Synthesis of telomere-mimic carbocyclic 5'-nor oligodeoxynucleotides.

Telomere-mimic S-ODNs have been synthesized and examined their effects on the proliferation of human tumor cell lines by XTT assay. Furthermore, the guanosine derivatives of carbocyclic 5'-nor nucleoside were synthesized.     

Synthesis of substituted 5-fluoro-5,6-dihydropyrimidines.

The reaction of 5-substituted uracils with fluorine in acetic acid and other solvents and the following treatment with different alcohols yielded the corresponding 5-fluoro-5,6-substituted-5,6-dihydropyrimidines. Thymine gave 5-fluoro-5-methyl-6-alkoxy-5,6-dihydropyrimidines. 5-Halogeno uracils and 5-nitrol uracil were converted into 5-fluoro-5-halogeno-6-hydroxy-5,6-dihydropyramidines and the 5-nitroanalogue, respectively. The structures of the compounds were confirmed by mass spectrometry.     

Synthesis and properties of poly 5-methylthiouridylic acid.

In an effort to search for good methods for the enzymatic synthesis of polynucleotide analogs with antitemplate activity, 5-methylthiouridine-5'-diphosphate (ms5UDP) has been synthesized and investigated as a substrate for polynucleotide phosphorylase. While ms5UDP was polymerized at a very low rate to give a 6% yield of polynucleotides by the polynucleotide phosphorylase of Micrococcus luteus, it was utilized more efficiently by the corresponding enzyme of Escherichia coli resulting in a 15% yield of poly (5-methylthiouridylic) acid. Results of the co-polymerization of ms5UDP and UDP revealed that the ratio of 5-methylthiouridylate to uridylate residues in the polynucleotide product was lower than the ratio of ms5UDP to UDP in the substrate mixture. The 5-methylthio group conferred only minute changes on the conformation of the modified polyuridylic acid, and the complexes formed between poly-(5-methylthiouridylic) acid and poly(adenylic) acid possessed slightly higher Tm values than did the unmodified counterparts. Poly(5-methylthiouridylic) acid was a potent inhibitor of calf thymus DNA polymerase alpha.     

Synthesis and overproduction of the 5A protein of insertion sequence IS5.

We have demonstrated both the synthesis and overproduction of the 5A protein encoded by the longest open reading frame of the bacterial insertion sequence IS5. Expression was obtained in vitro and in Escherichia coli maxicells from plasmids containing IS5 in either orientation, as well as in vitro from a restriction fragment containing exclusively IS5 DNA. When IS5 was cloned in the appropriate orientation downstream of a strong tac promoter, production of the 5A protein was increased to 10 to 20% of the total protein synthesized in vitro.     

Synthesis of 5-thio-D-galactose

A synthesis of 5-thio-D-galactose, in the form of its crystalline, anomeric methyl glycopyranosides, is described. Compounds prepared as intermediates included ethyl 2,3-di-O-(tert-butyldimethylsilyl)-5,6-O-carbonyl-β-D-galactofuranoside, the corresponding 5,6-dideoxy-5,6-epithio derivative, and ethyl 2,3,6-tri-O-acetyl-5-S-acetyl-5-thio-β-D-galactofuranoside. On methanolysis, the latter afforded methyl 5-thio-α-D-galactopyranoside which, in turn, was transformed into methyl 5-thio-β-D-galactopyranoside. Acetolysis proved to be less satisfactory for incorporation of the sulfur atom into a pyranose ring-form. Characteristics of the ¹³C-n.m.r. spectra of derivatives of 5-thio-D-galactose are described, including the fact that ¹J_C,H values for the anomeric pyranosides differ by only 1–3 Hz, as compared with ≈ 10 Hz for their oxygen analogs.     

Synthesis of 5-Methylmercaptouracil

5-Methylmercaptouracil was prepared by the reaction of uracil with dimethyl sulfoxide and monochloromethyl ether. At the same time the formation of methylal, methyl sulfide, methyl disulfide, methyl methanethiolsulfonate, methyl chloride and paraformaldehyde were observed. Acetyl chloride was successfully used instead of monochloromethyl ether. Uracil reacted with dimethyl sulfoxide and phenacyl bromides, resulting in the formation of 5-bromouracil. The mechanism of these reactions were discussed.     

