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Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
oligosaccharide synthesis

Synthetic chemistry is the base for all the other biochemical, and immunological studies described in the next sections, such as the study of specificity of lectin, development of influenza virus adhesion inhibitors, etc. Various oligosaccharides and glycoconjugates (with espetial emphazise onto sialosides) serving as versatile tools for the above mentioned studies have been synthesized. Two examples are shown below.

α2-6 Sialooligosaccharides. General problem of sialylation, in particular, of primary alcohols sialylation is achievement of high α-specificity, as all the natural sialosides have α-configuration. The particularity of glycosylation by sialic acid derivatives, in contrast to derivatives of common hexapyranoses, is the lack in their structure of the so-called co-participating groups, which determine glycosylation stereochemistry and provide the necessary configuration of the formed glycosidic bond. We have developed two approaches to the synthesis of 2-6 sialooligosaccharides. The first approach is based on co-participation of acetoxyacetyl groups introduced as protective groups to positions 4 and 9. The second method is the return to the synthesis under conditions of Koenigs-Knorr reaction, described for the first time in 1971, i.e. the use of Ag2CO3 as catalyst-promoter.

Sialylation of mono- and disaccharide diols under the Koenings-Knorr conditions
Sialylation of mono- and disaccharide diols under the Koenings-Knorr conditions
Sialylation of mono- and disaccharide diols under the Koenings-Knorr conditions

Scheme 1. Sialylation of mono- and disaccharide diols under the Koenings-Knorr conditions,
sp = (CH2)3NHCOCF3.

The results are shown in Table 1. It can be seen that sialylation proceeds stereoselectively and with high yields. In addition, it is necessary to mention the simplicity of such methodology.

Table 1. Sialylation of saccharides under the Koenigs-Knorr conditions

acceptor

product solvent time [days] α-anomer [%] α/β
5 9 H2Cl2 3 91 α
5 9 ClCH2CH2Cl 7 76 3/1
6 10 H2Cl2 7 76 6/1
6 10 ClCH2CH2Cl 7 52 3/1
7 11 ClCH2CH2Cl 9 88 10/1
8 12 ClCH2CH2Cl 7 48 α

 

a2-3 Sialooligosaccharides. 3-Aminopropyl glycosides 5-13 were synthesized using the protected Neu5Acα2-3Gal bromide as the glycosyl donor at the key stage.

 

Neu5Acα2-3Galβ-O(CH2)3NH2
5
Neu5Acα2-3Galβ1-4GlcNAcβ-O(CH2)3NH2
(3'SLN) 6
Neu5Acα2-3Galβ1-4(6-HSO3)GlcNAcβ-O(CH2)3NH2
(6-Su-3'SLN) 7
Neu5Acα2-3Galβ1-3GalNAcα-O(CH2)3NH2
(SiaTF) 8
Neu5Acα2-3Galβ1-3(6-HSO3)GalNAcα-O(CH2)3NH2
(6-Su-SiaTF) 9
Neu5Acα2-3Galβ1-3GlcNAcβ-O(CH2)3NH2
(SiaLec) 10
Neu5Acα2-3Galβ1-3(6-HSO3)GlcNAcβ-O(CH2)3NH2
(6-Su-Lec) 11
          Fucα1-4
GlcNAcβ-O(CH2)3NH2
Neu5Acα2-3Galβ1-3
(SiaLea) 12
Neu5Acα2-3Galβ1-4
GlcNAcβ-O(CH2)3NH2
Fucα1-3
(SiaLex) 13
We suggest a block synthesis strategy based on the use of acetylated bromide (14) as a glycosyl donor. Once obtained, this building block further permits to synthesise in divergent manner a wide set of complex oligosaccharides 513 containing Neu5Acα2-3Gal motif, including fuco- and sulfo-derivatives, which are indispensable compounds from the glycobiological viewpoint.
Glycosylation of glycosyl acceptors 17, 20, 23, and 29 with acetylated bromide 14 (see Scheme 2), which was obtained by treating the corresponding peracetate with HBr in the quantitative yield, was the key stage in the synthesis of spacered oligosaccharides 513 (Scheme 2).


Glycosylation was performed in methylene chloride in the presence of silver triflate, tetramethylurea, and molecular sieve 4 at room temperature for 3-15 h. Donor 14 was taken in a 1.5-fold excess, except for glycosylation of acceptor 15, when a two-fold excess of the glycosyl donor was used. The yields of Neu5Acα2-3Galβ-glycosylation products were 5685%; by-products (<10%, presumably the corresponding Neu5Acα2-3Galα-isomers) were easily removed by chromatography. The AgOTf-promoted glycosylation of diol 23 proceeded non-regiospecifically (56% of the 1-3 isomer, 11% of the 1-4 isomer, and 9% of the bis-glycosylation product), so the preparative synthesis was performed in the presence of Hg(CN)2 as the promoter. Under these conditions, the target 1-3 bound trisaccharide 24 was obtained in 75% yield. The presence of the free hydroxyl group at C4 in trisaccharide 24 permitted to obtain protected tetrasaccharide 27 by fucosylation. Deblocking of 27 gave rise to free spacered tetrasaccharide SiaLea 12 identical to that obtained earlier by another method. To synthesize isomeric tetrasaccharide SiaLex 13, the chloroacetyl derivative 29 was converted to trisaccharide 30, which was subjected to mild dechloroacetylation followed by fucosylation of the resulting acceptor 31.

 

This synthetic strategy enabled to synthesize oligosaccharides containing both sialic acid in position-3 of galactose and sulfate in position-6 of hexosamine, e.g. trisaccharides 7, 9, and 11, that are of great biological interest being the fragments of mucin type glycoproteins. These mucins take part, in particular, in the interaction with L-selectin and are receptors for pathogenic bacteria and viruses. Selective deblocking of the hydroxyl group at C-6 of hexosamine in 18, 21 and 25 followed by sulfation of the corresponding trisaccharides 19, 22, and 26 by the SO3Py complex. Chemical synthesis of the resulting deblocked trisaccharides 7, 9, and 11 has not been reported earlier.
Deprotection of synthesized oligosaccharides derivatives was performed by conventional methods (see, for example, ref. 6), namely sequential hydrogenolysis and treating with sodium methylate and alkali. Some amount of dimeric products, in which the carboxy group of sialic acid of one oligosaccharide molecule acylated the spacer group of the second molecule, was formed in several cases at the final stage of saponification. These dimers were quantitatively converted to the target compounds by treating with 1 N alkali for 24 h at r.t. De-N-acetylation was not observed under these conditions.
To summarize, Neu5Acα2-3Gal donor 14 is effective in 2-3 sialooligosaccharide synthesis. The advantages of this glycosyl donor are the simplicity of preparation from the corresponding peracetate, compatibility with various protecting groups, and convenient conditions of glycosylation the reactions proceed at room temperature for 3-15 h.

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