Koenigs-Knorr Glycosylation with Neuraminic Acid Derivatives

Earlier we reported a convenient and efficient method of preparing α2-6 sialooligosaccharides in conditions of Koenigs-Knorr reaction. The use of Ag2CO3 allowed carrying out α2-6 sialylation of galacto-4,6-diol of monoand disaccharides with chloride of acetylated N-acetylneuraminic acid methyl ester as glycosyl donor. In this study we applied this approach to other derivatives of neuraminic acid, namely, Neu5Gc, 9-deoxy-9-NAc-Neu5Ac, Neu5Acα2-8Neu5Ac, and Neu5Acα2-8Neu5Acα2-8Neu5Ac as glycosyl donors; eight compounds were synthesized: Neu5Gcα-O(CH2)3NH2 (8), Neu5Gcα2-6Galβ1-4GlcNAcβ-O(CH2)3NH2 (10), 9-deoxy-9-NAc-Neu5Ac-O(CH2)3NH2 (15), 9-deoxy-9-NAc-Neu5Acα2-6Galβ1-4GlcNAcβ-O(CH2)3NH2 (17), Neu5Acα28Neu5Acα-O(CH2)3NH2(23) Neu5Acα2-8Neu5Acα-OCH3 (24), Neu5Acα2-8Neu5Acα-OCH2(p-C6H4)NHCOCH2NH2 (25), and Neu5Acα2-8Neu5Acα2-8Neu5Acα-O(CH2)3NH2 (32). These sialosides were used for characterization of siglecs and other carbohydrate-binding proteins.


Introduction
Sialic acid family comprises over 40 neuraminic acid versions and monosaccharide derivatives; the number of biologically relevant sialic acid oligosaccharides is also very high. Sialic acids are a part of the most important molecules of life, since they occupy the terminal position on cell membrane glycoproteins and glycolipids. Given their location and ubiquitous distribution, sialic acids can mediate or modulate a wide range of physiological and pathological processes and play a role not only in the protection and adaptation of life, but also in being utilized by life-threatening infectious microorganisms [1]. Several families of sialic acidbinding proteins have been discovered over the last few decades, including mammalian selectins and siglecs [2]. Only a limited number of sialomolecules are available as the tools for the study of mammalian and microbial lectins and sialo-modifying enzymes. Due to this, the search of simple and efficient synthetic ways for sialosaccharides is very relevant. The methods of sialylation described in the literature [3][4][5] rarely give high yields and α-stereoselectivity but suggest complex modifications of donors; introduction and removal of substituents at C-3, C-4, C-5, and C-9 in order to improve sialylation stereoselectivity presume complex multistage synthesis of such compounds. In case of the use of 2-xantates, thiophosphates and various imidates, sialylation usually proceeds at −50-70 • C and requires strong Lewis acids as promoters, this posing the corresponding limitations to the repertoire of protective groups in glycosyl acceptor. Recently we have described a simple and efficient method of the synthesis of α-2-6 sialooligosaccharides in conditions of Koenigs-Knorr reaction [6] when the simplest possible sialyl donor is used at room temperature ang gives high α-stereoselectivity. It was interesting to study the applicability of this approach to other neuraminic acid derivatives (Neu5Gc, 9-deoxy-9-NAc-Neu5Ac, Neu5Acα2-8Neu5Ac, and Neu5Acα2-8Neu5Acα2-8Neu5Ac). Here, we describe practical chemical syntheses of several spacerarmed sialic acid glycosides, with especial interest to Nglycolyl-neuraminic acid version playing role in cancer immunology and transplantation, and also α2-8 oligosaccharides known as key motif of polysialic acids, numerous gangliosides, and serving as affinity ligand for siglec-7 [7].
Glycosylation was performed in dry CH 2 Cl 2 in the presence of Ag 2 CO 3 or Ag 2 CO 3 /AgOTf (silver trifluoromethanesulfonate) and molecular sieves 4Å (MS 4Å) at room temperature for 1-7 days; the reaction conditions and yields are given in the Table 1. In this study we have used equivalent amounts of glycosyl donor (Sug-Cl) and acceptor or excess of acceptor. The corresponding glycals were found to be the main side products in all glycosylation reactions. Deprotection resulted in the mixture of the target product with glycal and unreacted acceptor, easily separated using cation-exchange chromatography on Dowex H + resin [11]: elution with water gave glycal (acid), whereas the product (amino acid) had been retained on the column and was completely eluted with 1 M aqueous pyridine; unreacted glycosyl acceptor (amine) was eluted with 1 M NH 4 OH. In the case of disialic and trisialic glycosides partial lactonization occurred during purification on Dowex H + , so the lactone was treated with aqueous NaOH to give corresponding acid.
All Sug-Cl (see Table 1) have demonstrated considerably less activity in glycosylation compared to chloride of acetylated N-acetylneuraminic acid methyl ester (3) [6,8]; in several cases Ag 2 CO 3 promoted glycosylation (without AgOTf) either proceeded very slowly or did not proceed at all. The optimal of AgOTf added to Ag 2 CO 3 ratio (in respect to the reaction duration and acceptable α/β anomer ratio) was found to be 10% mol using chloride of Neu5Ac (3) as glycosyl donor and HO(CH 2 ) 3 NHCOCF 3 as acceptor; larger amount gave more β-isomer and glycal; preferable acceptor/donor ratio was not less than 2. Glycosylation at −10 • C did not improve the α/β ratio compared to room temperature.
In case of p-amidoglycyl-benzyl alcohol glycosylation with Neu5Acα2-8Neu5Ac the use of 0.1 mol of AgOTf per glycosyl chloride gave only 10% of 25; 0.6 ratio led to increase of conversion (yield of α + β increased from 20% to 32%), but it was accompanied with the increase of β-anomer formation (Scheme 3, no. 10 and no. 11 in Table 1).
Trisialic chlorides 31 reacted with HO(CH 2 ) 3 NHCOCF 3 only in the presence of AgOTf; increase of mol/mol ratios from 0.1 to 0.5 on chloride gave 32 (∼25% yield) accompanied by the increase of conversion degree and reduction of α/β ratio (Scheme 4, no. 12 and no. 13 in Table 1).