Synthesis of 5-deoxy-5-fluorosporaricin A

The structures of the O-specific side-chains in the lipopolysaccharides of Salmonella greenside, group Z, and Salmonella adelaide, group O, have been investigated. The former proved to be identical with that of Escherichia coli O 55. The latter, which was more extensively studied, was composed of repeating units having the structure

in which Col is colitose (3,6-dideoxy-l-xylo-hexose). This was also shown to be the biological repeating-unit. The same structure has been proposed for the O-antigen of E. coli O 111. The biological repeating-unit for the S. greenside O-antigen was also defined. The structural studies also confirmed that both lipopolysaccharides contain the hexose region typical for the Salmonella core.     

Synthesis of 5-thio-L-altrose

Treatment of 1,2,3-tri-O-acetyl-5,6-anhydro-D-galactofuranose with thiourea gave 1,2,3-tri-O-acetyl-5,6-dideoxy-5,6-epithio-L-altrofuranose, acetolysis of which gave 1,2,3,6-tetra-O-acetyl-5-S-acetyl-5-thio-L-altrofuranose. Deacetylation of the latter gave 5-thio-L-altrose.     

Synthesis of 5-Formyluridines

Abstract

5-Formyl-2′,3′-O-isopropylideneuridine and 5-formyl-2′,3′,5′-tri-O-acetyluridine were prepared by a new procedure involving palladium-catalyzed coupling of 5-iodouridine with styrene, followed by reaction with acetic anhydride or acetone and ozonolysis of the resulting 5-styryluridine derivatives.     

Synthesis and conformation control of peptide ribonucleic acid containing 5'-amino-5'-deoxyribopurinenucleosides.

A novel nucleic acid model, i.e. peptide ribonucleic acid (PRNA), tethering 5'-amino-5'-deoxypyrimidine ribonucleoside as a recognition site for nucleic acids, has been designed and synthesized. We have demonstrated that the recognition behavior of PRNA with complementary oligopurinenucleotides can be controlled externally through the orientational switching of the pyrimidine nucleobase of PRNA induced by added borates. We extend this methodology of controlling the nucleobase orientation and recognition behavior of novel mono and oligomeric PRNAs containing 5'-amino-5'-deoxypyrimidine and/or purinenucleosides. In case of the PRNA oligomer containing pyrimidine-purine mixed sequence, efficient orientational switching of nucleobases induced by added borates was also observed.     

Synthesis and chirality analysis of carbocyclic 5'-nor nucleosides.

Achiral carbocyclic "DL-like" 5'-nor nucleosides have been synthesized and analyzed by the chiral capillary electrophoresis to elucidate the "D-like" monomers.     

5''-Halogeno-2'',3''-cyclic sulphite isomers in the preparation of 5''-halogeno nucleosides. Synthesis of 5''-deoxyuridine and 5''-deoxy-5-fluorouridine.

When uridine (Ia) is reacted with thionyl chloride in hexamethylphosphoric triamide a mixture of isomeric 5'-chloro-2',3'-sulphites is formed, which can be separated to individual epimers IIa and IIIa, in 45% and 15% yields, respectively. Analogously, crystalline epimers IIb (37%) and IIIb (17%) can be obtained from 5-fluorouridine (Ib). Both isomers IIa, IIIa (or IIb, IIIb) afford a single 5'-chloro derivative IVa (or IVb, respectively) if treated with 0.1N sodium methoxide. From the mixture of sulphites IIa and IIIa (or IIb and IIIb) crystalline 5'-chlorouridine IVa is formed in 84.5% yield, calculated per starting uridine Ia (or crystalline 5'-chloro-5-fluorouridine IVb, 85.5% per starting 5-fluorouridine Ib, respectively). On reduction of 5'-chlorouridine IVa with tributyltin hydride 5'-deoxyuridine (Va) is formed in 79% yield. During the reduction of 5'-chloro-5-fluoro derivative IVb to 5'-deoxy-5-fluorouridine (Vb, 57%) a partial reductive elimination of 5-fluorine takes place under formation of 5'-deoxyuridine (Va, 9%).     

