Chemical and chemo-enzymatic synthesis of the alpha-Neu p5Ac-(2-->6)- beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp element that is part of N-linked carbohydrate chains of human lutropin.

In the framework of a project aimed at the elucidation of the nature of the functional importance of the N-glycosylation of the alpha-subunit of the glycoprotein hormones human lutropin and human chorionic gonadotropin, the structural element alpha-Neu p5Ac-(2-->6)-beta-D-GalpNac-(1-->4)- beta-D-GlcpNAc-(1-->2)-alpha-D-Manp, 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-alpha/beta-D-glucopyranosyl trichloroacetimidate with allyl 3,4,6-tri-O-benzyl-alpha-D-mannopyranoside gave after deacetylation allyl (3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl) -(1-->2)-3,4,6-tri-O-benzyl-alpha-D-mannopyranoside. Ethyl 3-O-benzyl-2-deoxy-2-phthalimido-l-thio-beta-D-glucopyranoside was converted into the galacto-derivative ethyl 4,6-di-O-acetyl-3-O-benzyl-2-deoxy-2-phthalimido-1-thio-beta-D -galactopyranoside via an oxidation-reduction route, as well as via SN2-type substitution with acetate. The use of this galacto thioglycoside, after its conversion into the corresponding bromide, as GaIN donor for condensation with the mentioned disaccharide derivative yielded after deacetylation allyl (3-O-benzyl-2-deoxy-2-phthalimido-beta-D-galactopyranosyl)-(1-->4) -(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1-->2) -3,4,6-tri-O-benzyl-alpha-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-alpha-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 stereose lective sialylation of synthetically prepared beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) CH2CH2CH3 with CMP-Neu5Ac and rat liver alpha-2,6-sialyltransferase was accomplished affording the same tetrasaccharide structural element.

Synthesis of Hexp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) (CH2)7 CH3 probes for exploration of the substrate specificity of glycosyltransferases: Part II, Hex = 3-O-methyl-beta-D-Gal, 3-deoxy-beta-D-Gal, 3-deoxy-3-fluoro-beta-D-Gal, 3-amino-3-deoxy-beta-D-Gal, beta-D-Gul, alpha-L-Alt, or beta-L-Gal.

Seven analogues of the trisaccharide beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH 2)7CH3 have been synthesized as potential substrates for glycosyltransferases involved in the chain-termination of N-acetyllactosamine-type N-glycans. These compounds include: 3-O-methyl-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp -(1-->O) (CH2)7CH3, 3-deoxy-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1 -->O) (CH2)7CH3, 3-deoxy-3-fluoro-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-M anp- (1-->O)(CH2)7Ch3, 3-amino-3-deoxy-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Ma np- (1-->O)(CH2)7CH3, beta-D-Gulp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-- >O)(CH2)7CH3, beta-L-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH 2)7CH3, and alpha-L-Altp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1- ->O) (CH2)7CH3. All trisaccharides were obtained by condensation of suitably modified glycosyl donors based on imidates or thioglycosides with the same disaccharide acceptor, octyl 3,4,6-tri-O-benzyl-2-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranosyl)-alpha-D-mannopyranoside, followed by deprotection.

Exploring the substrate specificities of alpha-2,6- and alpha-2,3-sialyltransferases using synthetic acceptor analogues.

The acceptor specificities of rat liver Gal(beta 1-4)GlcNAc alpha-2,6-sialyltransferase, recombinant full-length human liver Gal(beta 1-4)GlcNAc alpha-2,6-sialyltransferase, and a soluble form of recombinant rat liver Gal(beta 1-3/4)GlcNAc alpha-2,3-sialyltransferase were studied with a panel of analogues of the trisaccharide Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-O)(CH2)7CH3. These analogues contain structural variants of D-galactose, modified at either C3, C4 or C5 by deoxygenation, fluorination, O-methylation, epimerization, or by the introduction of an amino group. In addition, the enantiomer of D-galactose is included. The alpha-2,6-sialyltransferases tolerated most of the modifications at the galactose residue to some extent, whereas the alpha-2,3-sialyltransferase displayed a narrower specificity. Molecular dynamics simulations were performed in order to correlate enzymatic activity to three-dimensional structure. Ineffective acceptors for rat liver alpha-2,6-sialyltransferase were shown to be inhibitory towards the enzyme; likewise, the alpha-2,3-sialyltransferase was found to be inhibited by all non-substrates. Modified sialyloligosaccharides were obtained on a milligram scale by incubation of effective acceptors with one of each of the three enzymes, and characterized by 500-MHz 1H-NMR spectroscopy.

