Chemical and chemo-enzymatic synthesis of the [formula omitted] element that is part of N-linked carbohydrate chains of human lutropin

Abstract

In the framework of a project aimed at the elucidation of the nature of the functional importance of the N-glycosylation of the α-subunit of the glycoprotein hormones human lutropin and human chorionic gonadotropin, the structural element α- Neu p 5Ac-(2 → 6)-β- d-Gal p NAc-(1 → 4)-β- d-Glc p NAc-(1 → 2)-α- d-Man p , which is part of the carbohydrate chains of human lutropin, has been prepared by chemical and chemo-enzymatic synthesis in the form of its propyl glycoside. Condensation of 4-O- acetyl-3,6-di-O- benzyl-2-deoxy-2-phthalimido-α/β- d-glucopyranosyl trichloroacetimidate with allyl 3,4,6-tri-O- benzyl-α- d-mannopyranoside gave after deacetylation allyl ( 3,6-di-O- benzyl-2-deoxy-2-phthalimido-β- d-glucopyranosyl)-(1 → 2)-3,4,6-tri -O- benzyl-α- d-mannopyranoside . Ethyl 3-O- benzyl-2-deoxy-2-phthalimido-1-thio-β- d-glucopyranoside was converted into the galacto-derivative ethyl 4,6-di-O- acetyl-3-O- benzyl-2-deoxy-2-phthalimido-1-thio-β- d-galactopyranoside via an oxidation-reduction route, as well as via S N2-type substitution with acetate. The use of this galacto thioglycoside, after its conversion into the corresponding bromide, as GalN donor for condensation with the mentioned disaccharide derivative yielded after deacetylation allyl (3-O- benzyl-2-deoxy-2-phthalimido-β- d-galactopyranosyl)-(1 → 4)-(3,6-di -O- benzyl-2-deoxy-2-phthalimido-β- d-glucopyranosyl)-(1 → 2)-3,4,6-tri -O- benzyl-α- d-mannopyranoside . Methylsulfenyl bromide-silver triflate promoted sialylation of this trisaccharide derivative with O- ethyl S- [methyl(5-acetamido-4,7,8,9-tetra-O- acetyl-3,5-dideoxy- d -glycero-α- d -galacto- non-2-ulopyranosyl)onate] dithiocarbonate and subsequent deprotection resulted into the aimed tetrasaccharide structural element. Alternatively, this compound was prepared via a block synthesis, which, however, was not superior to the linear strategy. Finally, a stereoselective sialylation of synthetically prepared β- d-Gal p NAc-(1 → 4)-β- d-Glc p NAc-(1 → 2)-α- d-Man p- (1 → O)CH 2CH 2CH 3 with CMP-Neu5Ac and rat liver α-2,6-sialyltransferase was accomplished affording the same tetrasaccharide structural element.