Conformation of N-(purin-6ylcarbamoyl) glycine, a hypermodified base in tRNA

The crystal structure of the potassium salt of N-(purin-6ylcarbamoyl) glycine was determined from three-dimensional X-ray diffraction data. The N⁶-substituent is distal ($\text{trans}$) to the imidazole ring, forming an intramolecular hydrogen bond N(glycine) -H---N(1)adenine. This conformation of the N⁶-substituent is typical of ureidopurines, and blocks the two sites N⁶-H and N¹ of adenine that are normally utilized for complementary base-pairing in the double helical regions of nucleic acids; the internal hydrogen bonding further enhances the shielding of N¹. This blocking of N⁶-H and N¹ may be important in enhancing the single stranded conformation of the anticodon loop of tRNA and in preventing the modified adenosine adjacent to the anticodon from taking part directly in codon-anticodon interaction through the complementary base pairing.     

Conformation and possible role of hypermodified nucleosides adjacent to 3'-end of anticodon in tRNA: N-(purin-6-ylcarbamoyl)-L-threonine riboside

The crystal structure of N-(purin-6-ylcarbamoyl)-L-threonine riboside was determined from three-dimensional x-ray diffraction data. The N⁶-substituent is distal ($\text{trans}$) to the imidazole ring, leading to a bifurcated hydrogen interaction involving two intramolecular contacts with the hydrogen on N(threonine): a hydrogen bond to N(1) of adenine and a close contact to the hydroxyl oxygen of threonine. The conformation of the molecule and the internal hydrogen bond completely block the two sites N⁶-H and N¹ of adenine from taking part in the Watson-Crick base pairing. This inability to base pair according to the Watson-Crick scheme appears as a common structural feature in all modified bases adjacent to the 3′-end of anticodons. These results, along with Crick's hypothesis for codon recognition, suggest that the hypermodified bases adjacent to the anticodons may be important in (i) preventing the misreading of the codons by bases adjacent to anticodons and (ii) promoting a single stranded conformation for the anticodon loops.     

Determination of N-(9-( -D-ribofuranosyl)purin-6-ylcarbamoly)threonine at the picomole level in transfer-RNA

An assay has been developed for quantitation of the modified nucleoside, t⁶A, in tRNA at the pmole level. For tRNA from a variety of species, the content of t⁶A was found to be 0.18–0.25 mole %. These values lend support to the suggestion that t⁶A is located at the 3′-end of the anticodon in tRNA's whose codons begin with adenosine. Essentially no t⁶A was found in Mycoplasma sp. (Kid) tRNA which is deficient in many modified nucleosides. In the rat, no organ specific differences were found. The amount of t⁶A in Novikoff hepatoma tRNA was essentially the same as in tRNA from normal rat liver.     

Biosynthesis of N-(purin-6-ylcarbamoyl)-l-threonine riboside. Incorporation of l-threonine in vivo into modified nucleoside of transfer ribonucleic acid

l-[U-(14)C]Threonine is incorporated into N-(purin-6-ylcarbamoyl)-l-threonine riboside of rat liver and Escherichia coli tRNA. A pathway is suggested for the biosynthesis of this nucleoside.     

Metabolism of N-(purin-6-yl)glycine by Soya Bean Callus

The growth of cytokinin-dependent soya bean callus has beenshown to be accelerated by adding N-(purin-6-yl)glycine to themedium. Two biologically active peaks were detected when thecallus was cultured with N-(purin-6-yl)glycine. These two peaksco-chromatographed with 6-(2, 3, 4-trihydroxy-3-methylbutylamino)purineand zeatin respectively. When ¹⁴C labelled N-(purin-6-yl)glycinewas applied to the callus, radioactivity was found with boththese compounds irrespective of whether or not the N-(purin-6-yl)glycinewas labelled in the side chain or in the 8-position of the purinering. Small amounts of zeatin appear to be produced from N-(purin-6-yl)glycinewhich could explain why this compound stimulates the divisionof soya bean callus. N-(purin-6-yl)glycine, soya bean callus, metabolism, radioactivity, cytokinins     

Radioimmunoassays for the modified nucleosides N[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine and 2-methylthioadenosine.

