the presence of a suitable sialyltransferase. The sialylated aryl thioglycoside is then chemically derivatized by standard procedures, to provide a derivative suitable for use as a donor in chemical syntheses of sialylated oligosaccharides. The derivatized sialylated aryl thioglycosides are prepared in high yields, due to reduced number of chemical and purification steps involved in the process. Derivatized aryl thioglycosides useful as building blocks for the synthesis of biologically active sialylated oligosaccharides are also disclosed.

Description

METHOD FOR THE SYNTHESIS OF SIALYLATED OLIGOSACCHARIDE DONORS

FIELD OF THE INVENTION

The invention relates to oligosaccharides containing siaiic acid, in particular to thioglycosides comprising a sialylated residue of β-D-gaiactose and to the synthesis of such thioglycosides and derivatives thereof.

REFERENCES

The following references are cited in this application and are referred to at the relevant places using reference numerals put into square brackets:

1. Jennings, H. J. Curr. Top. Microbiol. Immunol. 1990, 150, 97.

2. Eichler, E., Jennings, H. J., Whitfield, D. M. J. Carbohydr. Chem., 1997, 16, 385.

3. (a) Pozsgay, V., Jennings, H. J., Kasper, D. L. J. Carbohydr. Chem., 1987, 6, 41. (b) Paulsen, H., Tietz, H. Carbohydr. Res., 1985, 144, 205. (c) Murase. T..

Kameyama, A., Kartha, K. P. R., Ishida, H., Kiso, M. Hasegawa, A. J. Carbohydr. Chem. 1989, 8, 265. (d) Otsubo, N.; Ishida, H.; Kiso, M. Tetrahedron Lett. 2000, 41, 3879.

4. (a) Schwarz, J. B., Kuduk, S. D., Chen, X. T., Sames, D., Giunz, P. W., Danishefsky, S. J., J. Am. Chem. Soc. 1999, 121, 2662. (b) Duclos Jr., R.I.

Caώohydr. Res. 2000, 328, 489.

5. (a) Gilbert, M.; Brisson, J. -R.; Karwaski, M. -F.; Michniewicz, J., Cunningham, A. -M.; Wu, Y.; Young, N. M.; Wakarchuk, W. W. J. Biol. Chem. 2000, 275, 3896. (b) Gilbert, M.; Watson, D. C; Wakarchuk, W. W. Biotechnol. Lett, 1997, 19, 417. 6. Yu, R. K., Koerner, T. A. W., Ando, S., Yohe, H. C. Prestegard, J. H. J. Biochem. 1985, 98, 1367.

7. Garreg, P. J. Adv. Carbohydr. Chem. Biochem. 1997, 52, 179.

8. (a) Jiang, L; Hartley, R. C; Chan, T. H. Chem. Commun. 1996, 2193. (b) Wang, Z. G.; Douglas, S. P.; Krepinsky, J. J. Tetrahedron Lett. 1996, 39, 6985. (c) Ito Y.; Kanie, O.; Ogawa, T. Ang. Chem. Int. Ed. 1996, 35, 2510 (d) Ito, Y.; Ogawa, T. J. Am. Chem. Soc. 1997, 119, 5562. (e) Dreef-Tromp, C. M.; Willems, H. A. M.; Westerduin, P.; van Veelen, P.; van Boeckel, C. A. A. Biorg. Med. Chem. Lett. 1997, 7, 1175. (f) Eggenweiler, H. M.; Bayer, E.

Innovation Perspect. Solid Phase Synth. Comb. Libr. Collect 1996, 4, 363.

9. Douglas, S. P.; Whitfield, D. M.; Krepinsky, J. J. J. Am. Chem. Soc. 1995, 117, 2116.

10. Mehta, S.; Whitfield, D. M. Tetrahedron Lett. 1998, 39, 5907.

11. (a) Kunz, H.; Harreus, A. Liebigs Ann. Chem. 1982, 41. (b) Sato, S.;

Nunomura, T.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1988, 29, 4097. (c) Sato, S.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1988, 29, 5267. (d) Nunomura, S.; Ogawa, T. Tetrahedron Lett. 1988, 29, 5681.

12. Nukada, T., Berces, A., Whitfield, D.M. J. Org. ChemA9SS, 64, 9030.

13. Ito, Y., Paulson, J. C, J. Am. Chem. Soc. 1993, 115, 1603.

14. (a) Demchenko, A. V., Boons, G-. J., Tetrahedron. Lett. 1998, 39, 3065. (b) Demchenko, A. V., Boons, G-. J., Chem. Eur. J. 1999, 5, 1278.

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19. Douglas.S. P., Whitfield, D. M., Krepinsky, J.J. J. Am. Chem. Soc. 1991, 113, 5095.

BACKGROUND OF THE INVENTION

The N-acetylneuraminic acid (NeuδAc) residue is an important constituent of a large number of biologically active oligosaccharides. It is normally attached to the 3- or the 6-position of a galactose residue, in an α-configuration. The disaccharide α-D-Neu5Ac- (2→3)-β-D-Gal is an important constituent of biologically important structures, such as GMι, GM₂, sialyl Lewis X and sialyl Lewis A, to name a few. Such oligosaccharides are ubiquitously found in cell membrane glycoproteins and gangliosides. Particular gangliosides are known to be tumor-associated antigens and there is a growing demand for efficient syntheses of these antigenes, to be used as cancer vaccines. Similarly, Neu5Ac-containing carbohydrate epitopes expressed on the surface of leukocytes play a key role in the recruitment of leukocytes to the sited of inflammation.

In view of their presence in biologically important oligosaccharides, a significant amount of work has been done towards the chemical synthesis of α-D-Neu5Ac-(2→3)-β-D-Gal disaccharides. Several research groups have published multistep syntheses of building blocks containing this disaccharide as part of their syntheses of larger oligosaccharides. However, in chemical syntheses the key glycosylation step rarely proceeds in greater then 60% yield and all syntheses require at least 10 chemical steps (see US 5,527,901). A number of enzymatic syntheses of the α-D-Neu5Ac-(2→3)-β-D-Gal glycosidic linkage have also been proposed (see, for example, US 5,908,766; US 5,882,901; US 5,876,980; US 5,872,096; US 5,728,554; US 5,409,817; US 5,374,541; US 5,352,670; Auge, C; Le Narvor, C; Lubineau, A. Carbohydr. in Eur. 1999 27, 22). However, the products of these syntheses require several chemical steps to be transformed into oiigosaccharide donors. Furthermore, most of the available (α-2,3) sialyltransferases used for enzymatic sialylation do not effectively use monosaccharide derivatives of D-galactose as substrates, resulting in relatively poor yields of the enzymatic glycosylation step. As a result, the proposed processes of enzymatic sialylation of galactose derivatives are not commercially viable and a strong need for an effective yet simple synthesis of α-D-Neu5Ac-(2→3)-β-D-Gal disaccharide exists.

Pathogenic bacteria, such as Campylobacter jejuni, Neisseha meningitidis and Group B Streptococcus have sialylated oligosaccharides as integral parts of their outer surface. It is widely believed that these sialic acid residues mimic host cell surface oligosaccharides. This leads to reduced immunogenicity and hence evolutionary advantages for these pathogens [1]. As part of the Institute for Biological Sciences of the Canada National Research Council program to develop protective vaccines against these microorganisms, a program has been initiated to synthesize some of their sialylated oligosaccharides [2]. Since the program required a large number of structural variants and derivatives of sialylated oligosaccharides to be synthesized, a building block approach was taken to satisfy this requirement. As a part of this effort, a highly efficient chemoenzymatic route to Neu5Ac-α-(2→3)-D-Galp disaccharide donors has been developed.

Such donors were previously prepared by a chemical method involving a three step preparation of a sialic acid glycosyl donor and a six step synthesis of a D -Galp acceptor. These monosaccharides were coupled in 60% yield and three additional steps of functional group manipulations were required to make a disaccharide donor. The whole process required nine chromatographic separations. A number of research groups have employed similar strategies in their syntheses of sialylated oligosaccharides [3]. Some recent syntheses have an improved efficiency [4], but such procedures still require too many synthetic steps and too many chromatographic separations. SUMMARY OF THE INVENTION

According to one aspect, the invention provides a highly efficient method for preparing thioglycosides comprising a sialylated residue of β-D-galactose. The method comprises the step of preparing, preferably by a chemical synthesis, a thioglycoside comprising a non-sialylated residue of β-D-gaiactose, and enzymatically sialylating the thioglycoside with a sialic acid in the presence of a suitable sialyltransferase. The sialylated thioglycoside so prepared is then derivatized by standard procedures, to provide a derivative suitable for use as a donor in chemical syntheses of sialylated oligosaccharides. The derivatized sialylated thioglycosides are prepared in high yields, due to reduced number of chemical and purification steps involved in the process.

According to another aspect, the invention provides novel derivatized thioglycosides comprising a sialylated residue of β-D-galactose, which can be used as building blocks in the synthesis of biologically active sialylated oligosaccharides.

In a preferred embodiment, aryl thioglycosides of galactose and lactose are enzymatically sialylated to afford glycoside donors, with minimal chemical steps and chromatographic purification. In the case of galactose, the method involves a two step preparation of a non-sialylated aryl thioglycoside donor, which process requires one simple chromatographic separation. This is followed by enzymatic glycosylation of the product so obtained using sialic acid, CTP and glycosides as substrates and CMP- Neu5Ac synthetase and sialyl transferase as catalysts [5], to produce the Neu5Ac-α- (2→3)-D-Galpβ(1→)SAr disaccharides (see Fig. 1) in >80% isolated yield, after gel filtration or reverse phase purification. These disaccharides so obtained are then derivatized by standard procedures (see Fig. 2), to provide derivatives suitable for use as donors in a chemical glycosylation. This process have been successfully scaled up from 10 mg to 100 mg, followed by 1g, with no diminution in yield.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 represents a general reaction scheme for the chemoenzymatic synthesis of Neu5Ac-α-(2→3)-D-Galpβ(1-→)-SAr disaccharide donors according to the invention.

