Bromopyridone Nucleotide Analogues, Anoxic Selective Radiosensitizing Agents That Are Incorporated in DNA by Polymerases.

Ionizing radiation is frequently used to kill tumor cells. However, hypoxic solid tumor cells are more resistant to this treatment, providing the impetus to develop molecules that sensitize cells to ionizing radiation. 5-Bromo-2'-deoxyuridine (BrdU) has been investigated as a radiosensitizing agent in the lab and clinic for almost 5 decades. Recent reports that BrdU yields DNA interstrand cross-links (ICLs) in non-base-paired regions motivated us to develop radiosensitizing agents that generate cross-links in duplex DNA selectively under anoxic conditions. 4-Bromo- and 5-bromopyridone analogues of BrdU were synthesized and incorporated into oligonucleotides via solid-phase synthesis. Upon irradiation, these molecules yield DNA interstrand cross-links under anaerobic conditions. The respective nucleotide triphosphates are substrates for some DNA polymerases. ICLs are produced upon irradiation under anoxic conditions when the 4-bromopyridone is present in a PCR product. Because the nucleoside analogue is a poor phosphorylation substrate for human deoxycytidine kinase, a pro-nucleotide form of the 4-bromopyridone was used to incorporate this analogue into cellular DNA. Despite these efforts, the 4-bromopyridone nucleotide was not detected in cellular DNA. Although these molecules are improvements over previously reported nucleotide analogues designed to be hypoxic radiosensitizing agents, additional advances are needed to create molecules that function in cells.

DNA interstrand cross-linking upon irradiation of aryl halide C-nucleotides.

γ-Radiolysis kills cells by damaging DNA via radical processes. Many of the radical pathways are O2 dependent, which results in a reduction in the cytotoxicity of ionizing radiation in hypoxic tumor cells. Consequently, there is a need for chemical agents that increase DNA damage by ionizing radiation under O2-deficient conditions. Modified nucleotides that are incorporated in DNA and produce highly reactive σ-radicals are useful as radiosensitizing agents. Aryl halide C-nucleotides (4-6) were incorporated into oligonucleotides by solid-phase synthesis. Duplex DNA containing 4-6 forms interstrand cross-links upon γ-radiolysis under anaerobic conditions or UV irradiation. Deep Vent (exo(-)) DNA polymerase accepted the nucleotide triphosphate of C-nucleotide 6 as a substrate and preferentially incorporated it opposite pyrimidines, but no further extension was detected. Incorporation of 6 in extended products by Deep Vent (exo(-)) during PCR or by Sequenase during copying of single stranded DNA plasmid was undetectable. Aryl halide nucleotide analogues that produce DNA interstrand cross-links under anaerobic conditions upon irradiation are potentially useful as radiosensitizing agents, but further research is needed to identify molecules that are incorporated by DNA polymerases and do not block further polymerization for this approach to be useful in cells.

In vitro reconstruction of the chain termination reaction in biosynthesis of the Escherichia coli O9a O-polysaccharide: the chain-length regulator, WbdD, catalyzes the addition of methyl phosphate to the non-reducing terminus of the growing glycan.

The Escherichia coli O9a O-polysaccharide (O-PS) represents a model system for glycan biosynthesis and export by the ATP-binding cassette (ABC) transporter-dependent pathway. The polymannose O9a O-PS is synthesized using an undecaprenol-diphosphate-linked acceptor by mannosyltransferases located at the cytoplasmic membrane. An ABC-transporter subsequently exports the polymer to the periplasm where it is assembled onto lipopolysaccharide prior to translocation to the cell surface. The chain length of the O9a O-PS is regulated by the dual kinase/methyltransferase activity of the WbdD enzyme and modification of the polymer is crucial for binding and export by the ABC-transporter. Previous biochemical data provided evidence for phosphorylation/methylation at the non-reducing end of the O9a O-PS but the structure of the terminus has not been determined. Here, we describe the exploitation of a synthetic O9a O-PS repeating unit carrying a fluorescent tag as an acceptor for in vitro phosphorylation and methylation by a purified soluble form of WbdD. Phosphorylation of the acceptor was evident by both a mobility shift in thin layer chromatography and radiolabeling of the acceptor using [γ-(33)P]ATP. Methylation of the acceptor was dependent on phosphorylation and was demonstrated by radiolabeling using S-[methyl-(3)H]adenosyl-methionine as a substrate, in the presence of ATP. NMR spectroscopic and mass spectrometric methods were used to determine the precise structure of the terminal modification, leading to the conclusion that WbdD catalyzes the addition of a novel methyl phosphate group to the 3-position of the non-reducing terminal mannose of the O9a O-PS repeating unit.

