guanosine-diphosphate and phosphoramidite

GDP D-glycero-alpha-D-manno-Heptopyranose has been prepared in good overall yield from 2,3,4,6,7-penta-O-acetyl-D-glycero-D-manno-heptopyranose by a short-step synthesis. Phosphitylation using the phosphoramidite procedure afforded the alpha-anomer in high selectivity. Subsequent oxidation and partial deprotection gave the acetylated phosphate derivative, which was subjected to the coupling reaction with GMP-morpholidate to furnish the acetylated heptose nucleoside diphosphate in good yield. De-O-acetylation and final purification afforded the target GDP D-glycero-alpha-D-manno-heptopyranose, which serves as the substrate of the heptosyl transferase in Aneurinibacillus thermoaerophilus DSM 10155 and occurs as an intermediate in the biosynthesis of GDP 6-deoxy-heptose in Yersinia pseudotuberculosis.

Topics: Acetylation; Bacillaceae; Diphosphates; Glycoproteins; Guanosine Diphosphate; Heptoses; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Models, Chemical; Organophosphorus Compounds; Oxidation-Reduction; Yersinia pseudotuberculosis

Leishmania and other trypanosomatids are early eukaryotes that possess unusual molecular features, including polycistronic transcription and trans-splicing of pre-mRNAs. The spliced leader RNA (SL RNA) is joined to the 5' end of all mRNAs, thus donating a 5' cap that is characterized by complex modifications. In addition to the conserved m7GTP, linked via a 5'-5'-triphosphate bound to the first nucleoside of the mRNA, the trypanosomatid 5' cap includes 2'-O methylations on the first four ribose moieties and unique base methylations on the first adenine and the fourth uracil, resulting in the cap-4 structure, m7Gpppm3(6,6,2')Apm2'Apm2' Cpm2(3,2')U, as reported elsewhere previously. A library of analogs that mimic the cap structure to different degrees has been synthesized. Their differential affinities to the cap binding proteins make them attractive compounds for studying the molecular basis of cap recognition, and in turn, they may have potential therapeutic significance. The interactions between cap analogs and eIF4E, a cap-binding protein that plays a key role in initiation of translation, can be monitored by measuring intrinsic fluorescence quenching of the tryptophan residues. In the present communication we describe the multistep synthesis of the trypanosomatid cap-4 structure. The 5' terminal mRNA tetranucleotide fragment (pm3(6,6,2')Apm2'Apm2'Cpm2(3,2')U) was synthesized by the phosphoramidite solid phase method. After deprotection and purification, the 5'-phosphorylated tetranucleotide was chemically coupled with m7GDP to yield the cap-4 structure. Biological activity of this newly synthesized compound was confirmed in binding studies with eIF4E from Leishmania that we recently cloned (LeishIF4E-1), using the fluorescence time-synchronized titration method.

Topics: Animals; Binding Sites; Eukaryotic Initiation Factor-4E; Fluorescence; Guanosine Diphosphate; Guanosine Triphosphate; Organophosphorus Compounds; RNA Cap Analogs; RNA Caps; RNA, Messenger; RNA, Protozoan; RNA, Spliced Leader; Trypanosomatina