8-Azido-3,6-dioxaoctanoyl-AEEA

In order to elucidate the biological activities of cordycepin (3'-deoxyadenosine) and related 3'-deoxyribonucleosides on eukaryotic DNA replication, the inhibitory effects of triphosphate derivatives of 3'-deoxyadenosine(3'-dATP), 8-azido-3'-deoxyadenosine(8-N3-3'-dATP), 3'-deoxyguanosine(3'-dGTP), 3'deoxyuridine(3'dUTP), 5-fluoro-3'deoxyuridine(5-F-3'-dUTP), 3'-deoxycytidine(3'-dcTP), and 5-fluoro-3'-deoxycytidine(5-F-3'dCTP) on DNA primase and replicative DNA polymerases alpha, delta, and epsilon purified from cherry salmon (Oncorhynchus masou) testes or calf thymus were examined. All analogs, except 8-N3-3'-dATP, showed strong inhibitory effects on DNA primase, but none of them inhibited DNA polymerases alpha, delta, or epsilon. Kinetic analysis revealed that the inhibition modes of them were competitive with respect to the incorporation of natural substrate that had the corresponding base moiety and non-competitive with respect to other substrates. Based on the kinetic data, we compared the affinities of 3'-dNTPs between DNA primase and RNA polymerases I and II, since 3'-dNTPs also inhibit eukaryotic RNA polymerases. Although the Ki values for DNA primase were much larger than those for RNA polymerases, the Ki/K(m) values, which indicate the affinity of the analog to the enzyme, were very similar.

The linker-equipped disaccharide, 8-amino-3,6-dioxaoctyl 2,6-dideoxy-2-acetamido-3-O-β-D-galactopyranosyluronate-β-D-glucopyranoside (10), was synthesized in eight steps from acetobromogalactose and ethyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-1-thio-β-D-glucopyranoside. The hydroxyl group present at C-4(II) in the last intermediate, 8-azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2-trichloroacetamido-3-O-(benzyl 2,3-di-O-benzyl-β-D-galactopyranosyluronate)-β-D-glucopyranoside (9), is positioned to allow further build-up of the molecule and, eventually, construction of the complete hexasaccharide. Global deprotection (9→10) was done in one step by catalytic hydrogenolysis over palladium-on-charcoal.

3-Deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an essential component of LPS in the outer leaflet of the Gram-negative bacterial outer membrane. Although labeling of Escherichia coli with the chemical reporter 8-azido-3,8-dideoxy-d-manno-oct-2-ulosonic acid (Kdo-N3) has been reported, its incorporation into LPS has not been directly shown. We have now verified Kdo-N3 incorporation into E. coli LPS at the molecular level. Using microscopy and PAGE analysis, we show that Kdo-N3 is localized to the outer membrane and specifically incorporates into rough and deep-rough LPS. In an E. coli strain lacking endogenous Kdo biosynthesis, supplementation with exogenous Kdo restored full-length core-LPS, which suggests that the Kdo biosynthetic pathways might not be essential in vivo in the presence of sufficient exogenous Kdo. In contrast, exogenous Kdo-N3 only restored a small fraction of core LPS with the majority incorporated into truncated LPS. The truncated LPS were identified as Kdo-N3-lipid IVA and (Kdo-N3)2-lipid IVA by MS analysis. The low level of Kdo-N3 incorporation could be partly explained by a 6-fold reduction in the specificity constant of the CMP-Kdo synthetase KdsB with Kdo-N3 compared with Kdo. These results indicate that the azido moiety in Kdo-N3 interferes with its utilization and may limit its utility as a tracer of LPS biosynthesis and transport in E. coli We propose that our findings will be helpful for researchers using Kdo and its chemical derivatives for investigating LPS biosynthesis, transport, and assembly in Gram-negative bacteria.