Analysis of O2'-methylnucleoside 5'-phosphates in snake venom hydrolysates of RNA: identification of O2'-methyl-5-carboxymethyluridine as a constituent of yeast transfer RNA.

Snake venom phosphodiesterase liberates the O2-methylnucleoside (Nm) constituents of RNA as the corresponding 5-nucleotides (PNm), which, in contrast to normal 5-nucleotides (pN), are resistant to dephosphorylation by venom 5-nucleotidase. This property provides the basis of a convenient and highly reproducible quantitative assay for Nm residues in RNA. The assay method involves: (1) hydrolysis of RNA with whole or partially-purified snake venom; (2) isolation of the pNm derivatives, as a group, by anion-exchange chromatography on DEAE-cellulose; (3) resolution of the individual pNm compounds by two-dimensional paper chromatography; (4) identification and quantitative measurement of pNm derivatives by ultraviolet absorption spectrophotometry. Using this procedure, the molar proportions of the Nm constituents of wheat embryo, yeast, and Escherichia coli tRNA have been determined. The close correspondence between the values measured by venom hydrolysis and those obtained by analysis of alkali-stable dinucleotide (Nm-Np) sequences attests to the validity of the venom assay, and further indicates that alkali-stable sequences larger than dinucleotides are not present in significant amounts in the tRNA of the above three organisms. During the present investigation, several ultraviolet-absorbing components, not immediately identifiable as ribose-methylated nucleotides, were isolated along with the expected O2-methylnucleoside 5-phosphates. Preliminary characterization of one of these compounds suggests that it is a derivative of a novel nucleoside, O2-methyl-5-carboxymethyluridine (cm5Um is released as part of an alkali-stable dinucleotide, cm5Um-Ap. The proportion of pU-2 in venom hydrolysates of yeast tRNA (0.02 mol percent, the same as the content of cm5Um-Ap in alkaline hydrolysates) suggests that O2-methyl-5-carboxymethyluridine may be confined to a single isoaccepting species of tRNA in yeast. In an allied study, reinvestigation of the alkali-stable dinucleotide sequences of baker's yeast tRNA has confirmed previous results concerning the sequence distribution of O2-methylribose in yeast tRNA (Gray, M. W. & Lane, B.G. (1967) Biochim. Biophys. Acta 134, 243-257).     

2'-O-methyl ribothymidine: a component of rabbit liver lysine transfer RNA

One of the lysine transfer RNAs of rabbit liver is shown to contain 2′-O-methyl ribothymidine in place of ribothymidine. This represents the first demonstration of the presence of 2′-O-methyl ribothymidine in a nucleic acid.     

Synthesis of 6-deoxy-5-thio- -glucose

Three routes were investigated for the conversion of -glucose into the title compound. In the first approach, reduction of the 5,6-thürane ring of 5,6-dideoxy-5,6-epithio-1,2-O-isopropylidene α- -glucofuranose (17) as well as that of its 3-O-allyl derivative (13) with lithium aluminium hydride was investigated; 17 afforded the corresponding 6-deoxy derivative besides di-, tri-, and poly-mers, whereas only polymers were formed from 13. In the second approach, the oxirane ring of

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was reduced by sodium borohydride and the resulting 6-deoxy derivative was converted into the 5-thiobenzoate; the corresponding hex-4-enofuranose was formed as a byproduct. In the third approach partial mesylation of methyl 5-thio-α- -glucopyranoside was attempted, but the 6-mesylate 27 could be isolated only in modest yield (28%) together with rearranged 2,5-thioanhydromannofuranoside derivatives. The mechanism of this rearrangement is discussed in detail. The 6-mesylate 27 was converted via the 6-iodo derivative into the title compound.     

Synthesis and biological activity of 5-azacytosine nucleosides derived from 4-thio-2-deoxy-L-threo-pentofuranose and 4-thio-2-deoxy-D-erythro-pentofuranose.

1-O-Acetyl-2-deoxy-3,5-di-O-toluoyl-4-thio-D-erythro-pentofuranose and 2-deoxy-1,3,5-tri-O-acetyl-4-thio-L-threo-pentofuranose were coupled with 5-azacytosine to obtain alpha and beta anomers of nucleosides.     

A facile synthesis of 5-thio- -fucose and 5-thio- -arabinose from -arabinose

5-Thio- -fucopyranose tetraacetate was synthesized in 11 steps from or -arabinose diethyl dithioacetal by one-carbon elongation at C-5. Highly diastereo-selective addition of MeLi in ether to a

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derivative was achieved to give the corresponding 6-deoxy-β- -altrofuranose isomer in good yield. A sulfur atom was introduced at C-5 of 6-deoxy- -altrofuranose derivatives via substitution of a 5-tosylate with KSAc in HMPA with inversion of configuration, giving 5-thio- -fucopyranose. A derivative was also prepared from 6-deoxy-β- -altrofuranose derivatives. 5-Thio- -arabinopyranose tetraacetate, the 5-demethyl analog of 5-thio- -fucose, was also synthesized from in 5 steps. 5-Thio- -arabinose showed weak inhibitory activity against α- -fucosidase from bovine kidney (K_{i = 0.77 mM}).     