Fig. 2 represents a reaction scheme showing derivatizing of disaccharide donors of Fig. 1.

Fig. 3 represents a reaction scheme showing the use of derivatives 5a or 5b of

Fig. 2 as donors in chemical glycosylation of the O-6 of D-Glcp acceptor 8 to yield a sialylated trisaccharide 9.

Fig. 4 represents a reaction scheme showing the use of derivative 7 of Fig. 2 to as a donor in chemical glycosylation of a polymer-bound diol 10, to prepare a precursor compound (12) of the Neu5Ac-α-(2→3)-D-Galpβ(1→4)-D-GlcNAcp-β-(1→) trisaccharide.

Fig. 5 represents a reaction scheme showing enzymatic sialylation of the phenyl thioglycoside of lactose (D-Galp-β-(1→4)-D-Glcp, 13), to prepare a trisaccharide phenyl thioglycoside 14 and various derivatives thereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a highly efficient method for preparing thioglycosides comprising a sialylated residue of β-D-galactose. Both alkyl and aryl thioglycosides can be used, but aryl thioglycosides are preferred. Particularly preferred are optionally substituted phenyl and naphthyl thioglycosides.

The method comprises the step of preparing, preferably by a chemical synthesis, a thioglycoside comprising a non-sialylated residue of β-D-galactose, and enzymatically sialylating the thioglycoside with a sialic acid in the presence of a suitable sialyltransferase. The sialylated thioglycoside so prepared is then derivatized, to provide a derivative suitable for use as a donor in chemical glycosylation syntheses of sialylated oligosaccharides. If desirable or necessary, the thioglycoside activating group can be converted into other activating group, such as a bromide or fluoride. All the chemical steps involved in the synthesis, derivatization and conversion of sialylated thioglycosides and their derivatives are carried out using standard procedures of carbohydrate chemistry, well known to those skilled in the art.

Certain sialylated thioglycosides according to the present invention, such as sialylated thioglycosides of galactose and lactose, in particular β-aryl thioglycosides of galactose or lactose are of substantial interest, as containing carbohydrate structures common to many biologically important oligosaccharides. These thioglycosides and derivatives thereof, when used as donors in chemical glycosylation processes, constitute building blocks in syntheses of such oligosaccharides.

In the following, the invention will be described in more detail, with references to the accompanying drawings and by way of specific examples.

Figure 1 shows a general reaction scheme for the chemoenzymatic synthesis of Neu5Ac-α-(2→3)-D-Galpβ(1→)-SAr disaccharide donors according to the invention. In the first step, an aryl thioglycoside of β-D-galactose is prepared by a standard chemical method, using a protected β-D-galactose. After removal of protecting groups, the thioglycoside is enzymatically sialylated with sialic acid in the presence of CMP-Neu5Ac synthetase and sialyl transferase as catalysts.

As can be seen from Fig. 2, a simple acetylation of aryl thioglycosides 4a and 4b prepared according to procedure outlined in Fig. 1 produces lactones 5a and 5b in 75% and 90% yields with the NeuδAc carboxyl esterified with D-Galp O-2 [6], The lactone can be used either directly as a glycosyl donor, or can be opened to the methyl ester 6 and the D-Galp O-2 acetylated to yield 7 in 71% yield from 5a. It would be readily apparent to those skilled in the art that other esters and other acyi groups could be prepared by the same procedure, as exemplified below. These aryl thioglycosides can be used directly as glycosyl donors. In cases where other activating groups may be desirable, these thioglycosides can be converted into bromides, fluorides or trichloroacetimidates, as required [7], using methods well known to those skilled in the art. This procedure is sufficiently simple to produce these donors on a large scale, thereby allowing for the commercial scale synthesis of bacterial antigens and other sialylated oligosaccharides.

As proof of principle, disaccharides 5a or 5b have been used as donors to glycosylate the O-6 of D-Glcp acceptor 8 to yield trisaccharide 9 in 90% and 86% yields respectively (see Fig. 3). In a more demanding application, the polymer bound diol 10 has been glycosylated with donor 7 to yield 11. The polymer is the soluble mono-methyl ether of polyethylene glycol (MeO-(CH₂CH₂O)_n-OH, MPEG) [8] and dioxyxylene (p-OCH₂-C₆H - CH₂O-, DOX) is the linker [9]. Routine cleavage of 11 from the polymer using Sc(OTf)₃/Ac₂O afforded the trisaccharide 12 in 36% overall yield (see Fig. 4) [10]. This trisaccharide is a synthon for the Neu5Ac-α-(2→3)-D-Galp-β-(1 →4)-D-GlcNAcp-β-(1 →) grouping found in many glycoconjugates.

In another embodiment of the invention, the phenyl thioglycoside of lactose (D-Galp-β- (1→4)-D-Glcp, 13) has been enzymatically sialylated to afford the trisaccharide 14 (see Fig. 5). Since glycosylations with O-2 acetylated lactose donors [11] are frequently complicated by orthoester formation and acyl transfer, the trisaccharide 14 was benzoylated to give 15 in 44% yield [12]. A previous work used a 2-O pivaloyl lactose derivative as glycosyl acceptor for enzymatic glycosylation and subsequently successfully used this to prepare synthetic GM₃ glycolipid by chemical glycosylation and functional group manipulations [13]. That synthesis is a good precedent, but the procedure according to the present invention is significantly simpler. Benzoylation of 14 produced the lactone 15 between D-Galp O-2 and the NeuδAc carboxyl, as for acetylation of the Neu5Ac-α-(2→3)-D-Galp disaccharides. The highly hindered NeuSAc O-7 was not benzoylated under these conditions. Subsequent esterification with methanol resulted in ester 16, and was followed by N- and O- acetylation [14] leading to the peracylated trisaccharide donor 17 in 51 % overall yield. The regiochemistry of the acyl groups in 15 and 17 was confirmed by ¹³C-¹H HMBC correlation experiments. Thus, a synthon for GM3 and derivatives has been efficiently prepared. It would be apparent to those skilled in the art that this procedure can be extended to larger oligosaccharides, for example those related to common gangliosides. EXAMPLES

2-Naphthyl 1-thio-β-D-galactopyranoside (3b)

1,2,3,4,6-penta-O-acetyl-β-D-galactopyranose (5 g; 12.8 mmol) was dissolved in dry CH2CI₂ (50 mL) and cooled to 0°C under an atmosphere of argon. To this solution. 2- naphthalene thiol (2.1 g; 13.0 mmol) was added, followed by boron trifluoride etherate (2.3 mL; 19.0 mmol). The mixture was left to stir and warm to room temperature (R.T.) for 4 h. The mixture was cooled in an ice bath and quenched with triethylamine. After concentration, the mixture was purified by chromatography to give 2b (6.1 g; 98% yield): [α]_D ²² 6.0° (c, 0.7, CHCI3); Anal. Calcd. for C₂4H26O9S: C, 58.77; H, 5.41. Found C, 59.10; H, 5.41. Then 2b (3.0 g; 6.1 mmol) was dissolved in methanol (150 mL) and freshly prepared sodium methoxide was added until pH = 10. After 4 h the reaction was neutralized with Rexyn 101(H) resin; filtered and evaporated. The residue was crystallized from absolute ethanol to give 3b [15] (1.9 g; 97% yield): [a]_D ²² -21.3° (c, 1.0, CH₃OH); ¹³C NMR (CD₃OD): δ 62.9 (C-6), 70.6 (C-4), 71.2 (C-2), 76.5 (C-3), 80.9 (C-5), 90.3 (C-1), 127.2, 127.6 (Ar-6, Ar-7), 128.6, 128.8 (Ar-5, Ar-8), 129.4 (Ar-4), 129.8 (Ar- 3), 130.6 (Ar-1), 133.7, 133.8 (Ar-10, Ar-9), 135.3 (Ar-2); ¹H NMR (CD₃OD): δ 3.55 (1H, dd, J_2,3 = 9.5 Hz, J₃ = 3.7 Hz, H-3), 3.65 (1 H, brt, H-5), 3.69 (1 H, bit, H-2), 3.76 (1 H, dd, J_5,6b = 5.1 Hz, J_6aβb = 11.7 Hz, H-6b) 3.83 (1 H, dd, J₅,_6a = 7.3 Hz, J_6a,_6b = 11.7 Hz, H-6a), 3.94 (1H, brd, J₃,₄2.9 Hz, H-4), 4.72 (1 H, d, J_1ι2 = 9.5 Hz, H-1); 7.46 (2H, m, Ar- 6, Ar-7), 7.63 (1H, dd, J₃,₄ = 8.6 Hz, J ₃ = 1.8 Hz, Ar-3), 7.79 (3H, m, Ar-4, Ar-5, Ar-8), 8.08 (1H, d, J_1ι3 = 1.8 Hz, Ar-1); Anal. Calcd. for C₁₆H₁₈O₅S: C, 59.61; H, 5.63. Found C, 59.25; H, 5.74.

Phenyl thioglycosides 2a, 13 and 3a were prepared as described for 2b and 3b above, see Ref. [16].

