ABSTRACT
Experimental Methods ........................................................................................... 253 General Methods ............................................................................................... 253
Methyl {4-[(Tert-Butyloxycarbonyl)glycylamido]benzyl 5-Acetamido4,7,8,9-Tetra-O-Acetyl-3,5-Dideoxy-d-Glycero-d-Galacto-Non-2Ulopyranosid}onate (3) ................................................................................ 253 4-[(Tert-Butyloxycarbonyl)glycylamido]benzyl 5-Acetamido-3,5Dideoxy-d-Glycero-d-Galacto-Non-2-Ulopyranosidonic Acid (5) .............256 4-(Glycylamido)benzyl 5-Acetamido-3,5-Dideoxy-d-Glycero-dGalacto-Non-2-Ulopyranosidonic Acid (6) .................................................256 4-[(4-Nitrophenoxy)adipoylglycylamido]benzyl 5-Acetamido-3,5Dideoxy-d-Glycero-d-Galacto-Non-2-Ulopyranosidonic Acid (8) ............. 257
Acknowledgment ................................................................................................... 257 References .............................................................................................................. 257
Benzyl and substituted benzyl glycosides of α-N-acetylneuraminic acid (Neu5Ac) are important tools in glycobiology because their binding af¥nity toward proteins that recognize sialic acid, including H1 and H3 subtypes of human in—uenza virus hemagglutinin, siglecs, and anti-glycan antibodies, can be ¥nely tuned.1-3
Here, we describe the synthesis of 4-(glycylamido)benzyl glycoside of Neu5Ac 6 having a free amino group, which can be used for direct coupling with a variety of labels, tags, and also with macromolecules or supports (ELISA plates, microchips, af¥nity matrices, beads) that carry an activated carboxylic group. By additional derivatization, glycoside 6 can be converted into a compound with an activated carboxyl group in the aglycon part, namely, into [4-(4-nitrophenoxy)adipoylglycylamido]benzyl glycoside 8, followed by coupling with various entities, including peptides and proteins, which have a free amino group. UV absorbance of 4-(glycylamido)benzyl residue (λmax 247 nm, ε = 17,000) allows performing a quantitative monitoring of reactions of 6 or 8.4 Finally, it should be noted that although α-glycosides 6 and 8 are the objects of primary interest, the correspondent β-glycosides are important as the negative control counterparts in studies of carbohydrate-protein interactions. Thus, the glycosides 6 and 8 were used for syntheses of different carbohydrate multimerics, sialoglycopolymers, sialoglycoclusters, and self-assembling sialoglycopeptides, to study in—uenza viruses binding to host cells.4-6
Earlier, a small-scale preparation of 4-[(tert-butyloxycarbonyl)glycylamido)]benzyl glycoside of peracetylated methyl ester of Neu5Ac 3 was described, which was based on Helferich reaction of the readily accessible glycosyl chloride 1 with the corresponding alcohol 2 in acetonitrile-dichloromethane mixture using HgBr2/Hg(CN)2 as a catalyst. The yield of 3 was ~60% (anomeric α/β mixture,~3/2, as revealed by 1H nuclear magnetic resonance [NMR]).1c We have developed an improved procedure where coupling of chloride 1 with alcohol 2 is performed in dichloromethane in the presence of Ag2CO3/AgOTf.7 Individual anomers of 3 are obtained by chromatography of the reaction mixture on silica gel in ~70% yield, α/β ~2:1. Stepwise deprotection of 3 (α or β), involving sequential Zemplén deacetylation (MeONa/ MeOH), hydrolysis of the methyl ester group (0.05 M NaOH), and N-deprotection (TFA), gives glycoside 6 (α or β) with 80%–85% yield (over three steps). A treatment of glycoside 6 (α or β) with a ¥vefold excess of bis(4-nitrophenyl) adipate 7 in DMF/ DMSO mixture gives two products, the monosialoside 8 (major, α or β, 75%–85%) and the disialoside 9 (minor, α or β, 15%–20%). The products can be puri¥ed by gelpermeation chromatography on Sephadex LH-20. The prepared glycoside 8 is stable for several months when stored at −20°C.