Synthesis of novel 5-aryl-2-thio-1,3,4-oxadiazoles and the study of their structure-anti-mycobacterial activities

The preparation of novel 5-aryl-2-thio-1,3,4-oxadiazoles 4a-41 and the computer-aided study of their in vitro anti-tubercular activity against Mycobacterium tuberculosis H37Rv (ATCC 27294) are reported. The average accuracy of the electronic-topological method and neural network methods applied to the activity prediction in leave-one-out cross validation is 80%.     

Synthesis and biological properties of 16(S)-amino-PGF2 alpha methyl ester

A synthesis of 16-amino-derivatives of PGF2 alpha is reported. Introduction of an amino group into position 16 of PGF2 alpha has decreased the sensitivity of the compound to metabolic degradation. 16(S)-amino-PGF2 alpha methyl ester shows high abortifacient activity with reduced diarrhoeic side effect.     

Melphalan potently substitutes the N-terminal Tyr of D-Ala2-Leu5-enkephalin methyl ester

In search of an affinity label of the opioid receptor, the nitrogen mustard melphalan, Mel, was built into the peptide chain of D-Ala2-Leu5-enkephalin (DALE) methyl ester in different positions. We report now that in contrast to the previous observations that an intact Tyr in position 1 is essential for opioid activity [(1980) Annu. Rev. Pharmacol. Toxicol. 20, 81-110], substitution of Tyr by Mel did not result in a loss of the binding affinity. Mel1, Leu5-enkephalin-OMe competed for the binding sites of [3H]naloxone as potently as DALE did; IC50 values for both compounds were 50 nM. Mel substitution has led to one order potency decrease in binding to the delta-sites. 0.5-1 microM of the compound irreversibly inactivates 50% of the binding sites of [3H]naloxone, and 5-10 microM of that of [3H]DALE. These results shed new light on the structural requirements established for opioid peptides. In addition, the new derivative can be used as an affinity label of the opioid receptor.     

Synthesis of leukotriene A4 methyl ester via acetylene intermediates

Rac-leukotriene A4 methyl ester has been synthesized from propargylic alcohol and 1-heptyne. The synthetic strategy involves the assembly of carbon chain by acetylenide anion condensations and the introduction of (Z)-double bonds by the triple bond hydrogenation.     

Synthesis of methyl 2-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside and methyl 2-O-alpha-D-talopyranosyl-alpha-D-talopyranoside

Treatment of methyl 3-O-benzyl-2-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-alpha-D- mannopyranoside (1) with tert-butyldiphenylsilyl chloride in N,N-dimethylformamide afforded methyl 3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-mannopyranoside (2). Oxidation of 2 with pyridinium chlorochromate, followed by reduction of the carbonyl group, and subsequent O-deacetylation afforded methyl 3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-alpha-D-mannopyranosyl- alpha-D- talopyranoside (5). Cleavage of the tert-butyldiphenylsilyl group of 5 with tetrabutylammonium fluoride in oxolane, followed by hydrogenolysis, gave methyl 2-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside (7). O-Deacetylation of 1 gave methyl 3-O-benzyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (8). Treatment of 8 with tert-butyldiphenylsilyl chloride afforded a 6,6'-disilyl derivative, which was converted into a 2',3'-O-isopropylidene derivative, and then further oxidized with pyridinium chlorochromate. The resulting diketone was reduced and removal of the protecting groups gave methyl 2-O-alpha-D-talopyranosyl-alpha-D-talopyranoside (15). The structures of both 7 and 15 were established by 13C-n.m.r. spectroscopy.     

Photobinding of 8-methoxypsoralen to transfer RNA and 5-fluorouracil-enriched transfer RNA.

The photobinding of [3H]8MOP to tRNA upon irradiation at 365 nm in the absence of O2 was determined by gel filtration. The maximum photobinding was found to be ca. 4 mol of 8MOP er mol of tRNA and 5FU-tRNA, with an overall quantum yield of 2.3 X 10(-3). The photobinding kinetics for 8MOP-tRNA showed an apparent induction period or sigmoidal kinetic curve, indicating a specific initial photobinding site on tRNA which was identified as 4-thiouridine at position 8 from the 5'-end of Escherichia coli tRNA. Photobinding of 8MOP to 5FU-tRNA proceeded without an apparent induction period. 8MOP-tRNA and 8MOP-5FU-tRNA adducts were characterized by absorption, fluorescence, and CD spectroscopy. A modified procedure was also developed to analyze the nucleoside composition in modified 8MOP-tRNA and 8MOP-5FU-tRNA. The results showed that 8MOP photochemically added mainly to pyrimidine bases. The photobinding of 8MOP changed the conformation (secondary in particular) of tRNA and inhibited aminoacyl-tRNA synthetase activity.