Polymer bound acceptor 10 was prepared by glycosylation of MPEGDOXOH [171 using 3,4,6-tri-0-acetyl-2-deoxy-2-Λ/-phthalimido-β-D-glucopyranosyl trichloroacetimidate [18] as donor. After deacetylation using 1 ,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in methanol, the primary hydroxyl was silylated according to Ref. [19]. Phenyl (5-Acetamido-3,5-dideoxy-D-g/ycero-α-D-gatacto-2-nonuiopyranosy!)- (2→3)-1 -thio-β-D-galactopyranoside (4a)

Phenyl 1-thio-β-D-galactopyranoside 3a (100 mg; 0.38 mmol) was sialylated with an α- 2,3-sialyltransferase (CST-06) from Campylobacter jejuni (CST-06 is a Campylobacter V^'e/^'ϋt7/^'α-2,3-sialyltransferase (CST-I) with Escheήchia coli maltose-binding protein fused at the N-terminus). The reaction was performed in a total volume of 10 mL. The reaction contained 3a (100 mg; 0.38 mmol), CTP (250 mg; 0.44 mmol), sialic acid (150 mg; 0.49 mmol), HEPES pH 7.5 (50 mM; 1.0 mL of 0.5 M HEPES), MgCI₂ (10 mM; 0.1 mL of 1 M MgCI₂), DTT (0.2 mM; 20 mL of 100 mM DTT), inorganic pyrophosphatase (10 mL; 10 U), CMP-NANA synthetase from Neisseria meningitidis NSY-04 (0.1 mL; 46 U) and CST-06 (2.6 mL; 0.56 U). The reaction was incubated at 32°C overnight. The reaction was 40 % complete as determined by TLC on silica with isopropanol/ butanol/0.1 M HCI (2:1 :1) as the developing solvent. Additional MgCI₂ (0.2 mL of 1 M MgCI₂), CST-06 (4.0 mL, 1.16 U) were added and the reaction was incubated at 32°C overnight. The reaction was 80 % complete. Additional CTP (60 mg), sialic acid (40 mg), MgCI₂ (0.2 mL of 1 M MgCI₂), NSY-04 (0.1 mL, 46 U), inorganic pyrophosphatase (5 mL, 5 U), CST 06 (4 mL, 1.16 U) were added. The reaction was incubated at 32°C overnight and was 95% complete. The reaction mixture was centrifuged and the supernatant was concentrated. The residue was placed on a Bio-Gel P2 column and eluted with water. Partially pure 5 was obtained (680 mg). A portion of this mixture (60 mg) was rechromatographed to obtain an analytically pure sample. The remaining amount (620 mg) was acetylated below: [α]_D ²² 1.3° (c, 0.2, H₂O); ¹³C NMR (D₂O): δ 21.7 (COCH₃), 39.3 (C-3"), 51.3 (C-5^N), 60.6 (C-6¹), 62.3 (C-9¹¹), 67.1 (C-2¹), 67.4 (C-4¹), 67.8 (C-7¹¹), 68.0 (C-4¹¹), 71.4 (C-δ"), 72.6 (C-6¹¹), 76.7 (C-3¹), 78.4 (C-5¹), 86.9 (C-1¹), 99.6 (C- 2¹¹), 127.6-131.9 (Ar), 173.5 (C-1¹¹), 174.7 (COCH3); ¹H NMR (D₂0): δ 1.63 (1 H, t, J₃a_,3e₊₃a,₄ = 24.2 Hz, H-3a"), 1.86 (1 H, s, COCH₃), 2.58 (1H, dd, J_3e,3a = 12.5 Hz, J_3e,4 = 4.4 Hz, H-3e"), 3.42 (H-7^M), 3.45 (H-9a", H-6"), 3.47 (H-2), 3.51 (H-4"), 3.54 (H-6a', H- 6b'), 3.56 (H-5¹), 3.68 (H-5¹¹), 3.69 (H-9b"), 3.70 (H-8¹¹), 3.83 (1H, d, J= 2.7 Hz, H-4¹), 3.96 (1 H, dd, J_2,3 = 9.2 Hz, J₃₄ = 3.1 Hz, H-3¹), 4.68 (1 H, H-V), 7.18-7.43 (5H, m, Ar); HRMS (FAB) Calcd. for C₂₃H₃₃NOι₃SNa: 586.1586; found m/z: 586.1401 (M+Na⁺). 2-Napthyl (5-Acetamido-3,5-dideoxy-D-g/ycero-α-D-ga/acto-2-nonulopyranosyl)- (2→3)-1 -thio-β-D-galactopyranoside (4b)

2-Napthyl 1 -thio-β-D-galactopyranoside 3b was sialylated with an α-2,3-sialyltransferase (CST-04) from Campylobacter jejuni (CST-04 is a Campylobacter jejuni α-2,3- sialyltransferase (CST-I) with CMP-NeuAc synthetase from Neisseria meningitidis fused at the N-terminus). The reaction was performed in a total volume of 500 mL. The reaction contained 3b (1.0 g; 3.1 mmol), CTP (2.0 g; 3.5 mmol), sialic acid (1.25 g, 4.0 mmol), HEPES pH 7.5 (50 mM; 25 mL of 1 M HEPES), MgCI₂ (30 mM; 15 mL of 1 M MgCI₂), DTT (0.2 mM; 0.1 mL of 1M DTT) and CST-04 (50 mL; 75 U). The reaction was incubated at 32°C and at 100 rpm overnight. The product formation was followed by TLC on silica with isopropanol/butanol/0.1 M HCI (2:1 :1) as the developing solvent. The reaction was 89 % complete as determined by capillary electrophoresis. The pH of the reaction was adjusted from 6.7 to 7.5 with 10N NaOH. Additional MgCI₂ (15 mL of 1 M MgCI₂), CTP (0.3 g, 0.5 mmol), sialic acid (0.2 g; 0.65 mmol) and CST-04 were added. After 90 min at 37°C the reaction was 97 % complete. The reaction mixture was centrifuged for 30 min at 10 K. The pellet was washed with water (100 ml) and recentrifuged. The supematants were combined and recentrifuged for 30 min at 14 K. The final volume of the reaction mixture was 680 mL. The product was purified by reverse phase column chromatography on C-18 silica gel (35-70 micron particles). The reaction mixture was divided into 4 parts, each part was diluted to 800 mL and loaded on a column of 100 g of C-18 silica gel, at the rate of 4 mUmin. The column was washed with water (150 mL), 10 % MeOH (150 mL) and 50% MeOH (400 mL), 100% MeOH (100%). The product 4b was obtained in the 50% MeOH fraction (1.7 g; 90% yield). [α]_D ²² 6.4° (c, 1.0, H₂O); ¹³C NMR (D₂O): δ 21.8 (COCH₃), 39.4 (C-3ⁿ), 51.4 (C- 5"), 60.8 (C-6¹), 62.4 (C-9¹¹), 67.3 (C-2¹), 67.5 (C-4¹), 67.8 (C-7¹¹), 68.1 (C-4¹¹), 71.5 (C-8^H), 72.7 (C-6"), 76.8 (C-3¹), 78.7 (C-5¹), 87.0 (C-1¹), 99.7 (C-2¹¹), 126.6-133.0 (Ar), 173.6 (C- 1"), 174.8 (COCH₃); ¹H NMR (D₂O): δ 1.63 (1 H, t, Jsa.se_÷sa^ = 24.4 Hz, H-3a"), 1.86 (1H, s, COCH₃), 2.59 (1 H, dd, J_3e,_3a = 12.7 Hz, J_3e,4 = 4.9 Hz, H-3e"), 3.41 (H-7^M), 3.45 (H- 9a"), 3.46 (H-6"), 3.51 (H-4^π), 3.53 (H-2), 3.56 (H-6a', H-6b'), 3.60 (H-5¹), 3.68 (H-5¹¹), 3.67 (H-9b"), 3.70 (H-8¹¹), 3.85 (1 H, d, J= 2.4 Hz, H-4¹), 3.99 (1 H, dd, J2.3 = 9-8 Hz, J₃,4 = 3.2 Hz, H-3^j), 4.78 (1 H, J₂,₃ = 9.8 Hz, H-1¹), 7.39-7.94 (7H, Ar). Anal. Calcd. for C₂₇H₃₅ Oι₃S: C, 52.76; H, 5.74; N, 2.28. Found C, 52.56; H, 5.76; N, 2.09.

Phenyl [(5-Acetamido-4 ,8,9-tetra-0-acetyl-3,5-dideoxy-D-g/ycero-α-D-ga/acto- 2-nonulopyranosyl-1 '→2-lactone)]-(2→3)-0-4,6-di-0-acetyl-1 -thio-β-D- galactopyranoside (5a)

Partially pure phenyl thioglycoside 4a (620 mg) was dissolved in pyridine (16 mL). Acetic anhydride (8 mL) was added at 0°C. After 24 h at room temperature, additional pyridine (4 mL) and acetic anhydride (2 mL) were added. The reaction mixture was stirred for another 20 h and concentrated. It was dissolved in dichloromethane and washed with water. The organic extracts were dried over magnesium sulfate and concentrated. The residue was column chromatographed with hexane/ethyl acetate/methanol (3:3:0.5) as eluant. The title compound 5a was obtained as a solid (193 mg; 75% yield from 3a): [α]_D ²² -31.3° (c, 0.3, CH₂CI₂); ¹³C NMR (CDCI₃): δ 20.5, 20.6, 20.7, 20.8, 20.9, 20.94, 23.1 (7COCH₃), 37.8 (C-3¹¹), 49.4 (C-5¹¹), 61.7 (C-6¹), 62.3 (C-9¹¹), 66.4 (C-4¹), 67.4 (C-7¹¹), 69.3 (C-4¹¹), 69.8 (C-8¹¹), 72.2 (C-2¹), 73.1 (C-6^π), 74.5 (C-5¹), 75.7 (C-3¹), 86.9 (C-1¹), 97.1 (C-2^M), 128.4, 129.1 , 132.7, 133.2, (Ar), 163.6 (C- 1"), 169.7, 169.8, 170.2, 170.3, 170.4, 170.8, 170.9 (COCH3); ¹H NMR (CDCI3): δ 1.91 , 2.03, 2.06, 2.12, 2.13, 2.25, 2.63 (21H, 7s, 7COCH₃), 1.94 (1H, t, H-3a"), 2.50 (1H, dd, J_3e,₃a = 13.2 Hz, J_3e,₄ = 5.4 Hz, H-3e"), 3.77 (1 H, dd, H-6^M), 3.93 ( 1H, t, J₄,₅₊₅,6 = 13.2 Hz, H-5), 4.03 (1 H, dd, J₂,₃ = 10.3 Hz, J₃,₄ = 2.9 Hz, H-3¹), 4.12 (H-9"), 4.15 (H-6a'), 4.19 (2H, H-6b', H-5"), 4.34 (1 H, dd, J₈,₉ = 2.9 Hz, J₉,₉ = 12.7 Hz, H-9b"), 4.68 (1 H, d, J_1ι2 = 9.8 Hz, H-1¹), 4.95 (1 H, t, _1ι2+2.₃ = 20.0 Hz, H-2¹), 5.19 (1 H, m, H-δ"), 5.34 (1H, dd, H- 7"), 5.42 (1 H, d, J _H,5 = 5.9 Hz, NHAc), 5.47 (1 H, m, H-4"), 5.53 (1 H, d, J= 2.0 Hz, H- 4¹), 7.33-7.58 (5H, m, Ar). MS(MALDI-TOF) Calcd. for C₃5H₄4NO₁₈SNa: 797.2200, found m/z: 798.3 (M+H⁺); 820.3 (M+Na⁺); 836.3 (M+K⁺); HRMS (FAB) found m/z: 797.2133 (M+Na⁺). Anal. Calcd. for C₃₅H4₃NO18S: C, 52.69; H, 5.43; N, 1.76. Found C, 53.52; H, 5.43; N, 1.64. Phenyl [Methyl (5-acetamido-4,7,8,9-tetra-0-acetyl-3,5-dideoxy-D-g/ycero-α-D- ga/acto-2-nonulopyranosyl)onate)]-(2→3)-0-4,6-di-0-acetyl-1 -thio-β-D- galactopyranoside (6)