2,3-Anhydrosugars in glycoside bond synthesis: mechanism of 2-deoxy-2-thioaryl glycoside formation.

A series of investigations probing the mechanism of the 2,3-anhydrosugar migration-glycosylation reaction were performed using a thioglycoside with the D-lyxo stereochemistry as the substrate. Among the work reported are the results of quantum mechanical calculations, NMR studies, the measurement of alpha-deuterium kinetic isotope effects, and the synthesis of a series of substrate analogues. All studies point to a consistent finding: that the reaction proceeds through an oxocarbenium ion intermediate, not an episulfonium ion as previously suggested. It is proposed that the high stereoselectivity of the reaction arises from a preferred "inside attack" of the nucleophile onto the oxocarbenium ion intermediate.

2,3-Anhydrosugars in glycoside bond synthesis. Application to 2,6-dideoxypyranosides.

We describe here the first use of 2,3-anhydrosugars as glycosylating agents for the preparation of 2-deoxypyranosides. In particular, the methodology was used to assemble 2,6-dideoxysugar glycosides. Glycosylation of a panel of alcohols with one of two 6-deoxy-2,3-anhydrosugar thioglycosides (8 and 9) in the presence of a Lewis acid afforded 2,6-dideoxy-2-thiotolyl glycoside products in generally excellent yields with an exclusively syn relationship between the aglycon and the C-3 hydroxyl group. Removal of the 2-thiotolyl group can be achieved upon reaction with tri-n-butyltin hydride and AIBN to give the corresponding 2,6-dideoxy pyranosides. Once developed, the method was applied to the synthesis of oligosaccharide moieties in the natural products apoptolidin and olivomycin A.

Synthesis of 8-azidooctyl glycoside derivatives of the O-chain repeating unit of Escherichia coli O9a lipopolysaccharide and a methylated analog.

Described is the synthesis of 8-azidooctyl glycoside derivatives of the Escherichia coli serotype O9a O-chain tetrasaccharide repeating unit and the terminal tetrasaccharide motif in this polysaccharide, which contains a methyl group on O-3 of the distal mannopyranose residue. The assembly of these compounds involved the sequential addition of monosaccharide residues from the reducing to the nonreducing end of the molecule using glycosyl trichloroacetimidate donors. Both compounds were initially prepared as p-methoxyphenyl glycosides, which were converted to the corresponding 8-azidooctyl derivatives at a late stage in the synthesis.

Stereoselective synthesis of 2-deoxy-furanosides from 2,3-anhydro-furanosyl thioglycosides.

The stereocontrolled synthesis of 2-thiotolyl-furanosides from 2,3-anhydro-furanosyl thioglycosides through a rearrangement-glycosylation process is reported. The efficiency of this reaction is high, providing 70-95% yields of the products. Treatment of the resulting 2-deoxy-2-thiotolyl-glycosides with hydrogen and Raney nickel affords the corresponding 2-deoxy-furanosides with a 1,3-syn relationship.

Recent advances in the synthesis of 2-deoxy-glycosides.

Glycosides of 2-deoxy-sugars, monosaccharides in which the hydroxyl group at C-2 is replaced with a hydrogen atom, occur widely in natural products and therefore have been the subject of intense synthetic activity. The report summarizes recent advances in this area, with a particular focus on work published since an earlier review on the topic, in 2000 (Marzabadi, C. H.; Franck, R. W. Tetrahedron2000, 56, 8385-8417).