Chrom P column). The benzene fraction was collected (~15 mg) and was found to contain 73 % C6H5D by mass spectrometry.Isolation of Hydrazobenzene and Diphenyl. Acetonitrile (5 ml)was added to the yellow residue from a (0.2 M) phenyldiazene de-composition reaction after distillation to dryness. Thin layer chromatography showed four spots after developing with CHC13pentane, 23:19 v/v. The main component (R¡ 0.86) was shown to be hydrazobenzene by uv spectrum and formation of benzidine on treatment with 0.1 % HC104 in CH3CN.Thick layer chromatography of the solution of the yellow residue gave nine distinct zones after development with CHCl3-pentane, 25:75 v/v. The component with the greatest R¡ value (0.63) was colorless and was shown to be diphenyl by its uv spectrum.Isolation of Azobenzene. The solution from a phenyldiazene decomposition (0.0135AT) was evaporated to dryness. The residue (in «-pentane) was chromatographed on an alumina (acid, Woelm) column (3 mm diameter, 3 cm long). Elution with «-pentane saturated with acetonitrile (2 ml) yielded a solution of azobenzene (by uv spectrum).Reaction with Hydroxide Ion. Distilled phenyldiazene solution in acetonitrile (10.4 ml, (1.97 ± 0.08) X 10~4 M) was mixed with degassed phosphate buffer (10 ml, pH 7.1, 0.05 M) introduced through a breakseal. A spectrum indicated (a) little change in the concentration of 3 and (b) only a trace of benzene. Sodium hydroxide solution (2.5 ml, 1 M) was introduced through a second breakseal. Sodium phosphate crystallized as a white solid. A spectrum showed that all of the phenyldiazene had reacted.Benzene was extracted with «-pentane (5 ml, two times) after open-ing to air and adding water (6 ml). The extract was distilled and analyzed by uv spectroscopy. After correcting for incomplete recovery (measured with controls), the yield of benzene was found to be 79 ± 8%.Reaction with Perchloric Acid. Six milliliters of phenyldiazene solution (1.4 X \0~i M) was mixed with 1.5 µ of degassed 70% aqueous perchloric acid (11.8 IV). Changes in the spectrum of the reaction solution were followed (Figure 7).Reaction with Benzenesulfonyl Chloride and Methyl Chloroformate. Phenyldiazene solution (1.4 X 10-4 M, 4.6 ml) was mixed with 0.35 ml of benzenesulfonyl chloride in acetonitrile (1.83 X 10~3 M). The reaction was followed by spectroscopic means. Degassed triethylamine solution in acetonitrile (5 X 10-2 , 0.5 ml) was introduced after 24 hr. Phenyldiazene solution (1.4 X 10~4 M, 4.2 ml) was mixed with 0.1 ml of degassed methyl chloroformate solution in acetonitrile (0.59 M). After five spectra of the reaction solution had been taken, 5 µ of degassed triethylamine (3.6 X 10-2 mmole) was introduced (1 hr after mixing). Other than the normal bimolecular disappearance of 3, no reaction was observed in either case.Reaction with Azobenzene. Phenyldiazene solution (1.7 X 10-4 M, 4.4 ml) was mixed with 0.25 ml of azobenzene solution in acetonitrile (1.34 X 10-3 M). The reaction was followed for 24 hr. The change in the concentration of 3 was that expected...

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A new chemical method for radioactive labeling of single-stranded regions of RNA has been used to probe the three-dimensional structure of E. coli tRNA(fMet) in solution. The procedure involves conversion of cytosine residues to N(4)-[(14)C]methylcytosines by treatment with (14)CH(3)NH(2) and sodium bisulfite at pH7. Ribonuclease digestion of the modified tRNA produces (14)C-labeled oligonucleotides which comigrate with the corresponding unlabeled oligonucleotides, facilitating structural analysis. By this procedure, E. coli tRNA(fMet) has been found to contain only six reactive cytosines: C(1), C(16), C(17), C(35), C(75) and C(76). In addition, slow reaction at C(m) (33) was observed. These results are in excellent agreement with previously reported data on the sites of exposed cytosine residues in tRNA(fMet) obtained by two other chemical methods. The methylamine-bisulfite procedure is recommended for studying the ordered structure of more complex polyribonucleotides such as viral and ribosomal RNAs.

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chemischen Verschiebung . !~~= & H ( P ) -~H ( N ) entnommen werden [vgl. (3,) und (341. Auch die Substitution der Aminogruppe scheint eine Rolle zu spielen [vgl. (3b) und (3c)l. Gaschromatographisch lieR sich erkennen, daR uberwiegend (95%) eines der beiden Stereoisomeren von (3) gebildet wird. Die h d e r u n g der chemischen Verschiebung 6 PbOl "1 prof. D~. R. west