The lactone 5a (170 mg; 0.22 mmol) was dissolved in methanol (4.5 mL). 4- (Dimethylamino)pyridine (0.5 mL of a 10% solution; 0.05 mmol) was added at 0°C. The reaction was stirred for 24 h at room temperature and concentrated under reduced pressure. The residue was column chromatographed with hexane/ethyl acetate/methanol (3:3:0.5) as eluant. The title compound 6a was obtained as a solid (145 mg; 81% yield). ;[α]_D ²² -6.5° (c, 0.46, CH₂CI₂); ¹³C NMR (CDCI₃): δ 20.6, 20.7, 21.4, 23.2 (COCH₃), 37.7 (C-3"), 49.4 (C-5¹¹), 53.2 (OCH₃), 62.2 (C-9¹¹), 62.4 (C-6¹), 66.7 (C- 7"), 67.8 (C-2¹), 67.9 (C-4¹, C-8¹¹), 68.7 (H-4¹¹), 72.4 (H-6¹¹), 74.4 (C-5¹), 75.1 (C-3¹), 87.1 (C-1¹), 96.9, (C-2¹¹), 109.7 (C-1¹¹), 127.4-132.9 (Ar), 167.6 (C-1¹¹), 170.2, 170.4, 170.6, 170.7 (COCH₃); ¹H NMR (CDCI₃): δ 1.71 (1 H, t, J_3a,3e₊3a.4 = 24.4 Hz, H-3a"), 1.86, 1.98, 2.02, 2.05, 2.07, 2.09, 2.18, 2.26 (24H, 8s, 8COCH3), 2.59 (1 H, dd, J_3e,3a = 12.7 Hz, _{3e 4} = 4.9 Hz, H-3e"), 3.66 (1H, dd, J₅,₆ = 10.8 Hz, J₆,7 = 2.2 Hz, H-6"), 3.86 (3H, s, OCH₃), 3.94 ( 1H, t, J₄,₅₊₅,₆ = 12.1 Hz, H-5¹), 3.99 (1 H, dd, J₈,₉ = 5.4 Hz, J₉,₉ = 12.3 Hz, H-9a"), 4.07 (3H, m, H-6a', H-6b', H-5"), 4.37 (1 H, dd, J₈,₉ = 2.1 Hz, J₉,₉ = 12.3 Hz, H- 9b"), 4.66 (1 H, dd, J₂,₃ = 9 8 Hz, J₃,₄ = 3.4 Hz, H-3¹), 4.88 (1 H, m, H-4ⁿ), 4.90 (1 H, d, _1>2 = 9.8 Hz, H-1¹), 4.96 (1H, d,

10.3 Hz, NHAc), 5.08 (1H, t, _,2+2,3 = 19.5 Hz, H-2¹), 5.38 (1H, dd, J₆,₇ = 2.2 Hz, J₇,₈ = 8.8 Hz, H-7 ), 5.56 (1 H, m, H-8"), 7.29 (3H, m, Ar), 7.54 (2H, m, Ar); HRMS (FAB) Calcd. for C₃₆H₄₇NOi₉SNa: 852.2360, found m/z: 852.2418 (M+Na⁺); Anal. Calcd. for C₃₆H₄₇NOι₉S: C, 52.11 ; H, 5.71 ; N, 1.69. Found C, 51.96; H, 5.64; N, 1.52.

Phenyl [Methyl (5-acetamido-4,7,8,9-tetra-0-acetyl-3,5-dideoxy-D-g/ycero-α-D- ga/acfo-2-nonulopyranosyl)onate)]-(2→3)-0-2,4,6-tri-0-acetyl-1-thio-β-D- galactopyranoside (7)

Alcohol 6 (110 mg; 0.13 mmol) was treated with pyridine (5 mL) and acetic anhydride (2.5 mL) at 10°C. The reaction was stirred for 16 h at room temperature and concentrated under reduced pressure. The residue was column chromatographed with hexane/ ethyl acetate/ methanol (3:3:0.5) as eluant. The title compound 7 was obtained as a solid (114 mg; 88% yield): [α]_D ²² 1.3° (c, 0.54, CH CI₂); ¹³C NMR (CDCI₃): δ 20.7, 20.75, 20.78, 20.9, 21.5, 23.2 (COCH3), 37.6 (C-3^N), 49.1 (C-5ⁿ), 53.2 (OCH₃), 62.3 (C- 6'), 62.4 (C-9"), 67.1 (C-7"), 67.8 (C-4¹), 67.8 (C-8¹¹), 67.9(H-2'), 69.3 (C-4¹¹), 72.1 (C-6ⁿ), 72.3 (C-3¹), 74.3 (C-5¹), 85.6 (C-1¹), 96.8, (C-2^I!), 127.6, 128.7, 132.0, 133.0 (Ar), 167.9 (C-1¹¹), 169.6, 169.7, 170.27, 170.33, 170.5, 170.9 (COCH3); ¹H NMR (CDCI₃): δ 1.71 (1H, t, J_3a,₃e₊₃a,4 = 24.4 Hz, H-3a"), 1.86, 1.98, 2.02, 2.05, 2.07, 2.09, 2.18, 2.26 (24H, 8s, 8COCH₃), 2.59 (1H, dd, J_3e,₃a = 12.7 Hz, J_3e,4 = 4.9 Hz, H-3e"), 3.66 (1H, dd, J₅,e = 10.8 Hz, J₆,7 = 2.2 Hz, H-6^M), 3.86 (3H, s, OCH₃), 3.94 (1 H, t, J₄,₅₊ ,6 = 12.1 Hz, H-5¹), 3.99 (1H, dd, J_8|9 = 5.4 Hz, J₉,₉ = 12.3 Hz, H-9a"), 4.07 (3H, m, H-6a', H-6b', H-5"), 4.37(1H, dd, J₈,₉ = 2.1 Hz, J₉,₉ = 12.3 Hz, H-9b"), 4.66 (1 H, dd, J₂,₃ = 9.8 Hz, J₃,4 = 3.4 Hz, H-3¹), 4.88 (1 H, m, H-4^M), 4.90 (1 H, d, _1ι2 = 9.8 Hz, H-1¹), 4.96 (1 H, d, J= 2.9 Hz, H-4'), 5.04 ( 1H, d,

10.3 Hz, NHAc), 5.08 (1H, t, 4,2+2,3 = 19-5 Hz, H-2¹), 5.38 (1H, dd, J₆,₇ = 2.2 Hz, J₇,β = 8.8 Hz, H-7^M), 5.56 (1 H, m, H-8ⁿ), 7.29 (3H, m, Ar), 7.54 (2H, m, Ar); HRMS (FAB) Calcd. for C₃₈H₄₉NO₂oSNa: 894.2465, found m/z: 894.2424 (M+Na⁺); Anal. Calcd. for C₃₈H₄₉NO₂₀S: C, 52.35; H, 5.66; N, 1.61. Found C, 52.12; H, 5.37; N, 1.57.

2-Naphthyl [(5-Acetamido-4,7,8,9-tetra-0-acetyl-3,5-dideoxy-D-g/ycero-α-D-ga/acfo- 2-nonulopyranosyl-1'→2-lactone)]-(2→3)-0-4,6-di-0-acetyl-1-thio-β-D- galactopyranoside (5b)