Synthesis of Hex p-(1-->4)-beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->O)(CH2)7CH3 probes for exploration of the substrate specificity of glycosyltransferases: Part I, Hex = beta-D-Gal, 4-deoxy-beta-D-Gal, 4-O-methyl-beta-D-Gal, 4-deoxy-4-fluoro-beta-D-Gal, or beta-D-Glc.

Five trisaccharide derivatives designed for detailed exploration of the acceptor specificity of glycosyltransferases involved in termination of N-acetyllactosamine-type structures have been synthesized: beta-D-Gal p-(1-->4)-beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->0)(CH2)7CH3 (1), 4-deoxy-beta-D-Gal p-(1-->4)-beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->O)(CH2)7CH3 (2), 4-O-methyl-beta-D-Gal p-(1-->4)-beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->O)(CH2)7CH3 (3), 4-deoxy-4-fluoro-beta-D-Gal p-(1-->4)-beta-D-Glc pNAc-(1-->2)-alpha-D-Man p(1-->O)(CH2)7CH3 (4), and beta-D-Glc p-(1-->4)-beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->O)(CH2)7CH3 (5). A general disaccharide acceptor octyl 3,4,6-tri-O-benzyl-2-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D -glucopyranosyl)-alpha-D-mannopyranoside was synthesized by condensation of 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-alpha, beta-D-glucopyranosyl trichloroacetimidate with octyl 3,4,6-tri-O-benzyl-alpha-D-mannopyranoside, followed by deacetylation. 2,3,4,6-Tetra-O-acetyl-alpha-D-galactopyranosyl trichloroacetimidate and 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate were used as the glycosyl donors in the syntheses of 1 and 5. The modified galactosyl derivatives required subtle anomeric activation. Suitable donors for 2 turned out to be 2,3,6-tri-O-acetyl-4-deoxy-alpha-D-xylo-hexopyranosyl trichloroacetimidate and ethyl 2,3,6-tri-O-acetyl-4-deoxy-1-thio-alpha, beta-D-xylo-hexopyranoside; for 3, ethyl 2,3,6-tri-O-acetyl-4-O-methyl-1-thio-alpha, beta-D-galactopyranoside; and for 4, 2,3,6-tri-O-acetyl-4-deoxy-4-fluoro-alpha-D-galactopyranosyl trichloroacetimidate. It was concluded that thioglycosides were most appropriate for stereoselective coupling of activated synthons (carrying deoxy or O-methyl groups), whereas trichloroacetimidates gave high yields with deactivated (fluorine-containing) synthons.

Synthetic substrate analogues for UDP-GlcNAc: Man alpha 1-6R beta(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme.

UDP-GlcNAc:Man alpha 1-6R beta(1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complex N-glycans. We have tested a series of synthetic analogues of the substrate Man"'alpha 1-6(GlcNAc"beta 1-2Man'alpha 1-3)Man beta-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attaches N-acetylglucosamine in beta 1-2 linkage to the 2"'-OH of the Man"'alpha 1-6 residue. The 2"'-deoxy analogue is a competitive inhibitor (Ki = 0.13 mM). The 2"'-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3"'-, 4"'- and 6"'-OH groups are not essential for binding or catalysis since the 3"'-, 4"'- and 6"'-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3"'-position to pentyl and substituted pentyl groups causes competitive inhibition (Ki = 1.0-2.5 mM). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3"'-O-(4,4-azo)pentyl group and a 3"'-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man"'alpha 1-6 residue are essential for binding although the 2"'- and 3"'-OH face the catalytic site of the enzyme. The 4-OH group of the Man beta-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind.(ABSTRACT TRUNCATED AT 250 WORDS)

Free radical and cytotoxic effects of chelators and their iron complexes in the hepatocyte.

In a comparative screening study of chelators intended for clinical use eleven iron chelators have been tested for their ability to mobilize (59Fe) iron from 59Fe-labelled ferritin and from hepatocytes of rats labelled with 59Fe-transferrin. The toxic effects of the chelators were also studied using microsomal lipid peroxidation induced by Fe3+/ADP and NADPH. From these tests it was shown that 1,2-dimethyl 3-hydroxypyrid-4-one (L1) and mimosine were the most effective iron chelators in iron mobilization and did not catalyse lipid peroxidation. In conclusion it can be stated that besides to investigate the iron binding capacity of new chelators also their ability to catalyse lipid peroxidation has to be ruled out.