Preparation of Neuraminic Acid Glycosyl Chlorides
Derivatives (Sug-Cl) General procedure for 7, 14, 21 + 22, and 31. Peracetylated methyl ester of neuraminic acid derivative (1 mmol of 6, 13, 19 + 20 or 27-30) was dissolved in 10 ml freshly distilled chloroform followed by addition of dry methanol (0.81 ml, 20 mmol). The solution was cooled (ice + salt) and AcCl (2.84 ml, 40 mmol) was added dropwise at cooling and stirring. After 30 min the reaction mixture was thoroughly sealed then warmed slowly and kept at room temperature for 1.5-2 days. The reaction mixture was evaporated at reduced pressure (water jet pump) and coevaporated with dry toluene several times to neutral pH value of the solution. Chlorides obtained in this way were used without further purification assuming quantitative yield of the reaction (the substances were stored at −18 • C). procedure for 4, 8, 10, 15, 17,  23, 24, 25, and 32 (see Table 1  filtrate was evaporated, coevaporated with toluene, and dried in vacuo. Table 1 for yields).

Deprotection and Separation. (see
(a) Procedure for 4, 8, 15, and 17: the dry residue of treated reaction mixture (see above) was dissolved in 6 ml of dry MeOH and 0.3 ml 2 M MeONa/MeOH were added. The mixture was kept for 30 min at r.t., then evaporated followed by addition of 6 ml H 2 O. After 10-15 h (r.t.) the solution was evaporated, the residue was dissolved in ∼ 1 ml water and the solution was applied on Dowex 50x4-400 (H + ) ion-exchange resin column (1.5 × 6 cm). The resin was washed sequentially with water (50 ml), 1 M aq. pyridine (50 ml) and 1 M aq. NH 3 (50 ml). Target glycosides were completely eluted with 1 M pyridine. In the case of 10 and 17, nonreacted acceptor 16 was eluted with 1 M aq. NH 3 in deprotected form (11). Pure α-glycoside 8 and corresponding β anomer were obtained by HPLC separation (ODS C 18 , water).
(d) Procedure for 25 was the same as (c), with additional treatment with CF 3 COOH to remove Boc protection before the first Dowex chromatography. The mixture was dissolved in 0.3 ml CF 3 COOH, kept for 1 h at r.t., then coevaporated with toluene and dried in vacuo; the followed procedures were performed according to procedure (c).