Naphthyl thioglycoside 4b (100 mg; 0.16 mmol) was treated with pyridine (7 mL) and acetic anhydride (4 mL) at 0°C. The reaction was stirred for 24 h at room temperature and concentrated under reduced pressure. The residue was column chromatographed with hexane/ethyl acetate/methanol (3:3:0.5) as eluant. The title compound 5b was obtained as a solid (126 mg; 90% yield). [α]_D ²² -20.6° (c, 0.54, CH₂CI₂); ¹³C NMR (CDCI₃): δ 20.5, 20.6, 20.65, 20.8, 20.9, 21.0, 23.2 (7COCH₃), 37.7 (C-3^π), 49.3 (C-5ⁿ), 61.8 (C-6¹), 62.9 (C-9¹¹), 66.5 (C-4¹), 67.4 (C-7¹¹), 69.3 (C-4¹¹), 69.9 (C-8¹¹), 72.3 (C-2¹), 73.1 (C-6"), 74.6 (C-5¹), 75.6 (C-3¹), 86.9 (C-1¹), 97.1 (C-2"), 126.8, 127.6, 127.7, 128.7, 129.7, 130.4, 132.1, 132.8, 133.5 (Ar), 163.6 (C-1¹¹), 169.7, 169.9, 170.3, 170.4, 170.44, 170.8, 171.0 (COCH₃); ¹H NMR (CDCI3): δ 1.92, 2.02, 2.03, 2.13, 2.14, 2.28 (21 H, 6s, 7COCH₃), 1.97 (1 H, t, J_3a,3e₊3a,4 = 17.6 Hz, H-3a"), 2.52 (1 H, dd, J_3e,3a = 13.7 Hz, J_3e,₄ = 5.4 Hz, H-3e"), 3.80 (1H, d, ,6 = 10.7 Hz, H-6"), 3.93 ( 1H, t, 4,5+5,₆ = 12.7 Hz, H-5¹), 4.05 (1 H, dd, J₂,₃ = 10.3 Hz, J₃, = 2.9 Hz, H-3¹), 4.13 (H-9"), 4.17 (H-6a'), 4.21 (H-5^M), 4.24 (H-6b'), 4.36 (1 H, dd, J₈;_&= 2.9 Hz, J₉ ,₉> = 12.7 Hz, H-9b"), 4.76 (1 H, d, 4_,2 = 9.7 Hz, H-1¹), 5.0 (1H, t, 4,2+2.3 = 20.0 Hz, H-2¹), 5.23 (1 H, m, H-8"), 5.35 (1H, dd, J₆,₇ = 1.5 Hz, J₇.₈ = 6.4 Hz, H-7"), 5.47 (1H, m, H-4^π), 5.48 (1H, d, NHAc), 5.53 (1H, d, J= 2.9 Hz, H-4¹), 7.50-7.53 (2H, m, Ar), 7.61-7.63 (1 H, dd, Ar), 7.78-7.84 (3H, m, Ar), 8.06 (1 H, x, Ar). HRMS (FAB) Calcd. for C₃₉H₄₅NOι₈SNa: 870.2254, found m/z: 870.2252 (M+Na⁺). Anal. Calcd. for C₃₉H₄₅NO₁₈S: C, 55.25; H, 5.35; N, 1.65. Found C, 55.18; H, 5.45; N, 1.48.

Phenyl (5-Acetamido-3,5-dideoxy-D-g/ycero-α-D-ga/acfo-2-nonulopyranosyl)- (2→3)-β-D-galactopyranosyl-(1→4)-1-thio-β-D-glucopyranoside (14)

Thioglycoside 13 (100 mg; 0.23 mmol) was sialylated as above for 4b using CST-04.

The product was partially purified by P-2 gel permeation chromatography to yield 95% pure 14 (152 mg; 87% yield): [α]_D ²² -15.2° (c, 0.11 , CH₃OH); ¹³C NMR (D₂O): δ 22.9 (COCH₃), 40.2 (C-3'"), 52.5 (C-5^,n), 60.9 (C-6¹), 61.9 (C-6ⁿ), 63.4 (C-9"), 68.3 (C-4 ),

69.2 (C-2"), 69.4 (C-6¹¹¹), 70.2 (C-7¹¹), 72.3 (C-2¹), 72.6 (C-8^nl), 73.7 (C-4¹¹¹), 76.3 (C-3¹¹),

76.6 (C-3¹), 78.6 (C-4¹), 79.6 (C-5¹), 88.0 (C-1¹), 100.6 (C-2¹¹¹), 103.4 (C-1¹), 128.9, 130.1 , 132.5, 132.7 (Ar), 174.7 (C-1¹¹¹), 175.8 (COCH3); ¹H NMR (D₂O): δ 1.69 (1H, t, J_3a,4 =

12.7 Hz, J_3e,3a 12.2 Hz, H-3a'"), 1.92 (1H, s, COCH₃), 2.65 (1H, dd, J₃e,_3a = 12.2 Hz, J_3e,4 = 4.4 Hz, H-3e^,π), 3.30 (1 H, dd, 4,₂ = 9.8 Hz, J₂,₃ = 9.3 Hz, H-2¹), 3.39 (1 H, m, H-6^Mi),

3.44 (1H, m, H-7^m), 3.46 (1 H, m, H-2"), 3.52 (2H, m, H-5¹, H-4¹"), 3.53 (1H, m, H-9a'"), 3.57 (1 H, m, H-4¹), 3.58 (2H, m, H-3¹, H-5"), 3.62 (2H, m, H-6ab"), 3.71 (H-6a'), 3.76 (1H, m, H-9b'"), 3.77 (1 H, m, H-8¹"), 3.80 (1 H, m, H-5¹"), 3.85 (1H, m, H-4"), 3.86(1 H, m, H-6b') 4.01 (1H, dd, J_2,3 = 10.1 Hz, 4,4 = 3.6 Hz, H-3"), 4.42 (1H, d, 4,2 = 7.8 Hz, H- 1"), 4.71 (1 H, d, 4_,2 = 9.8 Hz, H-1¹), 7.28 (3H, m, Ar), 7.48 (2H, d, J = 6.8 Hz, Ar); HRMS(FAB) Calcd. for C₂₉H₄₃NOι₈SNa: 748.2099, found m/z: 748.2030 (M+Na⁺). Phenyl [(5-Acetamido-4,8,9-tri-0-benzoyM-3,5-dideoxy-D-g/ycero-α-D-ga/acto-2~ nonulopyranosyl-1'-→2-lactone)]-(2→3)-0-4,6-dι-0-benzoyl-β-D- galactopyranosyl-(1 →4)-2,3,6-tri-0-benzoyl-1 -thio-β-D-glucopyranoside (15)

Partially pure phenyl thioglycoside 14 (130 mg; 0.18 mmol) was dissolved in pyridine (20 mL). Benzoic anhydride (1.62 g, 40 eq.) was added and the mixture heated at 40°C. After 44 h the reaction was cooled and quenched with methanol and concentrated. The residue was column chromatographed with hexane/ ethyl acetate (6:4) as eluant. The title compound 15 was obtained as a slightly impure solid (141 mg; 50% yield). A small portion was rechromatographed with toluene/ ethyl acetate (6:4) as eluant to yield analytically pure material. [α]_D ²² -23.2° (c 0.75 CHCI₃); ¹³C NMR (CDCI₃): δ 22.8 (COCH₃), 37.6 (C-3^,M), 51.3 (C-5¹¹¹), 60.5 (C-6¹¹), 61.8 (C-6¹), 63.0 (C-9¹¹¹), 66.0 (C-7¹¹¹), 66.1 (C-4"), 69.2 (C-4^MI), 70.1 (C-2¹), 70.2 (C-8¹¹¹), 71.3 (C-5¹¹), 72.9 (C- 6"'),73.3 (C-3¹), 73.4 (C-2¹¹), 73.7 (C-3¹¹), 76.2 (C-4¹), 76.6 (C-5¹), 85.7 (C-1¹), 97.1 (C-2^ιn), 100.0 (C-1¹¹), 128.3, 130.0, 133.3 (Ar), 163.2 (C-1¹), 165.2, 165.3, 165.4, 166.1 , 167.2 (COPh), 172.9 (CON); ¹H NMR (CDCI3): δ 1.85 (3H, s, COCH3), 2.00 (1H, t, 4a,3e₊3a,4 = 13.4 Hz, H-3a"'), 2.47 (1 H, dd, 4e,3a = 12.2 Hz, 4e,4 = 5.5 Hz, H-3e'"), 3.23 (1 H, m, H-5¹), 3.50 (1H, m, H-6¹"), 3.53 (1 H, dd, J_6a,₆b = 11.0 Hz, J₅,_6b = 6.1 Hz, H-6b"), 3.66 (2H, m, H-5", H-6a"), 3.86 (1 H, m, H-7¹"), 3.98 (1 H, brt, ,4 = 9.5 Hz, 4,5 9.8 Hz, H-4¹), 4.08 (1H, brdd, 4,₅ = 10.4 Hz, J₅,_NH = 7.9 Hz, H-5¹"), 4.32 (2H, m, H-6a', H-9b"'), 4.48 (1H, d, 4_,2 = 7.6 Hz, H-1"), 4.58 (1H, brd, J_6a,_6b = 11.3 Hz, H-6a'), 4.84 (2H, m, H-8¹", H-2¹¹), 4.84 (1H, d, 4_,2 = 9.8 Hz, H-1¹), 4.88 (1 H, m, H-3^π), 4.99 (1 H, m, H-8¹"), 5.33 (1H, t, 4.₂ = 9.8 Hz, 4_,3 = 9.5 Hz, H-2¹), 5.53 (1H, brd, J₃,₄2.8 Hz, H-4^M), 5.80 (1H, t, J₂,₃ = 9.5 Hz, 4_,4 = 9.8 Hz, H-3¹), 5.86 (1 H, ddd, = 5.5 Hz, J_3b,₄ = 12.2 Hz, 4,5 = 10.4 Hz, H-4¹"), 6.14 (1H, d, 4_/H,5 = 7.9 HZ, NH), 7.07 (2H, t, J = 7.4 Hz, Ar), 7.16 (2H, t, J = 7.4 Hz, Ar), 7.27 (2H, m, Ar), 7.40 (12H, m, Ar), 7.48 (6H, m, Ar), 7.55 (5H, m, Ar), 7.89, 7.93, 7.95, 7.96, 7.99, 8.01 , 8.06, 8.10, 8.12 (18H, 9brd, J = 7.6 Hz, Ar). MS (FAB) Calcd. for C₈₅H₇₃NO₂₅SNa: m/z: 1562.4089, found m/z: 1563 (M+Na⁺), 956 (M-GlcSPh⁺). Anal Calcd. for C₈₅H₇₃NO₂₅S: C, 66.27; H, 4.78; N, 0.97. Found C, 66.37; H, 4.74; N. 0.76. Phenyl [Methyl (5-acetamido-4,8,9-tri-0-benzoyl-3,5-dideoxy-D-g/ycero-α-D- gafacto-2-nonulopyranosyl)onate)]-(2→3)-0-4,6-di-0-benzoyl-β-D- galactopyranosyl-(1 →4)-2,3,6-tri-0-benzoyl-1 -thio-β-D-giucopyranoside f 16)