Synthesis and biological properties of 2''-deoxy-5-vinyluridine and 2''deoxy-5-vinylcytidine.

Rapid and efficient syntheses for the preparation of 2'-deoxy-5-vinyluridine and 2'-deoxy-5-vinylcytidine are described starting from nucleoside precursors. Contrary to some previous reports, 2-deoxy-5-vinyluridine has be found to be quite stable under normal laboratory conditions but when tested in animals shows neither toxicity nor anti-leukemic (L1210 cells) or anti-parasitic (Plasmodium berghei) activity. 2'-Deoxy-5-vinylcytidine appears to polymerise readily. It is much less toxic to cell cultures than 2'-deoxy-5'vinyluridine but is almost as active against herpes virus replication (ID50:0.2 microgram/ml) for both type 1 and type 2 herpes virus (selectivity index:225).     

Synthesis of 5'-deoxy-5'-nucleosideacetic acid derivatives

The synthesis of several new 5'-deoxy-5'-nucleosideacetic acid derivatives by the reactions of alkoxycarbonylmethylene triphenylphosphoranes with nucleoside 5'-aldehydes is described.     

Synthesis of polynucleotide 5''-triphosphatase in vaccinia virus-infected HeLa cells.

Synthesis of polynucleotide 5'-triphosphatase, which is presumably involved in the initial modification in the series of reactions by which 5'-termini of vaccinia mRNA become capped and methylated, has been demonstrated in vaccinia virus infected HeLa cells. Synthesis of the enzyme is prevented by actinomycin D and cycloheximide, suggesting that both de novo DNA-dependent RNA and protein syntheses are required. On the other hand, cytosine arabinoside, an inhibitor of viral DNA replication, does not prevent induction of the enzyme. The latter observation, together with the kinetics of synthesis of the enzyme in vaccinia virus-infected HeLa cells, suggests that polynucleotide 5'-triphosphatase is an "early" or prereplicative viral protein. Immunologlobulin produced against the purified virion-associated polynucleotide 5'-triphosphatase as antigen neutralized the activity of the induced polynucleotide 5'-triphosphatase, thus indicating the identity of the two enzymes.     

Synthesis of methyl alpha- and beta-D-xylopyranoside-5-18O.

Methyl alpha- and beta-D-xylopyranoside-5-18O (5 and 6) were prepared by way of oxygen exchange between 18O-water and the periodate-oxidation product (1) obtained from 1,2-O-isopropylidene-alpha-D-glucofuranose. The isotopic enrichment of 5 and 6 was determined by hydrolysis of each to D-xylose-5-18O (3), conversion of the sugar into 1,2,3,4-tetrakis-O-(tert-butyldimethylsilyl)-beta-D-xylopyranose-5-18O (7), and determination of the 18O content of the latter by use of a quadrupole, mass spectrometer.     

Acetylation and cleavage of purine nucleosides. Synthesis of 6-azauridine, 5-fluorouridine, and 5-methyluridine.

Inosine (I) when acetylated with acetic anhydride in the presence of acetyl chloride in acetic acid solution (the so called "acid acetylation"), affords an acetylated nucleoside III (75%) along with cleavage products of the nucleoside (hypoxanthine, 19%). The reaction of I with acetyl chloride (7 days) results in the formation of hypoxanthine (95%) and triacetylribofuranosyl chloride (IV) isolated in the form of tetraacetylribofuranose (47%). The acetylated purine nucleoside affords a similar result by reaction with acetyl chloride or acetyl bromide. 2'-Deoxyuridine gives a diacetyl derivative (80%) by reaction with acetyl bromide. On treatment with acetyl bromide, the nucleoside bond of purine nucleosides is quantitatively cleavaged (4 h, 20 degrees C) with the formation of tri-O-acetyl-D-ribofuranosyl bromide (X). The halogenose X affords pure beta-anomers, namely, 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose (75%), the triacetyl derivatives of 5-methyluridine (XVIIa; 75%, referred to guanosine), 6-azauridine (XVIII; 71%), and 5-fluorouridine (XIXa; 75%).