Radioimmunoassays were established for the modified nucleosides N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine, t6A, and 2-methylthioadenosine, ms2A. The assays depended on the production of antisera specific for t6A and ms2A that have not been previously reported. The nitrocellulose membrane filtration and saturated ammonium sulfate RIA techniques were compared for efficiency. Various radioactive antigens were employed to establish which type of antigen would give the best binding. The tritium post-labeling procedure of Randerath and Randerath was used to obtain labeled nucleosides of high enough specific activity to be useful for RIAs when the labeled nucleoside was not available commerically. The specificity of the antibodies toward nucleosides and purified tRNAs is reported. Although the titer of the t6A antiserum was low, the specificity was very sharp. An interesting finding was that threonine, a major structural component of the side-chain modification of t6A, was completely infective as an inhibitor.     

Cytokinin activity of O6-substituted guanine and hypoxanthine derivatives

O⁶-Substituted guanine and hypoxanthine derivatives were prepared and tested for their cytokinin activity by the tobacco callus, radish cotyledons and lettuce seed bioassay systems. The results indicated that some derivatives of both types possess cytokinin activity.     

Synthesis and Comparative Cytokinin Activities of N-(Purin-6-yl)-d- and -l-amino Acid Methyl Esters

Six pairs of enantiomeric N-(purin-6-yl)amino acid methyl esters were synthesized and tested for their cytokinin activities by three bioassay systems, the growth of tobacco callus, the seed germination of lettuce and the fresh weight increase of excised radish cotyledons.l-(—)-Antipodes were as a whole more active than the corresponding d-(+)-isomers in the tobacco callus and seed germination tests, whereas an uniform tendency was not observed in the radish cotyledon expansion. The discussions were focused on the effects on the biological response of configurational arrangements around the asymmetric center at α-position to the adenine ring and on species difference of cytokinin receptor molecules.     

Biological activity of p-methylaminophenol,an essential structural component of N-(4-hydroxyphenyl)retinamide,fenretinide

Fenretinide, N-(4-hydroxyphenyl)retinamide (4-HPR), is a synthetic amide of all-trans-retinoic acid (RA), which inhibits cell growth, induces apoptosis, and is an antioxidant, and cancer chemopreventive and antiproliferative agent. These findings led us to investigate which structural component of 4-HPR contributes to these potent activities. Our approach was to examine 4-aminophenol (4-AP), p-methylaminophenol (p-MAP), and p-acetaminophen (p-AAP). It was found that vitamin E, 4-AP and p-MAP scavenge alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radicals in a 1:2 ratio, in contrast to 4-HPR and p-AAP, for which 1:1 and 1:0.5 ratios were observed relative to DPPH radicals. However, RA was inactive. Lipid peroxidation in rat liver microsomes was reduced by compounds (RA > p-MAP = 4-HPR > 4-AP) in a dose-dependent manner, while p-AAP was inactive. In addition, both p-MAP and 4-HPR are potent inhibitors of cell growth and inducers of apoptosis in HL60 cells. p-MAP exhibits the same level of antiproliferative activity as 4-HPR against HL60R cells, which are a resistant clone against RA, and it inhibits the growth of various cancer cell lines (MCF-7, MCF-7/Adr(R), HepG2, and DU-145) to an extent greater than 4-AP and p-AAP, but is less potent than 4-HPR. Thus, although the antioxidant activity of p-MAP is more potent than that of 4-HPR, p-MAP is less potent than 4-HPR in anticancer activity. These results suggest that both the anticancer and antioxidative activities shown by 4-HPR are due to the structure of p-MAP. The retinoyl residue or long alkyl chain substituent attached to an aminophenol may be significant for anticancer properties.     

Cytokinin activity of N-phenyl-N′-(4-pyridyl)urea derivatives

We have synthesized 35 N-phenyl-N′-(4-pyridyl)urea derivatives and tested their cytokinin activity in the tobacco callus bioassay. Among them, N-phenyl-N′- (2-chloro-4-pyridyl)urea is highly active, the optimum concentration of which is lower than 4 × 10^?9 M (0.001 ppm), 3 compounds, i.e. N-(2-methylphenyl)-N′-(2-chloro-4-pyridyl)urea, N-(3-methylphenyl)-N′-(2-chloro-4-pyridyl)urea and N-(3-chlorophenyl)-N′-(2-chloro-4-pyridyl) urea are as active as N⁶-benzyladenine (concentration for optimum yield: 4.4 × 10^?8 M or 0.01 ppm), and N-phenyl-N′-(2-methyl-4-pyridyl)urea and N-(2-chlorophenyl)-N′-(2-chloro-4-pyridyl)urea are as active as N-phenyl-N′-(4-pyridyl)urea (concentration for optimum yield: 4.7 × 10^?7 M or 0.1 ppm), while the activity of the other 29 compounds are not so remarkable and 11 of them are almost or completely inactive.