Lactone 15 (78 mg; 0.051 μmol) was converted to its methyl ester as described above for 6a (44 mg, 55%). [α]_D ²² -7.7°(c 0.64 CHCI₃); ¹³C NMR (CDCI3): δ 23.0 COCH₃), 37.7 (C-3¹¹¹), 51.7 (C-5¹¹¹), 52.5 (OCH₃), 61.3 (C-6^N), 63.1 (C-6¹), 64.3 (C-9¹¹¹), 67.0 (C-7¹¹¹), 68.2 (C-4¹¹), 68.9 (C-4¹¹¹), 69.9 (C-8¹¹¹), 70.0 (C-2¹¹), 70.5 (C-2¹), 71.1 (C-5^M), 74.4 (C-6^m), 74.4 (C-3"), 74.8 (C-3¹), 75.6 (C-4¹), 77.5 (C-5¹), 85.9 (C-1¹), 96.8 (C-2¹¹¹), 103.7 (C-1¹¹), 128.3, 129.7, 132.9 (Ar), 165.2, 165.3, 165.4, 165.5, 165.9, 166.0 (COPh), 167.5 (C-1¹), 172.8 (CON); H NMR (CDCI3): δ 1.92 (3H, s, COCH3), 1.94 (1 H, t, 4a,3e₊3a,4 = 12.8 Hz, H- 3a"'), 2.59 (1H, dd, 4_e,₃a = 12.5 Hz, 4e,₄ = 4.9 Hz, H-3e ), 3.25 (3H, s, OCH₃), 3.31 (1H, brs, OH), 3.54 (1H, dd, J_6a,_6b = 11 -3 Hz, ,66 = 6.4 Hz, H-6b"), 3.57 (1 H, m, H-6¹"), 3.70 (1 H, dd, J_6a,_6b = 11 -3 Hz, 4,₆a = 7.0 Hz, H-6a"), 3.78 (1 H, dd, 4,2 = 7.6 Hz, J₂,₃ = 10.1 Hz, H-2"), 3.86 (1H, brt, H-5ⁿ), 4.02 (1H, dd, 4,7 = 4.9 Hz. 4,8 = 8.2 Hz, H-7¹"), 4.14 (1H, m, H-5^IM), 4.14 (1H, m, H-5¹), 4.36 (1H, brt, J₃,₄ = 9.5 Hz, 4,59.5 Hz, H^'), 4.62 (1H, dd, 4,₃ = 10.1 Hz, 4,₄ = 3.0 Hz, H-3^π), 4.65 (1 H, dd, J_9at9b = 12.2 Hz, ,9b = 4.9 Hz, H-9b'"), 4.78 (1 H, dd, J_6a = 11.9 Hz, 4,6, = 5.2 Hz H-6b'), 4.89 (1H, d, 4,2 = 7.6 Hz, H-1¹¹), 4.97 (1 H, dd, J_9a,_9b = 12.2 Hz, 4,9a = 3.9 Hz, H-9a'"), 5.02 (1H, d, 4,2 = 9.8 Hz, H-1¹), 5.19 (1 H, m, H-6a'), 5.19 (1 H, m, H-4"), 5.25 (1 H, ddd, 4a,4 = 4.9 Hz, J_3b = 12.5 Hz, 4,₅ = 10.7 Hz, H-4¹"), 5.49 (1 H, t, 4,2 = 9.8 Hz, 4,3 = 9.5 Hz, H-2¹), 5.83 (1H, m, H-8¹"), 5.86 (1H, t, 4_,3 = 9-5 Hz, 4,₄ = 9.8 Hz, H-3¹), 6.09 (1H, d, J_NH,₅ = 7.9 Hz, NH), 7.07 (2H, t, J = 7.9 Hz, Ar), 7.13 (2H, t, J = 7.3 Hz, Ar), 7.26 (5H, m, Ar), 7.48 (20H, m, Ar), 7.74, 7.89, 7.93, 7.95, 7.99, 8.01 , 8.01 , 8.03 (16H, 8brd, J = 7.6 Hz, Ar). MS (FAB) Calcd. for C₈₆H₇₇NO₂₆SNa: m/z: 1594.4351 , found m/z: 1594.3 (M+Na⁺), 618 (Bz₃MeNeu5Ac"). Anal. Calcd. for C₈₆H₇₇NO₂₆S: C, 65.69; H, 4.94; N, 0.89. Found C, 66.13; H, 4.91 ; N, 0.77. Phenyl [Methyl (5-diacetamido-7-0-acety!-4,8,9-trϊ-0-benzoyl-3,5-dideoxy-D- g/ycero-α-D-ga/acto-2-nonulopyranosyl)onate)]-(2→3)-0-2-0-acetyl-4,6-di-0- benzoyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-0-benzoyl-1-thio-β-D- glucopyranoside (17)

Diol 16 (29 mg; 18 μmol) was dissolved in isoprenyl acetate (4 mL) to which was added p-toluenesulphonic acid (2 mg) and heated at 65°C for 16 h. The mixture was neutralized with a few drops of triethylamine and after concentration was purified by column chromatography eluting with ethyl acetate/hexanes (6:4) to give 17 (29 mg; 92% yield); [α]_D ²² 2.3° (c 0.26 CHCI₃); ¹³C NMR (CDCI3): δ 20.9, 21.3 (OCOCH3), 26.8, 28.0 (NCOCH₃), 38.6 (C-3'"), 56.2 (C-5¹¹¹), 52.8 (OCH₃), 60.7 (C-6¹¹), 63.1 (C-6¹), 63.1 (C-9¹¹¹), 67.6 (C-7¹¹¹), 67.6 (C-4¹¹), 67.6 (C-4¹¹¹), 69.4 (C-8¹¹¹), 70.6 (C-2¹¹), 70.5 (C-2¹), 70.9 (C-5¹¹), 70.0 (C-6¹¹¹), 72.0 (C-3"), 74.6 (C-3¹), 77.0 (C-4¹), 77.2 (C-5¹), 86.0 (C-1¹), 97.2 (C-2¹¹¹), 101.6 (C-1¹¹), 128.3, 129.6, 133.0 (Ar), 165.0, 165.2, 165.4, 165.8, 166.0 (COPh), 167.4 (C-1¹), 169.8, 170.1(COCH₃), 173.3, 174.2 (CON); ¹H NMR (CDCI3): δ 1.59 (1H,. t, 4a,₃e_+3a,₄ = 12.8 Hz, H-3a'"), 2.04, 2.07, 2.26, 2.33 (12H, s, COCH₃), 2.63 (1 H, dd, 4e,3a = 12.5 Hz, 4e,₄ = 5.2 Hz, H-3e'"), 3.46 (3H, s, OCH₃), 3.40 (1H, dd, J_6a,₆b = 11.0 Hz, 4,₆* = 8.2 Hz, H-6b"), 3.48 (1 H, dd, J_6a,₆b = 11.0 Hz, 4,6a = 5.5 Hz, H-6a"), 3.76 (1 H, brt, H- 5"), 3.99 (1H, m, H-5¹), 4.10 (1H, brt, 4,4 = 9-5 Hz, 4,59.5 Hz, H-4¹), 4.27 (1H, dd, J_9a,_9b = 12.5 Hz, ,₉* = 6.1 Hz, H-9b"'), 4.46 (1 H, dd, J_6a = 11.9 Hz, ,6*, = 6.1 Hz H-6b'), 4.53 (1H, t, 4,₅ = 10.4 Hz, 4,₆ = 10.1 Hz, H-5¹"), 4.77 (1 H, dd, J₂,₃ = 10.1 Hz, 4,4 = 3.0 Hz, H-3"), 4.77 (1 H, dd, 4,₆ = 10.1 Hz, 4,7 = 2.1 Hz, H-6^IM), 4.91 (1 H, d, 4,2 = 7.9 Hz, H-1"), 4.91 (1 H, m, H-9a'"), 4.91 (1 H, m, H-1¹), 4.91 (1 H, m, H-6a'), 5.03 (1H, brd, ,₄ = 3.1 Hz, H-4"), 5.06 (1 H, dd, 4,₂ = 7.9 Hz, 4,₃ = 10.1 Hz, H-2^I!), 5.38 (1H, t, 4,2 = 9.2 Hz, 4_,3 = 9.5 Hz, H-2¹), 5.39 (1 H, m, H-7¹"), 5.66 (1 H, ddd, a,4 = 5.2 Hz, J_3b,₄ = 12.8 Hz, 4_,5 = 10.7 Hz, H-4¹"), 5.73 (1 H, t, 4,₃ = 9.5 Hz, 4,4 = 9.8 Hz, H-3¹), 5.86 (1H, m, H-8¹"), 7.00 (2H, t, J = 7.9 Hz, Ar), 7.06 (2H, t, J = 7.3 Hz, Ar), 7.19 (3, m, Ar), 7.32 (14H, m Ar), 7.45 (8H, m Ar), 7.73, 7.78, 7.79, 7.79, 7.81 , 7.89, 7.91 , 7.98 (16H, 8brd, J = 7.6 Hz, Ar). MS (FAB) Calcd. for C₉₂H₈₃NO₂₉SK: m/z: 1720.4667, found m/z: 1720.2 (M+Na⁺), 1588.4 (M-SPh⁺), 1114.3 (Bz₅Ac₄MeNeu5AcGal⁺). Methyl [(5-Acetamido-4,7,8,9-tetra-0-acetyl-3,5-dideoxy-D-g/ycerø-α-D-ga/acto-2- nonulopyranosyl-1"→2⁹-lactone)]-(2→3)-0-(4,6-di-0-acetyl-β-D-galactopyranosyl)- (1→6)-0-2,3,4-tri-0-benzyl-α-D-glucopyranoside (9)

1. Preparation from 5a.

A mixture of compound 5a (22 mg; 0.03 mmol) and methyl 2,3,4-tri-O-benzyl-α-D- glucopyranoside 8 (14 mg; 0.03 mmol) and 4A molecular sieves in anhydrous dichloromethane (0.7 mL) was stirred under argon for 1 h. The reaction mixture was cooled to 0-5°C and treated with N-iodosuccinimide (17 mg; 0.08 mmol) and triflic acid (2.2 μL, 0.02 mmol). The reaction mixture was stirred at room temperature for 4 h, cooled to 0°C, quenched with triethylamine, filtered and concentrated. The residue was column chromatographed with hexane/ethyl acetate/ethanol (3:3:0.5) as eluant. The title compound 9 was obtained as a foam (28 mg; 90% yield).

2. Preparation from 5b

A mixture of compound 5b (15 mg; 0.02 mmol) and 8 (14 mg; 0.03 mmol) and 4A molecular sieves in anhydrous dichloromethane (0.5 mL) was stirred under argon for 1 h. The reaction mixture was cooled to 0-5°C and treated N-iodosuccinimide (17 mg; 0.08 mmol) and triflic acid (1.7 μL; 0.02 mmol). The reaction mixture was stirred at room temperature for 4 h, cooled to 0°C and quenched with triethylamine, filtered and concentrated. The residue was column chromatographed with hexane/ethyl acetate/ethanol (3:3:0.5) as eluant. The title compound 9 was obtained as a foam (19 mg; 86% yield). [α]_D ²² -5.6° (c, 0.54, CH₂CI₂); ¹³C NMR (CDCI₃): δ 20.4, 20.5, 20.6, 20.61, 20.7, 20.8, 23.1 (7COCH₃), 37.8 (C-3¹¹¹), 49.4 (C-5¹¹¹), 55.5 (OCH3), 61.1 (C-6¹), 62.0 (C-9"¹), 66.1 (C-4^M), 67.1 (C-7^MI), 68.9 (C-6^M), 69.4 (C-4¹¹¹, C-8¹"), 70.0 (C-5¹), 70.8 (C-5¹¹), 72.8 (C-6¹¹¹), 73.1 (Bn), 73.3 (H-2ⁿ), 74.0 (C-3"), 74.9 (Bn), 75.7 (Bn), 77.4 (C-4¹), 79.3 (C-2¹), 81.9 (C-3¹), 97.0 (C-2¹¹¹), 97.9 (C-1¹), 100.6 (C-1¹¹¹), 127.5-138.8 (Ar), 163.5 (C-1¹¹), 169.5, 169.6, 169.8, 170.4, 170.43, 170.6, 170.9 (7COCH₃); ¹H NMR (CDCI3): δ 1.90, 2.02, 2.03, 2.04, 2.09, 2.19 (21 H, 6s, 7COCH₃), 1.89 (1H, t, 4a,3e₊3a,4 = 24.9 Hz, H-3a'"), 2.44 (1 H, dd, 4_e,₃a = 13.5 Hz, 4e,4 = 5.6 Hz, H-3e'"), 3.42 (3H, s, OCH₃), 3.53 (1 H, dd, 4_,2 = 3.4 Hz, 4,₃ = 9.3 Hz, H-2¹), 3.65-3.69 (2H, m, H-6¹", H-4¹), 3.73 (1 H, dd, 4,6 = 4.9 Hz, 4,6 = 10.8 Hz, H-6a"), 3.81 (1 H,dd, 4,5 = 10.3 Hz, 4,₆ = 4.4 Hz, H-5¹), 3.87 (1 H, t, 4,5+5,6 = 13.7 Hz, H-5^M), 3.92 (1 H, dd, 4,3 = 10.8 Hz, 4,₄ = 2.9 Hz, H-3^M), 4.01 (1 H, t, 4,3+3.4 = 18.6, Hz, H-3¹), 4.08-4.18 (5H, m, H-6'a, H-6'b, H-5¹". H-6¹¹, H-9¹"), 4.24 (1H, dd, 4,9 = 2.9 Hz, 4.9 = 12.2 Hz, H-9b'"), 4.63 (1H, d, 4,2 = 3.4 Hz, H-1¹), 4.69 (2H, d, 2CHHC₆H₅), 4.77 (1H, d, CHHC₆H₅), 4.77 (1 H, t, 4,2+2,₃ = 20.3 Hz, H-2"), 4.83 (1H, d, CHHC₆H₅), ), 4.93 (1H, d, CHHC₆H₅), 5.0 (1H, d, CHHC₆H₅), 5.09 (1H, m, H-8¹"), 5.32 (1H, dd, 4,₈ = 7.8 Hz, H-7¹"), 5.40 (1 H, ddd, H-4^IM), 5.44 (1 H, d, J= 2.5 Hz, H-4ⁿ), 5.54 ( 1 H, d, J =10.3, NHAc), 7.20-7.30 (15H, m, Ar); MS (MALDI-TOF) Calcd. for C₅₇H₆₉NO₂₄Na: 1174.4105, found m/z: 1174.8 (M+Na⁺); 1190.8 (M+K⁺).

MPEG-DOXyl [Methyl (5-acetamido-4,7,8,9-tetra-0-acetyl-3,5-dideoxy-D-g/ycero-α- D-ga/acto-2-nonulopyranosyl)onate)]-(2→3)-0-(2,4,6-tri-0-acetyl-β-D- galactopyranosyl)-(1→4)-6-0-fe/*-butyldiphenylsilyI-2-deoxy-2-phthalimido-β-D- glucopyranoside (11)

A mixture of compound 7 (20 mg; 0.02 mmol) and MPEG-DOXyl 6-O-tert- butyldiphenylsilyl-2-deoxy-2-phthalimido-β-D-glucopyranoside 10 (100 mg; 0.02 mmol) was dried over drierite/CaCI₂ overnight under vacuum. The reaction flask was opened under argon and freshly activated 4A molecular sieves were added. Anhydrous dichloromethane (0.8 mL) was added and the reaction mixture was stirred for 1 h. It was cooled to 0°C and treated with N-iodosuccinimide (18 mg; 0.08 mmol), followed by triflic acid (2.0 μl; 0.2 mmol). The reaction mixture was warmed to room temperature, stirred for 1 h. At this point TLC indicated complete exhaustion of the glycosyl donor 7. The reaction mixture was cooled to 0°C and diisopropylethylamine (2 drops) was added, followed in 10 min by excess ferf-butyl methyl ether (75 mL). The reaction was vigorously stirred for 15 min in order to precipitate the polymer. The polymer was filtered and recrystallized from absolute ethanol (75 mL). The white precipitate was collected by filtration and rinsed with diethyl ether (2x10 mL) and dried in vacuo to afford the polymer bound trisaccharide 11 (70 mg; 70 % yield). The product was characterized as the cleaved compound 12. 4-O-Acetyl-DOXyl [Methyl (5-acetimido-4,7_s8,9-tetra-0-acetyI-3,5-dideoxy-D- g/ycero-α-D-ga/acto-2-nonulopyranosyl)onate)]-(2→3)-0-(2,4,6-tri-0-acetyl-β-D- galactopyranosyl)-(1→4)-3-0-acetyl-6-0-fert-butyldiphenylsilyl-2-deoxy-2- phthalimido-β-D-glucopyranoside (12)

The polymer bound trisaccharide 11 (70 mg; 0.014 mmol) was dissolved in dichloromethane (0.7 mL). Acetic anhydride (0.7 mL) was added, followed by scandium (III) trifluoromethane sulfonate (7 mg; 0.014 mmol). The reaction mixture was stirred under argon for 3.5 h, cooled to 0 °C, and treated with an excess of terf-butyl methyl ether (75 mL). The mixture was stirred for 15 min in order to precipitate the polymer.

The precipitate was filtered and the filtrate was concentrated. The residue was purified by column chromatography with hexane/ ethyl acetate/ethanol (3:3:0.5) as eluant. The title compound 12 was obtained (11 mg; 52 % yield). [α]_D ²² 6.1° (c, 0.31 , CHCI₃); ¹³C

NMR (CDCI₃): δ 20.2- 20.9 (COCH₃), 26.6 (C(CH₃)₃), 37.2 (C-3^m), 48.9 (C-5¹¹¹), 52.9 (OCH3), 55.0 (C-2¹), 61.2 (C-6^π), 62.3 (C-9¹¹¹), 63.1 (C-6¹), 65.7 (CH₂C₆H₅-DOX), 66.9 (C-4¹¹), 67.0 (C-7¹¹¹), 67.6 (C-8¹¹¹), 69.1 (C-4^ln), 69.9 (C-5¹), 70.0 (CH₂C₆H₅-DOX), 70.1 (C- 2"), 71.2 (C-3^M), 71.9 (C-6^MI), 75.4 (C-5"), 76.3 (H-4¹), 96.7 (C-1¹), 100.2 (C-1¹¹), 130.0- 140.0 (Ar); ¹H NMR (CDCI3): δ 1.10 (9H, s, C(CH₃) ₃ ), 1.67 (1 H, t, 4a,3e+3a,4 = 24.9 Hz, H-3a'"), 1.85, 1.96, 1.98, 2.01 , 2.04, 2.06, 2.08, 2.09, 2.12 (30H, 9s, IOCOCH3), 2.56 (1H, dd, 4e,₃a = 12.7 Hz, 4e, = 4.4 Hz, H-3e'"), 3.60 (1 H, dd, H-6¹"), 3.82 (OCH₃, H-5¹¹), 3.83 (H-5¹), 3.89 (H-9a'"), 3.90 (H-6'a), 3.93 (H-6a"), 3.96 (H-4¹), 3.97 (H-6b"), 4.02 (H- 5¹"), 4.13 (H-6'b), 4.22 (1 H, dd, 4,₂+₂,₃ = 19.1 Hz, H-2¹), 4.33 (H-9b'"), 4.40 (CHHC₆H₅), 4.52 (1H, dd, 4,₃ = 9.8 Hz, 4,4 = 2.9 Hz, H-3"), 4.74 (CHHC₆H₅), 4.85 (H-4^H, H-4¹"), 4.86 (H-1¹¹), 4.92 (H-2"), 5.0 (2H, s, CH₂C₆H₅), 5.06 (1 H, d, J = 10.8 Hz, NH), 5.33 (1 H, m, H-7^MI), 5.41 (1H, d, 4,₂ = 8.8 Hz, H-1¹), 5.51 (1H, m, H-8^m), 5.77 (1 H, t, 4,3+3,4 = 19.1 Hz, H-3¹), 7.07 (4H, dd, Ar-DOX), 7.38-7.80 (Ar). MS (MALDI-TOF) Calcd. for C₇₄H₈₈N₂O₃oSiNa: 1535.5088, found m/z: 1535.7 (M+Na⁺); 1551.7 (M+K⁺).

Although various particular embodiments of the present invention have been described hereinbefore for purposes of illustration, it would be apparent to those skilled in the art that numerous variations may be made thereto without departing from the spirit and scope of the invention, as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for preparing a thioglycoside comprising a sialylated residue of β-D- galactose, said method comprising the steps of: - providing a thioglycoside comprising a non-sialylated residue of β-D- galactose; and - enzymatically sialylating the residue of β-D-galactose with a sialic acid in the presence of a sialyltransferase.

2. A method according to claim 1 , further comprising the step of chemically derivatizing the sialylated thioglycoside, to provide a derivative suitable for use as a donor in a chemical glycosylation.

3. A method according to claim 1 , wherein the thioglycoside comprising a non- sialylated residue of β-D-galactose is prepared by a chemical synthesis.

4. A method according to claim 1 , wherein the thioglycoside is an alkyl or aryl thioglycoside.

5. A method according to claim 4, wherein the thioglycoside is an aryl thioglycoside.

6. A method according to claim 5, wherein the aryl thioglycoside comprising a non- sialylated residue of β-D-galactose is a β-aryl thioglycoside of galactose or lactose.

7. A method according to claim 6, wherein the aryl is an optionally substituted phenyl or naphthyl.

8. A method according to claim 1 , wherein the sialic acid is 5-N-acetylneuraminic acid.

9. A method according to claim 1 , wherein the sialyltransferase is an α(2,3)sialyltransferase.

10. A method according to claim 9, wherein the α(2,3)sialyltransferase is cloned from Campylobacter jejuni.

11. A method according to claim 5, wherein the aryl thioglycoside comprising a sialylated β-D-galactose residue is an aryl (5-acetamido-3,5-dideoxy-D-g/ycero-α- D-ga/acto-2-nonulopyranosyl)-(2→3)-1-thio-β-D-galactopyrano- side or a derivative thereof.

12. A method according to claim 11 , wherein the aryl is an optionally substituted phenyl or naphthyl.

13. A method according to claim 12, wherein the derivative is phenyl [(5-acetamido- 4,7,8,9-tetra-0-acetyl-3,5-dideoxy-D-g/ycero-α-D-ga/acto-2-nonulopyranosyl-

1'-→2-lactone)]-(2→3)-0-4,6-di-0-acetyl-1-thio-β-D-galactopyranoside.

14. A method according to claim 12, wherein the derivative is 2-napthyl [(5- acetam ido-4 ,7,8, 9-tetra- O-acetyl-3 , 5-d ideoxy-D-g/ycero-α-D-ga/acfo-2- nonulopyranosyl-1 '→2-lactone)]-(2→3)-0-4,6-di-O-acetyl-1 -thio-β-D- galactopyranoside.

15. A method according to claim 12, wherein the derivative is phenyl [methyl (5- acetamido-4,7,8,9-tetra-0-acetyl-3,5-dideoxy-D-g/ycero-α-D-ga/acto-2- nonulopyranosyl)onate)]-(2→3)-O-2,4,6-tri-0-acetyl-1-thio-β-D-galacto- pyranoside

16. A method according to claim 5, wherein the aryl thioglycoside comprising a sialylated β-D-galactose residue is an aryl (5-acetamido-3,5-dideoxy-D-g/ycero-α- D-ga/acfo-2-nonulopyranosyl)-(2→3)-β-D-galactopyranosyi-(1→4)-1-thio-β-D- glucopyranoside or a derivative thereof.

17. A method according to claim 16, wherein the aryl is an optionally substituted phenyl or naphthyl.

18. A method according to claim 17, wherein the derivative is phenyl [(5-acetamido- 4,8,9-tri-O-benzoyl-l-3,5-dideoxy-D-g/ycero-α-D-ga/acto-2-nonulopyranosyl-1^,→2- lactone)]-(2→3)-0-4,6-di-O-benzoyl-β-D-galacto- pyranosyl-(1→4)-2,3,6-tri-0- benzoyl-1-thio-β-D-glucopyranoside.

19. A method according to claim 17, wherein the derivative is phenyl [methyl (5- d iacetam id o-7- O-acety I-4 , 8 , 9-tri-O-benzoy I-3 , 5-d ideoxy-D-g/ycero-α-D-ga/acto-2- nonulopyranosyl)onate)]-(2→3)-0-2-0-acetyl-4,6-di-0-benzoyl-β-D- galactopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-1-thio-β-D-glucopyrano- side.

20. A thioglycoside or a derivative thereof, said thioglycoside comprising a sialylated residue of β-D-galactose.

21. A thioglycoside according to claim 20, wherein the glycoside is an alkyl or aryl thioglycoside.

22. A thioglycoside according to claim 21 , wherein the glycoside is an aryl glycoside.

23. A thioglycoside according to claim 22, wherein the glycoside is an aryl (5- acetamido-3,5-dideoxy-D-g/ycero-α-D-ga/acfo-2-nonulopyranosyl)-(2→3)-1-thio- β-D-galactopyranoside or a derivative thereof.

24. A thioglycoside according to claim 23, wherein the aryl is an optionally substituted phenyl or naphthyl.

25. A thioglycoside according to claim 24, wherein the derivative is phenyl [(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-g/ycero-α-D-ga/acto-

2-nonulopyranosyl-1'→2-lactone)]-(2→3)-O-4,6-di-0-acetyl-1-thio-β-D- galactopyranoside.

26. A thioglycoside according to claim 24, wherein the derivative is 2-napthyl [(5- acetamido-4,7,8,9-tetra-0-acetyl-3,5-dideoxy-D-g/ycero-α-D-ga/acto-2- nonulopyranosyl-1'-→2-lactone)]-(2→3)-O-4,6-di-0-acetyl-1-thio-β-D- galactopyranoside.

27. A thioglycoside according to claim 24, wherein the derivative is phenyl [methyl (5-acetam ido-4 ,7,8, 9-tetra-O-acety I-3 , 5-d ideoxy-D-g/ycero-α-D-ga/acfo-2- nonuiopyranosyl)onate)]-(2→3)-O-2,4,6-tri-0-acetyl-1-thio-β-D- galactopyranoside.

28. A thioglycoside according to claim 22, wherein the aryl thioglycoside comprising a sialylated β-D-galactose residue is an aryl (5-acetamido-3,5-dideoxy-D-g/ycetr>- α-D-ga/acto-2-nonulopyranosyl)-(2→3)-β-D-galactopy- ranosyl-(1 →4)-1 -thio-β-D- glucopyranoside or a derivative thereof.

29. A thioglycoside according to claim 28, wherein the aryl is an optionally substituted phenyl or naphthyl.

30. A thioglycoside according to claim 29, wherein the derivative is phenyl [(5-acetamido-4,8,9-tri-0-benzoyl-l-3,5-dideoxy-D-g/ycero-α-D-ga/acfo-2- nonulopyranosyl-1'→2-lactone)]-(2→3)-O-4,6-di-O-benzoyl-β-D-galacto- pyranosyl-(1→4)-2,3,6-tri-O-benzoyl-1-thio-β-D-glucopyranoside.

31. A thioglycoside according to claim 29, wherein the derivative is phenyl [methyl (5-diacetamido-7-0-acetyl-4,8,9-tri-0-benzoyl-3,5-dideoxy-D-g/yce/O-α-D-ga/acfo- 2-nonulopyranosyl)onate)]-(2→3)-0-2-O-acetyl-4,6-di-0-benzoyl-β-D- galactopyranosyl-(1→4)-2,3,6-tri-0-benzoyl-1-thio-β-D-glucopyranoside.

26. A thioglycoside according to claim 24, wherein the derivative is 2-napthyl [(5- acetamido-4,7,8,9-tetra-0-acetyl-3,5-dideoxy-D-g/ycero-α-D-ga/acfo-2- nonulopyranosyl-1'→2-lactone)]-(2→3)-O-4,6-di-0-acetyl-1-thio-β-D- galactopyranoside.

27. A thioglycoside according to claim 24, wherein the derivative is phenyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-g/ycero-α-D-ga/acto-2- nonulopyranosyl)onate)]-(2→3)-O-2,4,6-tri-0-acetyl-1-thio-β-D- galactopyranoside.

28. A thioglycoside according to claim 22, wherein the aryl thioglycoside comprising a sialylated β-D-galactose residue is an aryl (5-acetamido-3,5-dideoxy-D-g/ycero- α-D-ga/acto-2-nonulopyranosyl)-(2→3)-β-D-galactopy- ranosyl-(1 →4)-1 -thio-β-D- glucopyranoside or a derivative thereof.

30. A thioglycoside according to claim 29, wherein the derivative is phenyl [(5-acetamido-4,8,9-tri-O-benzoyl-l-3,5-dideoxy-D-g/ycero-α-D-ga/acfo-2- nonulopyranosyl-1'→2-lactone)]-(2→3)-O-4,6-di-0-benzoyl-β-D-galacto- pyranosyl-(1→4)-2,3,6-tri-0-benzoyl-1-thio-β-D-glucopyranoside.

31. A thioglycoside according to claim 29, wherein the derivative is phenyl [methyl (5-diacetamido-7-0-acetyl-4,8,9-tri-0-benzoyl-3,5-dideoxy-D-g/ycero-α-D-ga/acfo- 2-nonulopyranosyl)onate)]-(2→3)-O-2-0-acetyl-4,6-di-0-benzoyl-β-D- galactopyranosyl-(1-→4)-2,3,6-tri-O-benzoyl-1-thio-β-D-glucopyranoside.

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